Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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1 Forum Original Research Communication Sulfite Oxidase Catalyzes Single Electron Transfer at Molybdenum Domain to Reduce Nitrite to Nitric Oxide Jun Wang1,2#, Sabina Krizowski3#, Katrin Fisher-Schrader3, Dimitri Niks4, Jesús Tejero1,2, Courtney Sparacino-Watkins1,2, Ling Wang1,2, Venkata Ragireddy1,2, Sheila Frizzell1,2, Eric E. Kelley1,5, Yingze Zhang2, Partha Basu6, Russ Hille4, Guenter Schwarz3+, and Mark T. Gladwin1,2,+* From the 1Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, 15213 2Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, 15213 3Biochemistry, Institute of Biochemistry and Center for Molecular Medicine, Cologne University 4Department of Biochemistry, University of California at Riverside, Riverside, CA, 92521 5Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA, 15213 6Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, 15261. Address correspondence to: Mark T. Gladwin, MD, Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA 15261. Tel: 412-692-2210; Fax: 412-692-2260; E-mail: [email protected]; Guenter Schwartz, PhD, Institute of Biochemistry, Department of Chemistry Cologne University, Zuelpicher Str. 47, 50674 Koeln, Germany. Tel: +49-221-470 64 40; Fax +49-221470 50 92; E-mail: [email protected]

Running head: sulfite oxidase functions as nitrite reductase Word count: 5859 Reference number: 55 Number of grayscale illustrations: 3 Number of color illustration: 5

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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2 Abstract Aims: Recent studies suggest that the molybdenum enzymes xanthine oxidase, aldehyde oxidase and mARC exhibit nitrite reductase activity at low oxygen pressures. However, inhibition studies of xanthine oxidase in humans have failed to block nitrite-dependent changes in blood flow, leading to continued exploration for other candidate nitrite reductases. Another physiologically important molybdenum enzyme – sulfite oxidase (SO) – has not extensively been studied. Results: Using gas-phase NO detection and physiological concentrations of nitrite, SO functions as nitrite reductase in the presence of a one-electron donor, exhibiting redox-coupling of substrate oxidation and nitrite reduction to form NO. With sulfite, the physiological substrate, SO only facilitates one turnover of nitrite reduction. Studies with recombinant heme and molybdenum domains of SO indicate that nitrite reduction occurs at the molybdenum center via coupled oxidation of Mo(IV) to Mo(V). Reaction rates of nitrite to NO decreased in the presence of a functional heme domain, mediated by steric and redox effects of this domain. Using knock-down of all molybdopterin enzymes and SO in fibroblasts isolated from patients with genetic deficiencies of molybdenum cofactor and SO, respectively, SO was found to contribute significantly to hypoxic nitrite signaling as demonstrated by activation of the canonical NO-sGC-cGMP pathway. Innovation: Nitrite binds to and is reduced at the molybdenum site of mammalian SO, which may be allosterically regulated by heme and molybdenum domain interactions, and contributes to the mammalian nitrate-nitrite-NO signaling pathway in human fibroblasts. Conclusion: SO is a putative mammalian nitrite reductase, catalyzing nitrite reduction at the Mo(IV) center.

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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3 Introduction While nitrate and nitrite were traditionally considered to be inert and potentially toxic metabolites of nitric oxide (NO) oxidation, it is now increasingly appreciated that both nitrate and nitrite can be recycled to bioactive NO (28,10,12). Studies have suggested that this reductive mammalian nitrate-nitriteNO pathway mediates important signaling events and as such compliments the traditional oxidative Larginine-NO synthase-NO pathway (29). Nitrate is formed in blood from oxidation of NO by oxyhemoglobin or is derived from dietary sources, such as leafy green vegetables and water. While the majority of absorbed inorganic nitrate is ultimately excreted into the urine, up to 25% of plasma nitrate is taken up by the salivary glands and concentrated 10- to 20- fold in saliva, providing a route for enterosalivary recycling of nitrate (52,34,30). Symbiotic bacteria in the oral cavity effectively reduce nitrate to nitrite, which is further reduced to NO in the acidic stomach or absorbed systemically (50,34,30). Approximately half of plasma nitrite is derived from reduction of dietary nitrate by oral bacteria whereas half is derived from the oxidation of NO synthase-generated NO in plasma by ceruloplasmin (40,36). Nitrite is bioactive, and is reduced to NO to regulate blood pressure (10,12,13,50), hypoxic vasodilation (10,16,31), cellular cytoprotection (34) and mitochondrial respiration and function under hypoxic or exercise stress (41,24,51). A central current focus in this field aims to elucidate the processes and enzymes contributory to the one-electron and one-proton transfer reaction of nitrite to form NO (Eq. 1). NO2- + H+ + e-  NO + OH-

Eq. 1

Such proton and electron transfer reactions can be catalyzed by redox active hemoproteins, such as hemoglobin, myoglobin, neuroglobin, cytoglobin and plant hemoglobins (10,41,48,19,45,47), and can also be catalyzed by molybdenum cofactor (Moco-) containing enzymes, such as xanthine oxidase, aldehyde oxidase and mARC (26,2,42). However, despite evidence that these Moco enzymes can contribute to nitrite-dependent signaling in rodents, studies in humans have shown that infusions of allopurinol or oxypurinol, specific xanthine oxidoreductase inhibitors, do not sufficiently block nitrite-

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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4 mediated vasodilation (12). Therefore we evaluated SO, a third Moco-containing enzymes, as a potential nitrite reductase and further examined the catalytic mechanism in the purified enzyme system. In contrast to xanthine oxidase and aldehyde oxidase, eukaryotic nitrate reductase in plants and SO in animals and plants represent a distinct second family of Moco enzymes that catalyze oxygen atom transfer reactions (44). For plant nitrate reductase, nitrite-dependent NO synthesis has been reported (53). Given the high structural similarity between nitrate reductase and SO (9), it is likely that nitrite would react with the reduced molybdenum center of SO. Sulfite oxidase is a homodimer (110 kDa) with each monomer containing a N-terminal cytochrome b5-type heme, a central Moco domain, and a C-terminal dimerization domain. It is located in the mitochondrial intermembrane space of mammalian cells where it aerobically converts sulfite to sulfate while concomitantly reducing two molecules of cytochrome c, representing the last step in the enzymatic catabolism of cysteine. Mechanistic studies indicate that during the sulfite-SO-cytochrome c catalytic cycle, movement between the molybdenum and heme domain is required to enable efficient single-electron transfer from molybdenum via the heme b5 cofactor to cytochrome c (21). As nitrite-dependent NO synthesis is enhanced under hypoxia, we hypothesized that under hypoxic or ischemic conditions a fully reduced system would serve to switch sulfite-mediated cytochrome c reduction in mitochondria to a nitrite reduction reaction. Herein, we report that SO can function as source of bioactive NO by reducing nitrite at the molybdenum domain, coupled to a one-electron oxidation of Mo(IV)-to-Mo(V).

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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5 Results Reduced Human SO Converts Nitrite to NO. To determine if pre-reduced SO reduces nitrite to nitric oxide (NO) at physiological concentrations of nitrite (14,8), we performed measurements in a purge vessel under anaerobic conditions (helium gas purging through the reaction mixture). Rates were determined by integration of the peak NO generation rate over a 12-second interval (detailed methods are provided in the Supplementary Material). We validated these measurements using a purge and non-purge system with both fast (nitrite in acidified iodide) and slow (DETA-NONOate) NO donor standards and using SO holo enzyme preparations (Supplemental Figures 2-3). We use the purge vessel system for our kinetic measurements as this has been the standard in the field evaluating molybdenum enzymes as nitrite reductases (25-27, 51), allowing for comparisons of measured reaction kinetics. We

appreciate some

limitations using this method related to protein denaturation over time, exhaustion of the reducing agent, and potential oxidative damage to the Moco center. Our experiments used recombinant human SO ([Mo] = 0.63 μM), and phenosafranine (1 or 2electron donor) or sulfite (2-electron donor) as reducing substrates. To study the kinetics of SO-dependent nitrite reduction, first the concentration dependence of nitrite was assessed in the presence of a fixed concentration of phenosafranine (40 μM) and human SO. A concentration-dependent increase in the rate of NO formation was observed for nitrite (20 μM-100 mM) at pH 7.4 and fitted to the Michaelis-Menten equation (Fig. 1). When concentrations of nitrite are above 4 mM, the NO detected was over 1000 mV at the high-sensitivity mode, and thus the low-sensitivity mode was set to measure NO production when nitrite is in the range of 5-100 mM. Measured vmax, Km and kcat values are 34 nmoles-1mg-1, 80 mM and 1.9 s-1, respectively. At pH 6.5, the rate of NO formation also increases with the concentration of nitrite, although the kinetic parameters were not determined (Supplementary Fig. 1). It is notable that SO only catalyzes a single turnover reaction in the presence of sulfite, with no steady state NO formation observed. However, the relationship between the observed rates and the

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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6 concentration of nitrite shows a noticeable deviation from a lineal relationship and appears to indicate a possible reaction complex formation. We interpret this fact as the result of a reaction as follows: SO(IV) + Nitrite  [SO-Nitrite]  SO(V) + NO Where SO(IV) and SO(V) represent SO proteins with its molybdenum atom in the oxidation states IV and V, respectively (27,25). This kind of single turnover reaction can produce kobs vs [substrate] plots similar to Michaelis-Menten (17). We have renamed the calculated parameters Kd and ket, to indicate that they correspond to an apparent dissociation constant and an electron transfer rate for reactions in the presence of sulfite. Since sulfite is the only known physiological reducing substrate for SO, NO generation was next studied with sulfite as the reductant. In the absence of SO or in the presence of only sulfite or nitrite, NO formation was not detected, while the combination of nitrite and SO in the presence of either sulfite or phenosafranine resulted in significant NO generation, suggesting redox-coupling of substrate oxidation to nitrite reduction (Fig. 2A-B). It is important to note that the addition of nitrate does not lead to NO formation, suggesting that SO is not a nitrate reductase. NO generation with phenosafranine is ~10 folder greater than that observed with sulfite (Fig. 2B), indicating that phenosafranine is a more efficient electron donor for nitrite reduction. This different reactivity with a strict two-electron donor, sulfite, versus a one or two-electron donor like phenosafranine, prompted us to study the reaction mechanism in more details, as discussed below. To study the kinetics of SO-dependent nitrite reduction, first the concentration dependence of nitrite was assessed in the presence of a fixed concentration of sulfite (5 μM) and human SO. A concentration-dependent increase in the rate of NO formation was observed for nitrite (20 μM-10 mM) and a plot of kobs versus nitrite indicated a hyperbolic fit similar to Michaelis-Menten kinetics for both pH 6.5 and 7.4 (Fig. 2C-E), with vmax, Kd and ket values are 0.0361 nmoles-1mg-1, 1.6 mM and 0.002 s-1 at pH 7.4 and 0.0796 nmoles-1mg-1, 1.7 mM and 0.0044 s-1 at pH 6.5, respectively. The increases of vmax 6

