Journal of Biochemistry Advance Access published April 18, 2014

Mechanistic Studies on Formation of the Dinitrosyl Iron Complex of the [2Fe-2S] Cluster of SoxR Protein Mayu Fujikawa, Kazuo Kobayashi#*, and Takahiro Kozawa The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 5670047, Japan *K. Kobayashi, The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan

Fax: 81-6-6879-4889 E-mail: [email protected],

Running Title: Distinct Sensitivities of Nitrosylation

This work was supported by a Grant-in aid for Scientific Research (C) (23570136 to KK) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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Tel: 81-6-6879-8501

Summary

The transcriptional activation of SoxR in Escherichia coli occurs through direct modification of the [2Fe-2S] by nitric oxide (NO) to form a dinitrosyl-iron complex (DNIC). Here, we investigated the reactions of NO with [2Fe-2S] clusters of SoxR; the results were compared with those obtained for spinach ferredoxin (Fd). UV-visible absorption and ESR spectra of SoxR upon treatment with an NOdonor showed the formation of DNIC of SoxR efficiently, whereas those of Fd exhibited small changes.

change in absorption, consisting of a faster phase and a slower phase, was observed. The slower phase fraction was increased with increases in the [NO]/[SoxR] molar ratio, reaching a plateau at ~2 equivalents of NO. On the basis of these results, we propose that the faster phase corresponds to the reaction of the first NO molecule with [2Fe-2S] of SoxR, followed by the reaction of the second NO molecule. In the reaction of NO with Fd, no slower phase was observed. These results suggest that the reaction of the second equivalent of NO is an important process for the formation of DNIC.

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Upon pulse radiolysis of a deaerated solution of SoxR in the presence of sodium nitrite, a biphasic

Keywords: nitric oxide/pulse radiolysis/ iron-sulfur cluster/transcription factor/dinitrosyl iron complex

Abbreviations: Fd, ferredoxin; Ad, adrenodoxin, DNIC, dinitrosyl iron complex; NOC7, (3-[2-hydroxy-2nitrosohydrazino]-N-methyl-1-propanamine); SoxRox, oxidized SoxR; SoxRred, reduced SoxR; Mb, myoglobin; eaq-, hydrated electron, SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sGC, guanylate cyclase, RRE, Roussin’s red ester; rRRE, reduced Roussin’s red ester. Downloaded from http://jb.oxfordjournals.org/ at Istanbul University Library on April 23, 2014

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Nitric oxide (NO) is a key signaling molecule in a diverse array of cellular processes, ranging from signal transduction in the nervous system to blood pressure regulation (1-4) and involvement in the immune response (5, 6). On the other hand, NO is a highly reactive and toxic free radical gas that can freely diffuse into cells and attack cellular components (7-11). In activated macrophages, NO can be a powerful toxic agent that kills pathogenic bacteria and tumor cells (5, 6). To survive the deleterious effects of NO, most bacteria have evolved specific NO-sensor proteins that regulate the expression of enzymes required for rapid detoxification of NO (12-14). It has become apparent that there are multiple regulatory systems in prokaryotes that mediate

(22)—all of which contain an iron-sulfur cluster—are involved in coordinating physiological responses to NO. In addition, the biological activity of iron-sulfur proteins, such as mitochondrial aconitase (23), IRP (24), ferrochelatase (25), and AirSR (26) are controlled by NO binding. Therefore, iron-sulfur clusters are potential sites for both NO-derived physiological signal transduction and toxicity. Reactions of NO with cysteine-ligated iron-sulfur cluster proteins typically result in disassembly of the ironsulfur core and formation of a dinitrosyl iron complex (DNIC) (27-29). The identity and assignment of the DNIC are based on its observed g = 2.03 ESR signals (30), which have been used to follow DNIC formation from iron-sulfur clusters (31-33). However, other types of reaction products, such as an ESR-silent, thiolatebridged [Fe2(µ-SR)2(NO)4] species with a Roussin’s red ester (RRE) formula have also been observed (34, 35). More convincing evidence for the mechanism of DNIC formation and the intermediates along the reaction pathway is provided by data obtained using the synthetic model compounds [2Fe-2S] and [4Fe-4S] clusters (36, 37). SoxR, a member of the MerR family of transcription factors (38), regulates the oxidative stress response to superoxide in Escherichia coli (39). X-ray crystallographic structures have been obtained for the oxidized (active) state of E. coli SoxR-DNA complex (Fig. 1) (40). The [2Fe-2S] cluster of SoxR, essential for transcriptional activation of the soxS promoter (41), is coordinated to four cysteine residues (Cys-119, Cys122, Cys-124, and Cys-130). This CX2CXCX5C motif for the binding of the [2Fe-2S] cluster is different from that in other [2Fe-2S] proteins. Under conditions of oxidative stress, the metal center is oxidized and regulates the transcription of soxS (42). In turn, increased SoxS levels induce expression of various antioxidant proteins

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responses to NO (12, 15-18). The bacterial transcriptional regulators NsrR (19, 20), SoxR (21), and FNR

and repair proteins (43). Recently, we showed that direct oxidation of the [2Fe-2S] cluster of SoxR by O2- is an important mechanism for activating E. coli SoxR signaling (44). The E. coli SoxRS-controlled response is also activated by NO (21, 45, 46). In vivo study of phagocytosed bacteria within murine macrophages, the gene expression of soxS, the effect of a soxRS mutant, and the inhibitor of NO synthase suggested that induction of the SoxRS by NO counteracts most of the cytotoxic effects of NO produced by macrophages (45). The SoxS-induced enzymes such as endonuclease IV and glucose 6-phosphate dehydrogenase were shown to be required for resistance to macrophage killing (21, 45).

DNICs observed by ESR signals both in intact bacterial cells and in purified SoxR (33, 47, 48). However, little is known about the actual mechanism of cluster nitosylation. Hydrated electrons (eaq-) react with NO2- to form NO (49). With the pulse radiolysis technique, it is possible to produce NO within nanoseconds, allowing the reaction of NO to be followed spectrophotometrically through pulse radiolysis of aqueous solutions in the presence of nitrite under anaerobic conditions (50, 51). Spectral identification of products has been reported for the reactions of NO with O2- (50) and the iron complex of N-(dithiocarboxy)sarcosine (51). This method has also recently been used to characterize thionitrous acid (HSNO), by investigating the reaction of NO with SH· (52). The disassembly of an iron-cluster by NO is exceedingly rapid, and intermediate nitrosylation products are too unstable to observe (36, 44, 47, 48, 53-55). In the present study, we describe the application of pulse radiolysis to follow the reaction of NO with the [2Fe-2S] cluster of SoxR with nanosecond time resolution. The method aids in the identification of intermediate species and mechanistic pathways for nitrosylation of iron-sulfur clusters. Using this approach, we confirmed that NO reacts rapidly with the [2Fe-2S] cluster of SoxR. In addition, we present evidence that the sensitivity of iron-sulfur proteins to NO is much higher in SoxR than in spinach ferredoxin (Fd), an observation with possible wider implications for understanding the reaction of NO with iron-sulfur cluster-containing proteins.

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SoxR activation by NO occurs through direct modification of the [2Fe-2S] center to form protein-bound

Materials and methods Materials All chemicals were the highest purity available and were used without further