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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7 and ket with the lower pH are not unexpected based on Eq. 1 and have been reported for the nitrite reduction reactions catalyzed by hemoglobin, myoglobin and neuroglobin (10,41,46). However, the NO generation rates only increased for about 3 fold per unit change of pH, instead of the well-known 10-fold increase for a reaction of heme globins with nitrous acid. The reasons for different influences of pH, which was also observed in other Mo-dependent enzymes (25-27), are unknown but may involve a direct binding of nitrite or protonation of active site residues at the Mo center. As discussed and evaluated below, the low ket values using sulfite as the reductant suggest that this may not be a catalytic reaction. When sulfite concentration was examined for effects on NO formation, no significant differences in the rate of nitrite reduction to NO were observed (2 μM – 5 mM, pH 6.5) (Supplementary Fig. 4A). However, at pH 7.4, NO formation rates were decreased at sulfite concentrations greater than 1 mM, suggesting a substrate-mediated inhibitory effect on nitrite reduction (Supplementary Fig. 4B). These results suggest that both nitrite and sulfite react at the molybdenum active site. Finally, all results were reproduced with mouse SO (Supplementary Table 1 and Supplementary Fig. 4C-H). Nitrite Reduction to NO at the Molybdenum Domain of SO. To further assess whether nitrite is reduced at the molybdenum (Mo) center, we next evaluated reactions of nitrite with isolated recombinant Mo- and heme-domains of human SO. Recombinant Mo-domain was used for kinetic studies of NO generation as a function of nitrite or phenosafranine or sulfite concentration, and for quantification of nitrite reduction in the presence of phenosafranine or sulfite. The concentration dependence of nitrite was first determined at a fixed concentration of 40 μM phenosafranine. Upon addition of 0.25 μM Mo-domain to nitrite and phenosafranine, NO formation was measureable with a lowest-detectable NO concentration at 5 μM nitrite at pH 7.4 (Fig. 3A-B). MichaelisMenten kinetics were obtained showing vmax, Km and kcat values with 112 nmoles-1mg-1, 31 mM and 4.7 s-1 at pH 7.4, respectively, with significantly higher vmax and kcat, and lower Km values than observed with the holo-enzyme (Supplementary Table 1). At pH 6.5, when nitrite was in the range of 1-400 μM, NO

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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8 production by Mo-domain was faster than at pH 7.4 (Supplementary Fig. 5). When sulfite was used as the reducing substrate, the rates showed a similar dependence towards nitrite but were much lower than those observed in the presence of phenosafranine. The rates observed at pH 6.5 were higher than those at pH 7.4, as observed in the presence of phenosafranine (Fig. 4A-C), with vmax, Kd and ket values are 0.179 nmoles-1mg-1, 0.973 mM and 0.0075 s-1 at pH 7.4, and 0.323 nmoles-1mg-1, 0.496 mM and 0.0136 s-1 at pH 6.5. Our studies using phenosafranine or sulfite as reducing substrate show much faster nitrite reduction to NO by the Mo-domain than by holo-SO (Supplementary Table 1). Mechanistically, these data suggest that nitrite reacts with the Mo-domain, and that electron transfer between Mo- and hemedomains and/or steric hindrance of the heme domain reduces the reaction rates with nitrite by approximately a factor of three. All results could be reproduced with the mouse Mo-domain using sulfite as the reductant (Supplementary Figs. 6-7). To investigate the inhibitory effect of the heme domain, we evaluated rates of NO formation and nitrite reduction in the presence of the heme domain, the Mo-domain and holo-SO at the same concentrations (Fig. 4D-E). When 40 μM phenosafranine was mixed with 1 mM nitrite in an anaerobic vessel, addition of the isolated heme domain did not generate NO whereas addition of the Mo-domain and holo-SO resulted in NO formation (Fig. 4D), suggesting that the heme domain alone does not reduce nitrite to NO. When 10 μM nitrite was mixed with 50 μM sulfite under anoxic conditions, nearly zero nitrite was reduced in 60 min in the presence of holo SO or the Mo-domain (Fig. 4E). However, when 50 μM phenosafranine replaced sulfite, around 25% and 55% nitrite were reduced anaerobically in the presence of holo-SO and the Mo-domain, respectively, while the heme domain did not reduce nitrite (Fig. 4E). Furthermore, nitrite-mediated phenosafranine oxidation by both the Mo-domain and holo-SO confirmed that the catalytic activity of the Mo-domain is ~2.5-fold greater than observed with the holoenzyme (Fig. 4F). All these results provide further evidence that nitrite reduction occurs at the Modomain, with enhanced reaction rates in the presence of phenosafranine compared with sulfite. 8

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9 Effects of the Heme Domain on Rates of Nitrite Reduction. To determine if either the heme-iron center or the heme domain itself negatively modulates NO production within the holoenzyme, we quantified the NO generation rates in the presence of a heme-deficient mouse SO, H119A/H144A variant (Fig.5, Supplementary Fig. 8A-C). Loss of His119 and His144, which coordinate heme iron, results in an apoheme domain, with an overall preservation of the enzyme structure as confirmed by circular dichroism (CD) spectroscopy (Fig. 5A). Functionally, the H119A/H144A SO and holo-SO exhibited approximately 30% and 20% of the NO formation activity of the isolated Mo-domain, respectively, suggesting that there is both a steric and electronic inhibitory effect from the heme domain on Mo-domain-dependent nitrite reduction (Fig. 5B, Supplementary Fig. 8A-C). Catalytic Mo(IV) to Mo(V) Nitrite Reduction Cycle. Based on the observation that phenosafranine, a one- or two-electron donor, efficiently catalyzes nitrite reduction by SO, while sulfite (a strict 2-electron donor) does not, we studied reactions of pre-reduced SO with nitrite under anaerobic conditions, in the presence or absence of sulfite. Importantly, addition of sulfite did not promote additional NO generation beyond that of the fully pre-reduced SO (Fig. 6A). This result suggests that sulfite cannot enter into a catalytic cycle with nitrite reduction, i.e. sulfite only modulates one turnover of nitrite reduction. On the other hand, addition of phenosafranine to reduced SO did increase NO production, suggesting that a oneelectron donor can catalyze nitrite reduction by SO, forming a redox cycle of phenosafranine oxidation and nitrite reduction. We applied EPR spectroscopy to monitor changes at the Mo center during its reaction with sulfite, nitrite and phenosafranine. When 20 μM Mo-domain was mixed with 100 μM sulfite, the Mo center was EPR silent, reflecting the anticipated reduced Mo(IV) state (Fig. 6B-(a)). When excess nitrite (1 mM) was added and incubated for 1.5 h, a stable Mo(V) spectrum appeared (15) and no further change was observed (Fig. 6B-(b)), indicating a one-electron oxidation but inability to convert to the Mo(VI) oxidation state. Upon addition of 50 μM reduced phenosafranine to the solution and quickly freezing afterwards (15 s), the Mo(V) spectrum disappeared, suggesting a conversion of Mo(V) back to the EPR9

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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10 silent Mo(IV) (Fig. 6B-(c)). Upon thawing and incubation with original added nitrite (1 mM) again for another 1.5 h, more than 80% of the Mo(V) signal was recovered (Figure 6B-(d)). These data suggest that Mo(IV), formed initially from the two-electron reduction of Mo(VI) by sulfite, is then oxidized to Mo(V) by nitrite (model shown in Fig. 7). Importantly, nitrite cannot further oxidize Mo(V) to EPR-silent Mo(VI). As sulfite cannot reduce Mo(V) to Mo(IV), the reaction stops at Mo(V). Addition of phenosafranine is able to reduce Mo(V) to Mo(IV) allowing nitrite to be reduced again (Fig. 7). These data in aggregate explain the lack of redox cycling of nitrite and sulfite and evident catalytic cycling of nitrite and the one-electron donor phenosafranine (Fig. 7). SO Catalyzes Nitrite-NO-cGMP Signaling in Human Fibroblasts. Fibroblasts isolated from patients with Moco deficiency (MOCD), which produces inactive SO, xanthine oxidase and aldehyde oxidase, as well as fibroblasts from a SO-deficient (SOD) patient were used to study hypoxic nitrite exposure on cGMP generation. Control experiments were performed using fibroblasts from healthy individuals cultured and treated under identical hypoxic conditions (1% O2). Sulfite oxidase expression in MOCD or SOD fibroblasts was not detectable when 120 μg protein was loaded for Western Analysis assay (Fig. 8A), indicating the deficiency of SO in patients’ cells. Control experiments were performed using fibroblasts from healthy individuals cultured and treated under identical hypoxic conditions (1% O2). In the presence of a NO donor, DEA-NO, cGMP levels increased significantly (Fig. 8B). Addition of nitrite (5-100 µM) to the medium produced a dosedependent increase in cellular cGMP (Fig. 8B). For fibroblasts from either Moco- or SO-deficient patients treated with 50 µM nitrite, the cGMP generation was reduced by approximately 80% (Fig. 8C), indicating a classic nitrite-NO-soluble guanylyl cyclase-cGMP response. A time-dependent exposure of wild type fibroblasts to nitrite showed a linear increase of cGMP levels over time (120 min) with a rate of 4.3 fmol cGMP per mg and minute (Fig. 8D). In contrast, SO-deficient cells showed no significant cGMP production (Fig. 8E) in the same time frame. Similar exploration of cGMP production was performed under normoxia (20% O2). The basal level of cGMP at 20% O2 is higher than at 1% O2 (Fig. 8F), which 10

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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11 indicates higher oxygen availability for NO synthase-dependent NO formation in fibroblasts under normoxia. Treatment with nitrite increases cGMP production in fibroblasts even at 20% oxygen, but the fold-increase was less under normoxia compared with hypoxia (Fig. 8G), which is also supported by less NO generation under normoxia detected in the NO analyzer (Supplementary Fig. 9). Most importantly, the effect of nitrite on cGMP formation was abrogated in the Moco- or SO-deficient patient’s fibroblasts (Fig. 8F). These results demonstrate that SO represent the primary Moco-dependent enzyme required for enzymatic conversion of nitrite to NO in human fibroblasts, given that the observed nitrite-dependent cGMP formation is impaired in SO-deficient cells.

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12 Discussion Mammals utilize two pathways to produce NO and regulate blood pressure and blood flow, the Larginine-to-NO pathway and the nitrate-to-nitrite-to-NO pathway (29). These NO biosynthesis pathways respond differently to oxygen tension. The arginine-to-NO pathway requires oxygen as a substrate for NO formation; conversely, the nitrate-to-nitrite-to-NO pathway is potentiated under hypoxic conditions. The upstream pathways for conversion of nitrate to nitrite have been clearly defined since early studies first described the involvement of this pathway in the generation of NO in the stomach, a process shown to be important in regulating stomach mucosal integrity, mucosal host defense, and later mucosal blood flow (28,5,6). Since the nitrate-to-nitrite-to-NO pathway exhibits increased potency under lower oxygen tensions, a role for nitrite as an effector of hypoxic signaling and vasodilation has been considered (10,29,16). While aerobic NO production has been extensively investigated, enzymes that regulate nitriteto-NO reduction are less well understood. Several studies have investigated the possible enzymatic origins for mammalian nitrate and nitrite reduction and a number of enzyme systems have been reported to control nitrite reduction to NO under certain conditions. As mentioned, xanthine oxidase has been proposed as a mammalian nitrate and nitrite reductase (18). A number of studies have demonstrated that the therapeutic effects of nitrite on pulmonary hypertension require a functional xanthine oxidase system (2,55), however the inhibition of xanthine oxidase does not inhibit nitrite-dependent vasodilation in human circulation (12). Several other mammalian nitrite reductase candidates have been proposed, such as the other Mo-containing enzyme aldehyde oxidase and heme enzymes (cytochrome c, eNOS, deoxyhemoglobin, deoxymyoglobin, neuroglobin, cytoglobin), as well as one nitrite anhydrase (carbonic anhydrase) (41,19,1,39,4). However, it is likely that additional catalytic human nitrate and nitrite reductases have not yet been identified, such as SO, which has comparable or higher activity than xanthine oxidase and aldehyde oxidase when phenosafranine, a one- or two-electron donor, was used as the reducing substrate. When sulfite, a strict

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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13 two-electron donor and a known physiological substrate of SO, was used as the reductant, less NO was produced. These studies suggest that reduced SO can reduce nitrite to form NO, in a reaction analogous to that of xanthine oxidase and aldehyde oxidase. It is now clear that many heme-based and molybdopterinbased proteins have the capacity to reduce nitrite to NO. Enzymes that metabolize nitrite to NO are likely dependent on specific organ location and local oxygen tensions. For example, deoxyhemoglobin reduces nitrite to NO in the blood, and this pathway has been shown to regulate hypoxic vasodilation (10,11) and more recently, inhibition of platelet activation (43). Deoxymyoglobin plays a major role in heart (41,38) and smooth muscle, where it regulates mitochondrial respiration, myocardial energetics, and hypoxic vasodilation. Consistent with a role for heme globins in vasodilatory and platelet inhibitory effects of nitrite, oxypurinol does not inhibit nitrite-dependent vasodilation. On the other hand, treatment of rodents with tungsten, which replaces molybdenum in xanthine oxidase, aldehyde oxidase and SO, inhibits the protective effects of nitrite in vascular injury models, such as carotid wire injury and pulmonary arterial hypertension (2,54). Allopurinol can also inhibit the effects of nitrite in many pre-clinical disease models (15). Our present studies using fibroblasts for SO-deficient patients suggest that SO plays a dominant role in nitrite reduction and sGC activation in these cells. These data in aggregate suggest that a number of enzymes can reduce nitrite to NO and the dominant effect of one system likely depends on enzyme concentration and oxygen concentration in specific tissues. Future work with conditional knock-out of xanthine oxidase, aldehyde oxidase and SO will be required to fully explore the relative importance of each in specific tissues and diseases. In this study we examined the enzymatic reactions of human and mouse SO in vitro with phenosafranine, sulfite, and nitrite using NO detection, nitrite reduction and EPR methodologies revealing that SO can catalyze the reduction of nitrite to NO by the one-electron oxidation of Mo(IV) to Mo(V). We unequivocally demonstrated that only fully reduced Mo(IV) enzyme is capable of nitrite reduction, while

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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14 Mo(V) does not produce NO. This result indicates that both the electron donor and the redox potential of the enzymatic metal center play an important role in catalytic reactions, which determines the electron flow in SO. SO has two internal cofactors: heme and molybdenum, which have reduction potentials of 68 mV (heme), -239 mV (Mo(V)/Mo(IV)) and 38 mV (Mo(VI)/Mo(V)), respectively (9). Electrons flow from the Mo center to the heme center, and Mo(V) is the favorable oxidation state. In order to efficiently catalyze nitrite reduction in vivo, either an appropriate one-electron donor can reduce Mo(V) to Mo(IV) or an oxidant can oxidize Mo(V) to Mo(VI), allowing sulfite to react with SO again. Following the nitritedependent formation of Mo(V), the remaining electron may be transferred to the heme domain, which in turn requires one cycle of cytochrome c reduction to complete the catalytic cycling. Given that reduced SO was not found to react with nitrate, we conclude that SO cannot function as nitrate reductase. A recent study by Qui & Rajagopalan has engineered a SO enzyme variant with partial nitrate reductase activity using structure-guided mutagenesis (35). Plant nitrate reductases have three internal cofactors: FAD, heme and molybdenum cofactor (Moco). Reduction potentials of them are 272 to -187 mV, -123 to -174 mV, and -25 to +15 mV, respectively (9). This redox pattern is consistent with a downhill flow of electrons within the enzyme, i.e. electrons are readily transferred from heme to Mo for nitrate reduction. In addition, electrons from NAD(P)H, the physiological reductant, are passed to heme and Mo sites one by one via the FAD domain. However, the well-accepted flow of electrons in SO is from the Mo domain to the heme domain, and finally to cytochrome c, the physiological oxidant. Our studies also suggest that the heme domain reduces the rate of nitrite reduction to NO. The fact that the apo-heme domain also inhibits nitrite reduction to some extent suggests an important conformational component in the access of nitrite to sulfite-reduced SO. There is a flexible hinge (14 residues in human SO) that tethers N-terminal heme domain to Mo-domain (21). Previous studies have shown that this hinge controls domain motion during the catalytic cycle of SO, which adopts various conformations during intra-molecular electron transfer (IET) and catalysis (49). Thus domain motion appears to change domain distance and modulate activity, as supported by molecular dynamic studies that 14

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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15 propose a much shorter Mo-heme distance (19 Å) during IET as compared to the distance in the crystal structure of SO (32 Å) (37). Electronically, there are six possible redox states of SO, depending on oxidation states of molybdenum and heme iron. Under hypoxic, ischemic or reductive conditions, cytochrome c is fully reduced, which in turn will slow down re-oxidation of the heme iron and ultimately inhibit IET between Mo and heme. This can provide a possible mechanism for hypoxic and/or ischemic nitrite-dependent NO formation catalyzed by SO. In addition to hypoxia, sulfite accumulation would increase the steady-state levels of Mo(IV) reduced SO. However, such a condition is only known for Moco- or SO- deficiency, genetic disorders leading to rapidly neurodegeneration, mental retardation, and early childhood death. Recently, a first successful experimental treatment for Moco deficiency has been reported (50). Those patients lack Moco and consequently sulfite accumulates in large quantities. Due to the reconstitution of Moco biosynthesis in treated patients, SO activity returns and excess sulfite is removed with 72-96 hours post-treatment (50). In the present study, Moco- or SO-deficiency in isolated human fibroblasts caused significant decreases of nitrite-dependent cGMP generation, supporting a role for SO in nitrite-dependent intracellular NO signaling. This work has identified human SO as a new candidate nitrite reductase that leads to NO production in vitro and in vivo, which requires a one-electron donor to drive the catalytic cycle between Mo(IV) and Mo(V) in the purified enzyme system. SO contributes to NO-soluble guanylyl cyclase-cGMP signaling in human fibroblasts. Further studies are needed to elucidate the physiological mechanism and significance how SO function in nitrite-NO signaling in human cells and tissues.

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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16

Innovation

Nitrite binds to and is reduced at the molybdenum site of mammalian SO to form NO, and thus

contributes to the mammalian nitrate-nitrite-NO signaling pathway.

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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17 Materials and Methods Reagents and Standard Samples Preparation. All chemicals were purchased from Sigma-Aldrich (USA and Germany). Holo SO, isolated Mo and heme domains were reduced by sulfite or dithionite, and then purified by passing through a Sephadex G-25 gel filtration column and eluted with the indicated buffer anaerobically. Concentrations of the heme cofactor in holo SO and heme domain were determined by measuring absorbance of the heme Soret band using ε414 = 113 mM-1 cm-1 or ε423 = 164 mM-1 cm-1. Concentrations of the Mo cofactor in holo SO and isolated Mo-domain were determined by HPLC Form A analysis (22). Cloning, Expression and Purification of the Recombinant Proteins. Mature human and mouse SO were PCR cloned into the SacI and SalI sites of pQE-80L (Qiagen, Hilden, Germany). The resulting constructs were used to create the Mo and heme domain expression constructs, as well as the heme-deficient variant (H119A/H144A) using the same restriction sites. All constructs were expressed in E.coli strain TP1000 (33,46) for 48 h at 24°C. Protein expression from pQE80 was induced after 4 h incubation at 24°C with 100 µM IPTG. 500µM sodium molybdate was added to the culture to achieve sufficient levels of Moco production. His-tagged SO was affinity purified according to the instructions of the manufacturer (Macherery, Nagel). Protein purification included further anion exchange with a SourceQ15 column (GE Healthcare) using 50 mM Tris-acetate, pH 8.0, as buffer A. Proteins were eluted with a gradient from 100-500 mM NaCl in buffer A. Measurement of NO Formation by Ozone-based Chemiluminescence. Detailed methods are provided in the Supplementary Material. In summary, nitric oxide production was measured by ozone-based chemiluminescence using a Sievers Nitric Oxide Analyzer (NOA 280i) equipped with a liquid sampling purge system (GE Analytical Instruments, Boulder, CO). Sievers NO AnalysisTM Software, Liquid (Version 3.2) was used for data acquisition, at a rate of one data point per 0.25 second.

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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18 The standard glass purge vessel (15-mL) was used to mix and deoxygenate reagents. For enzyme reactions, the reaction buffer (20 mM BisTris buffer or 10 mM phosphate buffer pH 6.5 or 7.4) was maintained in the presence of nitrite for two minutes in the sealed purge vessel to equilibrate the temperature to 37°C and to void oxygen from the reagents prior to initiating the enzyme reaction. Next, reducing substrate (sulfite or phenosafranine) was injected into the purge vessel through the vessel’s septa using a Hamilton syringe. Once a stable baseline was established, SO was injected into the mixture and the reaction was monitored for several minutes, until the reaction completed. NO production rates were estimated using a calibration curve prepared by the nitrite/I3- method (see Supplementary Material for details). All the NO generation rates were plotted against the corresponding concentrations of nitrite, which fitted hyperbola function, i.e. y = (P1 •· x)/(P2 + x). The values of P1 and P2 correspond to νmax and Km values in the Michaelis-Menten equation, i.e. ν = Vmax • [S]/(Km + [S]). In the case of single turnover reactions (non-steady-state), a similar fit was used, where the P1 and P2 terms represent the apparent νmax and Km values, which we refer to as ket (rate of electron transfer) and Kd (apparent dissociation constant) to indicate the single turnover nature of the studied reaction. Nitrite Reduction Assay. Time-course nitrite reduction was evaluated by measuring the concentration of nitrite in the reaction mixture with NOA 280i. 10 µM SO or isolated Mo-domain was mixed with 10 µM nitrite, in the presence of sulfite (50 µM) or reduced phenosafranine (50 µM) inside the anaerobic glove box. Afterwards nitrite concentration was measured at 5, 10, 20, 30, 60 min. Time zero was defined as the same reaction in the absence of protein. Each data point represents the average of three separate measurements. Steady-state Kinetics of Nitrite Reduction by Phenosafranine. Steady–state kinetics were performed anaerobically in a glove box using plate reader (Biotek, Bad Friedrichshall, Germany). All data recorded are mean values of at least triplicate measurements. Initial velocities were determined by following the

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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19 oxidation of the artificial electron donor phenosafranine at 520 nm using an extinction coefficient of 29,000 M-1· cm-1. SO was assayed at room temperature in Bis-Tris/acetate buffer, pH 6.5. In the phenosafranine-dependent reduction of nitrite, 12 µM human holo SO or human SO Mo-domain was mixed with 250 µM phenosafranine and 0-75 mM nitrite. The reaction was started with 250 µM sodium dithionite and control experiments were performed without the addition of enzyme. Circular Dichroism Spectroscopy. Structural similarity between recombinant wild-type mouse SO and the heme-deficient H119A/H144A variant was determined by circular dichroism (CD) spectroscopy with a JASCO J-715 CD spectropolarimeter. All measurements were performed at 20°C and secondary structure was measured over a wavelength range of 180-250 nm. EPR Spectroscopy. Electron paramagnetic resonance (EPR) spectra were recorded at 150 K with the EWWIN 6.0 acquisition and lineshape analysis software (Scientific Software Services) on a Bruker ER 300 spectrometer. Double integration was performed with EWWIN 6.0 using the “very rapid” xanthine oxidase Mo(V) signal as an integration standard (32). All reactions were performed in 50 mM Tris-acetate buffer, pH 8.0 anaerobically. In these experiments, sulfite (100 μM) was added to 20 μM anaerobic Mo domain, the reaction solution was transferred to an argon-flushed EPR tube, frozen and an EPR spectrum recorded. The sample was then thawed, 1 mM nitrite was added, and the mixture incubated for 1.5 h at RT before being re-frozen and a second EPR spectrum recorded. The sample was then re-thawed, 50 μM dithionite-reduced phenosafranine was added and the sample got re-frozen within 15 s of mixing, and a third EPR spectrum was recorded. Finally, the same solution was once again thawed and the reaction mixture incubated for an additional 1.5 h at RT before being frozen for the recording of a final EPR spectrum. Western Blot Analysis. Fibroblast cells were washed with ice-cold PBS, homogenized in PBS with sonication. Then 120 µg total protein of the cell lysate was loaded for electrophoresis. The cell lysates were resolved using 10% Bis-Tris SDS-PAGE gels and transferred to high-quality polyvinylidene

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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20 difluoride (PVDF) membrane. The membrane was then probed with primary antibodies specific to sulfite oxidase (Eurogentec, Germany) and beta-actin (Santa Cruz, Germany). After incubation with the secondary antibodies coupled to horseradish peroxidase from Santa Cruz, the chemiluminescent signals were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Germany). Determination of cGMP production in human fibroblasts. Human fibroblast derived from SO- and Moco-deficient patients or healthy individuals were grown on 15 cm plates at 37°C with 5% CO2 in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco). Cells were grown to about 90% confluence before treatment with 1 µM sildenafil and 5–100 µM nitrite or 5 µM DEA-NONOate for 2 h at hypoxic conditions (1% O2) or aerobically (20% O2). Control samples were incubated either without 5 µM DEA-NONOate or nitrite addition. Time-dependent cGMP production was measured at 0, 15, 30, 60, 120 min with 100µM Nitrite. Cells were harvested and lysed with 100 µl HCl (0.1 M) and total protein concentration was determined by Bradford assay. Triplicates were used for each condition and cGMP concentration was detected with a cGMP enzyme-linked immunosorbent assay kit (Cayman Chemicals). Results are expressed as the means ±standard deviation. Statistical differences between two sets of data were calculated using a Student’s t test. Statistical significance was assumed when P was less than 0.001.

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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21 Acknowledgements We thank Dr. Jack Lancaster for helpful discussions on reaction kinetics in NOA purge systems. Dr. Gladwin receives research support from NIH grants R01HL098032, RO1HL096973, and PO1HL103455, the Institute for Transfusion Medicine and the Hemophilia Center of Western Pennsylvania. Dr. Tejero acknowledges the support from the Competitive Medical Research Fund of the University of Pittsburgh Medical Center (UPMC) Health System. Dr. Guenter Schwarz thanks the German Sciency Foundation (DFG) and the Fonds der Chemischen Industrie for research support.

Author Disclosure Statement No competing financial interests exist.

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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22

Abbreviations Used

CD = circular dichroism

DEA-NO = DEA-NONOate

IET = intra-molecular electron transfer

Moco = molybdenum cofactor

MOCD = molybdenum cofactor deficiency

NO = nitric oxide

SO = sulfite oxidase

SOD = sulfite oxidase deficiency

WT = wild type

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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23 References 1. Aamand R, Dalsgaard T, Jensen FB, Simonsen U, Roepstorff A, Fago A. Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation. Am J Physiol Heart Circ Physiol 297:H2068-2074, 2009. 2. Alef MJ, Vallabhaneni R, Carchman E, Morris SM, Shiva S, Wang Y, Kelley EE, Tarpey MM, Gladwin MT, Tzeng E, Zuckerbraun BS. Nitrite-generated NO circumvents dysregulated arginine/NOS signaling to protect against intimal hyperplasia in Sprague-Dawley rats. J Clin Invest 121:1646-1656, 2011. 3. Ascenzi P, Bocedi A, Sanctis D, Pesce A, Bolognesi M, Marden MC, Dewilde S, Moens L, Hankeln T, Burmester T. Neuroglobin and Cytoglobin: two new entries in the hemoglobin superfamily. BAMBED 32: 305-313, 2004. 4. Basu S, Grubina R, Huang J, Conradie J, Huang Z, Jeffers A, Jiang A, He X, Azarov I, Seibert R, Mehta A, Patel R, King SB, Hogg N, Ghosh A, Gladwin MT, Kim-Shapiro DB. Catalytic generation of N2O3 by the concerted nitrite reductase and anhydrase activity of hemoglobin. Nat Chem Biol 3:785-794, 2007. 5. Benjamin N, O'Driscoll F, Dougall H, Duncan C, Smith L, Golden M, McKenzie H. Stomach NO synthesis. Nature 368:502, 1994. 6. Bjorne HH, Petersson J, Phillipson M, Weitzberg E, Holm L, Lundberg JO. Nitrite in saliva increases gastric mucosal blood flow and mucus thickness. J Clin Invest 113:106-114, 2004. 7. Brody MS, Hille R. The Kinetic behavior of chicken liver sulfite oxidase. Biochemistry 38: 66686677, 1999. 8. Bryan N, Rassaf T, Maloney RE, Rodriguez CM, Saijo F, Rodriguez JR, Feelisch M. Proc Natl Acad Sci USA 101: 4308-4313, 2004. 9. Campbell WH. Nitrate reductase structure, function and regulation: bridging the gap between biochemistry and physiology. Annu Rev Plant Phys 50:277-303, 1999.

23

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 24 of 57

24 10. Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD,Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO, Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med 9:14981505, 2003. 11. Crawford JH, Isbell TS, Zhi Huang, Shiva S, Chacko BK, Schechter AN, Darley-Usmar VM, Kerby JD, Lang JD, Kraus D, Ho C, Gladwin MT, Patel RP. Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation. Blood 107: 566-574, 2006. 12. Dejam A, Hunter CJ, Tremonti C, Pluta RM,Hon YY, Grimes G, Partovi K, Pelletier MM, Oldfield E H,Cannon RO,SchechterAN, Gladwin MT. Nitrite infusion in humans and nonhuman primates: endocrine effects, pharmacokinetics, and tolerance formation. Circulation 116:1821-1831, 2007a. 13. Dejam A, Hunter CJ, Gladwin MT. Effects of dietary nitrate on blood pressure. N Engl J Med 356:1590, 2007b. 14. Feelisch M, Fernandez BO, Bryan NS, Garcia-Saura MF, Bauer S, Whitlock DR, Ford PC, Janero DR, Rodriguez J, Ashrafian H. Tissue Processing of Nitrite in Hypoxia: an intricate interplay of nitric oxide-generating and -scavenging systems. J. Biol. Chem. 283:33927-33934, 2008. 15. Ghosh SM, Kapil V, Fuentes-Calvo I, Bubb KJ, Pearl V, Milsom AB, Khambata R, MalekiToyserkani S, Yousuf M, Benjamin N, Webb AJ, Caulfield MJ, Hobbs AJ, Ahluwalia A. Enhanced Vasodilator Activity of Nitrite in Hypertension: Critical Role for Erythrocytic Xanthine Oxidoreductase and Translational Potential. Hypertension 61:00-00, 2013. DOI: 10.1161/HYPERTENSIONAHA.111.00933 16. Gladwin MT, Shelhamer JH, Schechter AN, Pease-Fye ME, Waclawiw MA, Panza JA, Ognibene FP, Cannon RO. Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans. Proc Natl Acad Sci USA 97:11482-11487, 2000. 17. Hurley JK, Medina M, Gomez-Moreno C, Tollin G. Further characterization by site-directed mutagenesis of the protein-protein interface in the ferredoxin/ferredoxin:NADP+ reductase system

24

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 25 of 57

25 from Anabaena: requirement of a negative charge at position 94 in ferredoxin for rapid electron transfer. Arch Biochem Biophys. 312:480-486, 1994. 18. Jansson EA, Huang L, Malkey R, Govoni M, Nihlen C, Olsson A, Stensdotter M, Petersson J, Holm L, Weitzberg E, Lundberg JO. A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis. Nat Chem Biol 4:411-417, 2008. 19. Jayaraman T, Tejero J, Chen BB, Blood AB, Frizzell S, Shapiro C, Tiso M, Hood BL, Wang X, Zhao X, Conrads TP, Mallampalli RK, Gladwin MT. 14-3-3 binding and phosphorylation of neuroglobin during hypoxia modulate six-to-five heme pocket coordination and rate of nitrite reduction to nitric oxide. J Biol Chem 286:42679-42689, 2011. 20. Jiang Z, Zhu X, Diamond MP, Abu-Soud HM, Saed GM. Nitric oxide synthase isoforms expression in fibroblasts isolated from human normal peritoneum and adhesion tissues. Fertility and Sterility 90:769-774, 2008. 21. Johnson-Winters K, Nordstrom AR, Emesh S, Astashkin AV, Rajapakshe A, Berry RE, Tollin G, Enemark JH. Effects of Interdomain Tether Length and Flexibility on the Kinetics of Intramolecular Electron Transfer in Human Sulfite Oxidase. Biochemistry 49:1290-1296, 2010. 22. Kuper J, Llamas A, Hecht HJ, Mendel RR, Schwarz G. Structure of the molybdopterin-bound Cnx1G domain links molybdenum and copper metabolism. Nature 430:803-806, 2004. 23. Lamy MT, Gutteridge S, Bray RC. Electron-paramagnetic-resonance parameters of molybdenum(V) in sulfite oxidase from chicken liver. Biochemical Journal 185:397-403, 1980. 24. Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg JO, Weitzberg E. Dietary inorganic nitrate improves mitochondrial efficiency in humans. Cell Metab 13:149-159, 2011. 25. Li H, Samouilov A, Liu X, Zweier JL. Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction. J Biol Chem 276:24482-24489, 2001. 26. Li H, Cui H, Kundu TK, Alzawahra W, Zweier JL. Nitric oxide production from nitrite occurs primarily in tissues not in the blood: critical role of xanthine oxidase and aldehyde oxidase. J Biol Chem 283:17855-17863, 2008. 25

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 26 of 57

26 27. Li H, Kundu TK, Zweier JL. Characterization of the magnitude and mechanism of aldehyde oxidasemediated nitric oxide production from nitrite. J Biol Chem 284:33850-33858, 2009. 28. Lundberg JO, Weitzberg E, Lundberg JM, Alving K. Intragastric nitric oxide production in humans: measurements in expelled air. Gut 35:1543-1546, 1994. 29. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov 7:156-167, 2008. 30. Lundberg JO. Nitrate transport in salivary glands with implications for NO homeostasis. Proc Natl Acad Sci USA 109:13144-13145, 2012. 31. Maher AR, Milsom AB, Gunaruwan P, Abozguia K, Ahmed I, Weaver RA, Thomas P, Ashrafian H, Born GV, James PE, Frenneaux MP. Hypoxic modulation of exogenous nitrite-induced vasodilation in humans. Circulation 117:670-677, 2008. 32. Mcwhirter RB, Hille R. The Reductive half-reaction of xanthine-oxidase -identification of spectral intermediates in the hydroxylation of 2-hydroxy-6-methylpurine. J Biol Chem 266:23724-23731, 1991. 33. Palmer T, Santini CL, Iobbi-Nivol C, Eaves DJ, Boxer DH, Giordano G. Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli. Mol Microbiol. 20:875-884, 1996. 34. Qin L, Liu XB, Sun QF, Fan ZP, Xia DS, Ding G, Ong HL, Adams D, Gahl WA, Zheng CY, Qi SR, Jin LY, Zhang CM, Gu LK, He JQ, Deng DJ, Ambudkar IS, Wang SL. Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane. Proc Natl Acad Sci USA 109:13434-13439, 2012. 35. Qiu JA, Wilson HL, Rajagopalan KV. Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction. Biochemistry 51: 1134−1147, 2012. 36. Raat NJ, Noguchi AC, Liu VB, Raghavachari N, Liu DL, Xu XL, Shiva S, Munson PJ, Gladwin MT. Dietary nitrate and nitrite modulate blood and organ nitrite and the cellular ischemic stress response. Free Radic Biol Med 47:510-517, 2009. 26

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 27 of 57

27 37. Rajapakshe A, Meyers KT, Berry RE, Tollin G, Enemark JH. Intramolecular electron transfer in sulfite-oxidizing enzymes: probing the role of aromatic amino acids. J Biol Inorg Chem 17:345-352, 2012. 38. Totzeck M, Hendgen-Cotta UB, Luedike P, Berenbrink M, Klare JP, Steinhoff HJ, Semmler D, Shiva S, Williams D, Kipar A, Gladwin MT, Schrader J, Kelm M, Cossins AR, Rassaf T. Nitrite regulates hypoxic vasodilation via myoglobin-dependent nitric oxide generation. Circulation 126: 325-334, 2012. 39. Schmidt M, Laufs T, Reuss S, Hankeln T, Burmester T. Divergent distribution of cytoglobin and neuroglobin in the murine eye. Neurosci. Lett. 374: 207-211, 2005. 40. Shiva S, Wang X, Ringwood LA, Xu X, Yuditskaya S, Annavajjhala V, Miyajima H, Hogg N, Harris Z L, Gladwin MT. Ceruloplasmin is a NO oxidase and nitrite synthase that determines endocrine NO homeostasis. Nat Chem Biol 2:486-493, 2006. 41. Shiva S, Huang Z, Grubina R, Sun JH, Ringwood LA, MacArthur PH, Xu XL, Murphy E, DarleyUsmar VM, Gladwin MT. Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration. Circ Res 100:654-661, 2007. 42. Sparacino-Watkins CE, Tejero J, Sun B, Gauthier MC, Thomas J, Ragireddy V, Merchant BA, Wang J, Azarov I, Basu P, Gladwin MT. J Biol Chem 289:10345-10358, 2014. 43. Srihirum S, Sriwantana T, Unchern S, Kittikool D, Noulsri E, Pattanapanyasat K, Fucharoen S, Piknova B, Schechter AN, Sibmooh N. Platelet Inhibition by Nitrite Is Dependent on Erythrocytes and Deoxygenation. PLoS ONE 7: e30380, 2012. 44. Stolz JF, Basu P. Evolution of nitrate reductase: Molecular and structural variations on a common function. Chembiochem 3:198-206, 2002. 45. Sturms R, DiSpirito AA, Hargrove MS. Plant and cyanobacterial hemoglobins reduce nitrite to nitric oxide under anoxic conditions. Biochemistry 50:3873-3878, 2011. 46. Temple CA, Graf TN, Rajagopalan KV. Optimization of expression of human sulfite oxidase and its molybdenum domain. Arch Biochem Biophys 383:281-287, 2000. 27

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 28 of 57

28 47. Tejero J, Gladwin MT. The globin superfamily: functions in nitric oxide formation and decay. Biol Chem 395:631-639, 2014. 48. Tiso M, Tejero J, Basu S, Azarov I, Wang X, Simplaceanu V, Frizzell S, Jayaraman T, Geary L, Shapiro C, Ho C, Shiva S, Kim-Shapiro DB, Gladwin MT. Human neuroglobin functions as a redoxregulated nitrite reductase. J Biol Chem 286:18277-18289, 2011. 49. Utesch T, Mroginski MA. Three-Dimensional Structural Model of Chicken Liver Sulfite Oxidase in its Activated Form. J Phys Chem Lett 1:2159-2164, 2010. 50. Veldman A, Santamaria-Araujo JA, Sollazzo S, Pitt J, Gianello R, Yaplito-Lee J, Wong F, Ramsden CA, Reiss J, Cook I, Fairweather J, Schwarz G. Successful treatment of molybdenum cofactor deficiency type A with cPMP. Pediatrics 125:E1249-E1254, 2010. 51. Webb A, Bond R, McLean P, Uppal R, Benjamin N, Ahluwalia A. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc Natl Acad Sci USA 101:13683-13688, 2004. 52. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, Rashid R, Miall P, Deanfield J,Benjamin N, MacAllister R, Hobbs AJ, Ahluwalia A. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension 51:784-790, 2008. 53. Yamasaki H, Sakihama Y. Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species. Febs Letters 468:89-92, 2000. 54. Zuckerbraun BS, Shiva S, Ifedigbo E, Mathier MA, Mollen KP, Rao J, Bauer PM, Choi JJ, Curtis E, Choi AM, Gladwin MT. Nitrite Potently Inhibits Hypoxic and Inflammatory Pulmonary Arterial Hypertension and Smooth Muscle Proliferation via Xanthine Oxidoreductase - Dependent Nitric Oxide Generation. Circulation 121:98-109, 2010.

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55. Zuckerbraun BS, George P, Gladwin MT. Nitrite in pulmonary arterial hypertension: therapeutic avenues in the setting of dysregulated arginine/nitric oxide synthase signalling. Cardiovasc Res

89:542-552, 2011.

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Figure legends

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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31

Fig. 1. Kinetics of NO generation from human holo-SO as a function of nitrite, in the presence of phenosafranine. Initial rates of NO generation were measured anaerobically at pH 7.4, 37°C, in 20 mM BisTris buffer. (A) NO generation traces with 0.63 μM SO and 40 μM reduced phenosafranine in the presence of 20 μM – 4 mM nitrite at pH 7.4. High sensitivity mode was used in the range of 20 μM – 4mM. (B) Similar measurements were performed as (A) at low sensitivity mode in the range of 5-100 mM nitrite. (C) Corresponding NO generation rates were calculated from the representative traces shown in (A) and (B).

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Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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32

Fig. 2. Representative traces and kinetics of NO generation from human holo-SO, in the presence of sulfite. Initial rates of NO generation were measured anaerobically at pH 6.5 and 7.4, 37°C, in 20 mM BisTris buffer anaerobically. (A) Traces show NO generation from the reaction of nitrite with sulfite, in the absence (black), or in the presence of SO (red), with SO (1.5 μM), nitrite (1 mM) and sulfite (10 μM) at pH 6.5. (B) Traces show NO generation by 0.63 μM SO reacting with various reductant and oxidant combinations at pH 6.5: sulfite + nitrite (red), phenosafranine (PHSN) + nitrate (black), PHSN + nitrite (blue). Concentrations of PHSN, sulfite, nitrate and nitrite are 40 μM, 40 μM, 1 mM and 1 mM, respectively. (C) NO generation traces with 0.63 μM SO and 5 μM sulfite in the presence of 20 μM – 10

32

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 33 of 57

33

mM nitrite at pH 6.5. (D) NO generation traces at 7.4. (E) Corresponding NO generation rates were

calculated as the representative traces are shown in (C-D).

33

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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34

Fig. 3. Kinetics of NO generation from Mo-domain as a function of nitrite, in the presence of phenosafranine. Initial rates of NO generation were measured anaerobically at pH 7.4, 37°C, in 20 mM BisTris buffer. (A) NO generation traces with 0.25 μM SO and 40 μM reduced phenosafranine in the presence of 5 μM – 1 mM nitrite at pH 7.4. High sensitivity mode was used in the range of 5 μM-1mM. (B) Similar measurements were performed as (A) at low sensitivity mode in the range of 2-100 mM. (C) Corresponding NO generation rates were calculated from the representative traces shown in (A)-(B).

34

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 35 of 57

35

Fig.4. NO generation and nitrite reduction by various types of engineered SO proteins. (A-B) Anaerobic NO generation by Mo-domain (0.25 μM) as a function of nitrite concentration (5 μM – 5 mM) in the presence of 5 μM sulfite, at pH 6.5 (A) and 7.4 (B), 37°C, in 20 mM BisTris buffer. (C) Corresponding NO generation rates were calculated from the representative traces shown in (A-B). (D) Anaerobic NO generation from the reaction of nitrite (1 mM) with the Mo-domain, holo-SO, or heme domain ([Mo] = 0.25 μM for Mo-domain; [Mo] = 0.63 μM for holo-SO; [heme] = 0.63 μM) at pH 7.4, 25°C, 20 mM BisTris buffer, with 40 μM PHSN was the reducing substrate. (E) Nitrite (10 μM) reduction is carried out in the anaerobic glove box at RT when nitrite reacts with: SO+sulfite (open circle, blue), SO+PHSN (dark circle, blue), Mo-domain+sulfite (open square, green), Mo-domain+PHSN (dark square,

35

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 36 of 57

36 green), heme domain+PHSN (dark triangle, red), PHSN (open diamond, black). Concentrations of sulfite, PHSN, SO, Mo-domain and heme-domain are 50, 50, 10, 10 and 10 μM, respectively. (F) Steady-state kinetics of nitrite reduction by PHSN catalyzed by human holo-SO and the isolated Mo-domain, recorded spectrophotometrically at 520 nm with at least triplicate measurements. 12 µM human holo SO or human SO Mo-domain was mixed with 250 µM phenosafranine and 0-75 mM nitrite. The reaction was started with 250 µM sodium dithionite and control experiments were performed without the addition of enzyme.

36

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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37

Fig. 5. Circular dichroism (CD) spectroscopy of wild-type mouse SO and H119A/H144A and NO generation by mouse SO proteins. (A) CD spectra of the secondary structure of wild-type mouse SO (—, solid line), H119A/H144A(…, dot line). (B) NO generation rates as a function of nitrite concentration catalyzed by wild-type mouse SO, molybdenum domain of mouse SO and the H119A/H144A mouse SO mutant. SO (—, solid line, square), H119A/H144A(…, dot line, open circle) and Mo domain (---,dash line, triangle).

37

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 38 of 57

38

Fig. 6. NO generation by pre-reduced human holo-SO; EPR spectroscopy of the human Modomain. (A) Anaerobic NO generation rates with 0.63 μM pre-reduced human SO and 1 mM nitrite, in the presence or absence of 5 μM sulfite at pH 6.5, 37°C, in 20 mM BisTris buffer. (B) Anaerobic EPR spectra of the Mo center in the isolated Mo-domain of human SO. (a) 20 μM Mo-domain + 100 μM sulfite, (b) 1 mM nitrite was added to (a), (c) 50 μM reduced PHSN was added to (b), (d) thawing and incubation of (c) for 1.5h.

38

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 39 of 57

39

Fig. 7. Proposed electron transfer in the Mo-domain and holo-SO. Proposed electron transfer between

the Mo center and (A) sulfite or nitrite, (B) PHSN or nitrite; (C) proposed electron transfer between the

two metal centers, Mo and heme-Fe, and a substrate (sulfite, nitrite or PHSN).

39

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 40 of 57

40

Fig. 8. Expression of SO in human fibroblast and cGMP formation from human fibroblast. (A) SO expression in fibroblasts from healthy individuals (WT), SO-deficient (SOD) or Moco-deficient (MOCD) patients. 120 µg protein from the cell lysate was loaded for electrophoresis. (B) Human fibroblast cells derived from WT were treated with 1 µM sildenafil, and then with various concentrations of nitrite or 5 µM DEA-NO followed by 2 h hypoxic incubation (1% O2). Cyclic GMP (cGMP) levels were determined in cell lysates. Data represent mean ±SD. (C) Human fibroblast cells derived from SOD or MOCD patients or WT were treated with 1 µM sildenafil, and then with 50 µM nitrite followed by 2 h hypoxic incubation (1% O2). Control samples are without nitrite treatment. (*P < 0.001, n= 3). (D) Human fibroblasts derived from WT were treated with 1 µM sildenafil, and subsequently with 100 µM nitrite

40

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 41 of 57

41 followed by hypoxic incubation for 0, 15, 30, 60 or 120 minutes. cGMP levels were determined accordingly (y = 4.296x + 197.52, R2 = 0.995). (E) The same kinetics of cGMP formation as shown in (D) was carried out in SO deficient fibroblasts (y = 0.199x + 268.86, R2 = 0.533). (F) Human fibroblast cells derived from SOD or MOCD patients or WT were treated with 1 µM sildenafil, and then with 50 µM nitrite under 2 h normoxia (20% O2). Control samples are without nitrite treatment. (*P < 0.001, n= 3). (G) Comparison of cGMP production in (C) and (F) after normalized to the WT control.

41

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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42 Supplementary Information Sulfite oxidase catalyzes single electron transfer reactions at molybdenum domain to reduce nitrite to nitric oxide Jun Wang1,2#, Sabina Krizowski3#, Katrin Fisher-Schrader3, Dimitri Niks4, Jesús Tejero1,2, Courtney Sparacino-Watkins1,2, Ling Wang1,2, Prafulla Ragireddy1,2, Sheila Frizzell1,2, Eric E. Kelley1,5, Yingze Zhang2, Russ Hille4, Partha Basu6, Guenter Schwarz3+, and Mark T. Gladwin1,2,+* 1

Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, 15213 2Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, 15213 3Biochemistry, Institute of Biochemistry and Center for Molecular Medicine, Cologne University 4Department of Biochemistry, University of California at Riverside, Riverside, CA, 92521 5Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA, 15213 6Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, 15261 * E-mail: [email protected]; [email protected] CONTENTS: - Supplementary Methods. Measurement of NO Formation by Ozone-based Chemiluminescence. - Supplementary Figure 1. Kinetics of NO generation by human SO holo enzyme as a function of nitrite concentration with phenosafranine as reductant. - Supplementary Figure 2. NO generation from nitrite/tri-iodide reaction and DETA-NONOate in a purge and non-purge systems. - Supplementary Figure 3. NO generation by human SO holo enzyme as a function of nitrite with phenosafranine as reductant in non-purge and purge systems. - Supplementary Figure 4. Kinetics of NO generation by human SO holo enzyme as a function of sulfite, and by mouse SO holo enzyme as a function of nitrite or sulfite. - Supplementary Figure 5. Kinetics of NO generation by recombinant human Mo-domain as a function of nitrite in the presence of phenosafranine as reductant. - Supplementary Figure 6. Kinetics of NO generation by recombinant human Mo-domain or by mouse Mo-domain as a function of nitrite or sulfite concentrations. - Supplementary Figure 7. Kinetics of NO generation from mouse Mo-domain as a function of sulfite concentration. - Supplementary Figure 8. NO generation by mouse SO proteins. - Supplementary Figure 9. NO generation by human holo SO and human SO Mo-domain aerobically and anaerobically. 42

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 43 of 57

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- Supplementary Table 1. Kinetic parameters for NO generation from nitrite reduction catalyzed by human and mouse SO, as well as recombinant Mo domains.

43

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 44 of 57

44 Supplementary

Methods

-

Measurement

of

NO

Formation

by

Ozone-based

Chemiluminescence. Nitric oxide production was measured by ozone-based chemiluminescence using a Sievers Nitric Oxide Analyzer (NOA 280i) equipped with a liquid sampling purge system (GE Analytical Instruments, Boulder, CO). The purge vessel, positioned in-line between the NOA and helium carrier gas. NO gas generated in the purge vessel is swept away at a rate of 200 mL/ min into the NOA detector. Inside the NOA’s gas-phase reaction chamber, NO rapidly reacts with ozone (O3) producing electronically excited NO2*, which emits a photon of light (hv) when it drops to ground state. Light generated from the reaction of NO and O3 is then filtered to include only red wavelength (>600nm) and detected by the photomultiplier tube. The NOA’s chemiluminescence signal (mV) directly monitors the rate of NO formation as a function of time. Chemiluminescence intensity, measured as voltage (V), is proportional NO concentration. Data was collected using the high sensitivity (0-1 V) setting, unless indicated as low sensitivity (0-10 V). Sievers NO AnalysisTM Software, Liquid (Version 3.2) was used for data acquisition, at a rate of one data point per 0.25 second. The standard glass purge vessel (15-mL) was used to mix and deoxygenate reagents. The purge vessel’s heating jacket was connected to a circulating water bath to control reaction temperature. For enzyme reactions, the reaction buffer (20 mM BisTris buffer or 10 mM phosphate buffer pH 6.5 or 7.4) was maintained in the presence of nitrite for two minutes in the sealed purge vessel to equilibrate the temperature to 37°C and to void oxygen from the reagents prior to initiating the enzyme reaction. Next, reducing substrate (sulfite or phenosafranine) was injected into the purge vessel through the vessel’s septa using a Hamilton syringe. Once a stable baseline was established, SO was injected into the mixture and the reaction was monitored for several minutes, until the reaction completed. 44

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 45 of 57

45 NO production rates were estimated using a calibration curve. Acidified nitrite was reacted with tri-iodide (I3-) to produce stoichiometric quantities of NO. A standard curve was used to convert the raw NOA signal (mV) into NO concentration.. Specifically, 50 μL nitrite (ranging from 0-1000 pmol) prepared in phosphate buffer (10 mM, pH 7.4) were sequentially injected with a Hamilton syringe into the purging I3- solution and the resultant peaks were recorded. The peaks obtained from these injections were then integrated for 0.2 min (peak max ± 0.1 min) with Origin 8.0 software. Plotting area under curve (AUC) against NO concentration (pmole) is linear, thus data were fit with a linear equation giving the following: y= 0.119x. NO generation rate (nmol· s-1· ) or NO amount (nmole) was calculated based on AUC of the highest 12-second NO signals, and analyzed. NO produced from the reaction of SO enzyme with nitrite was calculated using the same method. The raw data collected from the NOA experiments was analyzed with Origin software. Traces were smoothed by averaging data in a spanning 2 s. Because the peak max was identified the area under curve from time x to time x+12s was integrated, which was around the peak of the NO trace curve. The measured NOA response was converted to NO concentration using the linear equation derived from the I3- standard curve. The area under the curve (with dimensions of mV • min) was divided by the slope from the standard curve (0.119 mV • min • pmole-1 NO) to determine the NO concentration. Then the amount of NO was divided by 12s and the amount of protein used during each reaction (mg), and NO generation rates were attained (nmol· sec-1· mg-1). All the NO generation rates were plotted against the corresponding concentrations of nitrite, which fitted hyperbola function, i.e. y = (P1 •· x)/(P2 + x). The values of P1 and P2 correspond to νmax and Km values in the Michaelis-Menten equation, i.e. ν = Vmax • [S]/(Km + [S]). In the case of single turnover reactions (non-steady-state), a similar fit was used, where the P1 and P2 terms 45

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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46

represent the apparent νmax and Km values, which we refer to as ket (rate of electron transfer) and

Kd (apparent dissociation constant) to indicate the single turnover nature of the studied reaction.

46

47

human SO, pH 6.5 800

NO signal (mV)

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 47 of 57

nitrite 10 M 20 M 50 M 100 M 200 M 400 M 800 M

600 400

1 mM 2 mM

200 0 0

50

100

150

time (s)

Supplementary Figure 1. Kinetics of NO generation by human SO holo enzyme as a function of nitrite concentration with phenosafranine as reductant. Initial traces of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at pH 6.5, 37°C, 20 mM BisTris buffer in the presence of 0.63 μM human SO, 40 μM phenosafranine and 10 μM –2 mM nitrite.

47

48

B purge

500 pmol

400 250 pmol

300 200

125 pmol 50 pmol 20 2.5 5 10 pmol pmol pmol pmol

100

non-purge

200

NO signal (mV)

70

500 pmol

50

150 250 pmol

100 125 pmol

50

purge non-purge

60

40 30 20 y = 0.119 x R2 = 0.998

10

50 10 20 pmol 5 2.5 pmolpmol pmol pmol

0

0

0 0

4 8 Time (min)

0

12

5 10 Time (min)

0

15

F

E

D

200

200

250

purge

150

250

100 50 0 0

150

5

10

15

100 50 0 7.0

non-purge

150

30

purge non-purge

100

NO signal (mV)

200

100 200 300 400 500 NO (pmol)

7.1 7.2 Time (min)

7.3

200 150

50

DETA-NO (mM)

0 0

5

10

15

buffer 0.2 0.5 1 2 4

100 50 0 7.0

7.1 7.2 Time (min)

7.3

AUC (0.2 min)

NO signal (mV)

500

C

AUC

A

NO signal (mV)

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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20

10

0 0

1 2 3 DETA-NO (mM)

Supplementary Figure 2. NO generation from nitrite/tri-iodide reaction and DETANONOate in a purge or non-purge system. (A) traces of NO generation from nitrite-I3reaction in the range of 2.5 to 500 pmol nitrite in a purge vessel. (B) traces of NO generation from nitrite-I3- reaction in the range of 2.5 to 500 pmol nitrite in a non-purge vessel. (C) area under curve (AUC) versus NO as traces are shown in (A) and (B). (D) zoom-in traces of NO generation from DETA-NO in the range of 0.2 to 4 mM in a purge vessel. Inset shows the complete traces. (E) zoom-in traces of NO generation from DETA-NO in the range of 0.2 to 4 mM in a non-purge vessel. Inset shows the complete traces. (F) area under curve (AUC) versus NO as traces are shown in panels D and E.

48

4

49

purge

A

B

90

nitrite (mM) 1 0.4 0.2 0.1 0.05 0.02 buffer

60 30

non-purge

80

NO signal (mV)

NO signal (mV)

120

60

nitrite (mM) 1 0.4 0.2 0.1 0.05 0.02 buffer

40 20 0

0 0

2

4 6 8 Time (min)

0

10

C

5

10 15 20 25 Time (min)

D purge

80

90

NO signal (mV)

120

NO signal (mV)

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 49 of 57

nitrite (mM) 1 0.4 0.2 0.1 0.05 0.02 buffer

60 30

2.3

2.4 Time (min)

60

2.5

nitrite (mM) 1 0.4 0.2 0.1 0.05 0.02 buffer

40 20 0 8.0

0

non-purge

8.1 8.2 Time (min)

8.3

E

Supplementary Figure 3. NO generation by human SO holo enzyme as a function of nitrite with phenosafranine as reductant in non-purge and purge systems. Initial traces of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at pH 7.4, 37°C, 20 mM BisTris buffer in the presence of 0.63 μM human SO, 40 μM phenosafranine and 20 μM –1 mM nitrite in a non-purge system (A) and a purge system (B). (C) zoomed-in traces of panel A. (D) zoomed-in traces of panel B. (E) area under curve (AUC) of first seven-min integration versus nitrite concentration as traces were shown in panels A and B.

49

(A)

0.02

0.01

0.00 0

1000 2000 3000 4000 5000 sulfite (M)

human SO, pH 7.4 0.03

(B) 0.02

0.01

0.00 0

1000 2000 3000 4000 5000 sulfite (M)

NO genetation rate (nmol·sec-1·mg-1)

human SO, pH 6.5 0.03

NO generation rate (nmole·s-1·mg-1)

NO generation rate (nmole·s-1·mg-1)

50

(C) 0.08

0.04

20

10 0

20

40

60 80 time (s)

100

120

mouse SO, pH 6.5 0.06

(G)

0.04

0.02

0.00 0

1000

2000 3000 sulfite (M)

4000

(E) max = 0.0473 nmol s-1 mg-1

0.08

Kd = 1.2 mM

ket = 0.0026 s-1

0.04

ket = 0.0057s-1

0.00 0

2000 4000 6000 8000 10000 nitrite (M) nitrite

40

mouse SO, pH 7.4 0.12

max = 0.102 nmol s-1 mg-1

Kd = 948 M

NO signal (mV)

30

NO generation rate (nmole·s-1·mg-1)

(D)

NO generation rate (nmole·s-1·mg-1)

NO signal (mV)

10 M 20 M 30 M 50 M 100 M 200 M 400 M 800 M 1 mM 2 mM 3 mM 5 mM 8 mM 10 mM

mouse SO, pH 6.5

0.12

nitrite

40

NO generation rate (nmole·s-1·mg-1)

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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50 M 100 M 200 M 400 M 600 M 800 M 1 mM 2 mM 3 mM 5 mM 8 mM 10 mM

(F) 30

20

10 0.00 0

2000 4000 6000 8000 10000 nitrite (M)

0

20

40

60 80 time (s)

mouse SO, pH 7.4

0.06

(H)

0.04

0.02

0.00 0

1000 2000 3000 4000 5000 sulfite (M)

Supplementary Figure 4. Kinetics of NO generation by human SO holo enzyme as a function of sulfite, and by mouse SO holo enzyme as a function of nitrite or sulfite. Initial rates of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at pH 6.5 and 7.4, 37°C, 20 mM BisTris buffer. (A-B) NO generation rates by 0.63 μM human SO and 1 mM nitrite in the presence of 2 μM –5 mM sulfite at pH 6.5 (A) and at pH 7.4 (B). (CD): NO generation by 0.32 μM mouse SO and 5 μM sulfite in the presence of 10μM – 1mM nitrite at pH 6.5. (E-F): NO generation rates by 0.32 μM mouse SO and 5 μM sulfite in the presence of 50μM – 1mM nitrite at pH 7.4. (G): NO generation rates by 0.32 μM mouse SO and 800 μM nitrite in the presence of 2 μM – 4 mM sulfite at pH 6.5. (H): NO generation rates by 0.32 μM mouse SO and 1 mM nitrite in the presence of 2 μM – 5 mM sulfite at pH 7.4. 50

100

120

51

(A)

(B) nitrite (mM)

1000

low sentitivity: NO signal (mV)

high sentivity: NO signal (mV)

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 51 of 57

0.001 0.0025 0.005 0.01 0.02 0.05 0.1 0.2 0.4

800 600 400 200 0 0

50 100 time (s)

150

nitrite (mM)

16

1 2 5 10 20 100

12 8 4 0 0

50 100 time (s)

150

Supplementary Figure 5. Kinetics of NO generation by recombinant human Mo-domain as a function of nitrite in the presence of phenosafranine as reductant. Initial traces and rates of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at pH 6.5, 37°C, in 20 mM BisTris buffer. NO generation traces by 0.25 μM human Mo-domain and 40 μM phenosafranine in the presence of 1 μM – 0.4 mM nitrite recorded at the high sensitivity setup (A), and in the presence of 1 mM - 100 mM nitrite at the low sensitivity setup (B).

51

(A)

0.20 0.15 0.10 0.05 0.00 0

1000 2000 3000 4000 5000 sulfite (M)

human Mo-domain, pH 7.4

NO generation rate (nmole·s-1·mg-1)

human Mo-domain, pH 6.5

0.25

NO generation rate (nmole·s-1·mg-1)

.

NO generation rate (nmole·s-1·mg-1)

52

0.25

(B)

0.20 0.15 0.10 0.05 0.00 0

200

400 600 800 sulfite (M)

1000

mouse Mo-domain, pH 6.5 0.6

(C) 0.4

0.2

max = 0.493 nmol s-1 mg-1

Kd = 330 M

ket = 0.0207 s-1

0.0 0

2000 4000 6000 8000 10000 nitrite (M) nitrite

NO signal (mV)

120

2.5 M 5 M 10 M 20 M 30 M 50 M 100 M 200 M 400 M 800 M 1 mM 2 mM 3 mM 5 mM 8 mM 10 mM

80

40

0 0

20

40

60 80 time (s)

100

120

muse Mo-domain, pH 6.5 phosphate buffer

0.6

0.2

mouse Mo-domain, pH 7.4 max = 0.375 nmol s-1 mg-1

Kd = 694 M

ket = 0.0158 s-1

0.4

0.2

(E)

80

40

0.0 0

5 M 10 M 20 M 30 M 50 M 100 M 200 M 400 M 800 M 1 mM 2 mM 3 mM 5 mM 8 mM 10 mM

(F)

0.6

0

2000 4000 6000 8000 10000 sulfite (M)

0

20

40

60 80 time (s)

nitrite

120

10M 20M 30M 50M 100M 200M 400M 600M 800M 1mM

(H) NO signal (mV)

(G) 0.4

120

NO signal (mV)

(D)

NO generation rate (nmole·s-1·mg-1)

nitrite

NO generation rate (nmole·s-1·mg-1)

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 52 of 57

80

40

0.0 0

200

400 600 800 1000 nitrite (M)

0

20

40

60 80 time (s)

100

120

Supplementary Figure 6. Kinetics of NO generation by recombinant human Mo-domain or by mouse Mo-domain as a function of nitrite or sulfite concentrations. Initial rates of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at pH 6.5 and 7.4, 37°C, in 20 mM BisTris buffer (A-F) or in 10 mM phosphate buffer (G-H). (A-B) NO generation rates by 0.25 μM human Mo-domain and 1 mM nitrite in the presence of 2 μM – 5 mM sulfite at pH 6.5 and 7.4. (C-D) NO generation by 0.25 μM mouse Mo-domain and 5 μM sulfite in the presence of 2.5 μM – 10 mM nitrite at pH 6.5. (E-F) NO generation by 0.25 μM mouse Mo-domain and 5 μM sulfite in the presence of 5 μM – 10 mM nitrite at pH 7.4. (G-H) NO generation by 0.25 μM mouse Mo-domain and 5 μM sulfite in the presence of 10 μM –1 mM nitrite at pH 6.5 in 10 mM phosphate buffer.

52

100

120

B

C

NO generation rate (nmole·s-1·mg-1)

A

NO generation rate (nmole·s-1·mg-1)

53

NO generation rate (nmole·s-1·mg-1)

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 53 of 57

0.6

pH 6.5 0.4

0.2

0.0 0

1000 2000 3000 4000 5000 sulfite (M)

0.6

pH 7.4 0.4

0.2

0.0 0

2000

4000 6000 sulfite (M)

8000

0.6

pH 6.5, phosphate buffer 0.4

0.2

0.0 0

2000

4000 6000 sulfite (M)

8000

Supplementary Figure 7. Kinetics of NO generation from mouse Mo-domain as a function of sulfite concentration. Initial rates of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at 37°C. (A) NO generation by 0.25 μM mouse Modomain and 1 mM nitrite in the presence of 2 μM – 5 mM sulfite at pH 6.5, 20mM BisTris buffer. (B) NO generation rates by 0.25 μM mouse Mo-domain and 1 mM nitrite in the presence of 2 μM – 8 mM sulfite at pH 7.4, 20mM BisTris buffer. (C) NO generation rates by 0.25 μM mouse Mo-domain and 1 mM nitrite in the presence of 2 μM – 8 mM sulfite at pH 6.5, in 10 mM phosphate buffer.

53

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 54 of 57

54

A

nitrite

70

2.5 M 5 M 10 M 20 M 30 M 50 M 100 M 200 M 400 M 800 M 1000 M

60 50 40 30 20 10 0

20

40

60

80

B

100

120

nitrite

70

5M 10M 20M 50M 100M 200M 400M 800M 1000M

60 50 40 30 20 10 0

20

40

60

80

C

100

120

nitrite

70

10 M 20 M 30 M 50 M 100 M 20 M 400 M 800 M 1000 M

60 50 40 30 20 10 0

20

40

60

80

100

120

Supplementary Figure 8. NO generation by mouse SO proteins. Initial rates of NO generation were measured by chemiluminescence in the NO analyzer at pH 6.5, 37°C, in 20 mM BisTris buffer anaerobically. (A) NO generation by 0.25 μM Mo-domain of mouse SO and sulfite (5 μM) in the presence of 2.5 μM – 1 mM nitrite. (B) NO generation by 0.38 μM H119A/H144A and sulfite (5 μM) in the presence of 5 μM – 1 mM nitrite. (C) NO generation by 0.32 μM mouse SO in the presence of 10 μM – 1 mM nitrite.

54

55

40

nitrite (mM)

0.1 1 10

30

20

10 0

60 time (s)

120

hSO anaerobic aerobic

0.06

0.04

0.02

0.00 0

5 nitrite (mM)

10

D

C hSO-Mo, aerobic

hSO-Mo, anaerobic nitrite (mM)

0.1 1 10

60

40

20

0

60 time (s)

120

-1 -1 NO genetation rate (nmol·sec ·mg )

NO signal (mV)

hSO, anaerobic

hSO, aerobic

NO generation rate (nmole·s-1·mg-1)

B

A

NO signal (mV)

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 55 of 57

hSO-Mo anaerobic aerobic

0.3 0.2 0.1 0.0 0

5 10 nitrite (mM)

Supplementary Figure 9. NO generation by human holo SO and human SO Mo-domain aerobically and anaerobically. Initial rates of NO generation were measured by chemiluminescence in the NO analyzer at pH 7.4, 37°C, in 20 mM BisTris buffer anaerobically. (A) Traces of NO generation by human holo SO aerobically and anaerobically when nitrite is 0.1, 1, 10 mM. (B) NO generation rates were measured as shown in panel A. (C) Traces of NO generation by human SO Mo-domain aerobically and anaerobically when nitrite is 0.1, 1, 10 mM. (D) NO generation rates were measured as shown in Panel C.

55

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 56 of 57

56 Supplementary Table 1. Kinetic parameters for NO generation from nitrite reduction catalyzed by human and mouse SO, as well as recombinant Mo domains. vmax kcat (adjusted, Protein/domain pH buffer Km (mM) (adjusted, nmol· s-1· mg-1) s-1)

reference

(a)

human SO (wt)

7.4

Bis-Tris

34

80

1.9

this work

(a)

human SO Modomain

7.4

Bis-Tris

112

31

4.7

this work

XO

7.4

phosphate

0.92

2.25

0.28

1

XO

7.4

phosphate

8.31

2.46

2.49

1

XO

7.4

phosphate

2.87

2.6

0.85

1

XO

7.4

phosphate



0.585

0.693

2

AO

7.4

phosphate



16.4

0.244

2

AO

7.4

phosphate

13.5

3.3



3

AO

7.4

phosphate

8.5

2.7



3

AO

7.4

phosphate



16.4

0.244

2

protein/domain

pH

buffer

νmax (nmol· s-1· mg-1)

Kd (mM)

ket (s-1)

(b)

human SO (wt)

6.5

BisTris

0.0796

1.7

0.0044

this work

(b)

human SO (wt)

7.4

BisTris

0.0361

1.6

0.002

this work

(b)

mouse SO (wt)

6.5

BisTris

0.102

0.948

0.0057

this work

(b)

mouse SO (wt)

7.4

BisTris

0.0473

1.2

0.0026

this work

6.5

BisTris

0.323

0.496

0.0136

this work

7.4

BisTris

0.179

0.973

0.0075

this work

6.5

BisTris

0.493

0.33

0.0207

this work

7.4

BisTris

0.375

0.694

0.0158

this work

(b)

human SO Modomain (b) human SO Modomain (b) mouse SO Modomain (b) mouse SO Modomain

(a) Reducing substrate was phenosafranine; (b) Reducing substrate was sulfite. 56

Antioxidants & Redox Signaling Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide (doi: 10.1089/ars.2013.5397) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Page 57 of 57

57 References 1. Li H, Samouilov A, Liu X, & Zweier JL (2001) Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction: evaluation of its role in nitric oxide generation in anoxic tissues. J Biol Chem 276(27):24482-24489. 2. Maia LB, Moura JJG (2011) Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases. J Biol Inorg Chem 16(3):443-460. 3. Li H, Kundu TK, & Zweier JL (2009) Characterization of the magnitude and mechanism of aldehyde oxidase-mediated nitric oxide production from nitrite. J Biol Chem 284(49):33850-33858.

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Sulfite Oxidase Catalyzes Single-Electron Transfer at Molybdenum Domain to Reduce Nitrite to Nitric Oxide.

Recent studies suggest that the molybdenum enzymes xanthine oxidase, aldehyde oxidase, and mARC exhibit nitrite reductase activity at low oxygen press...
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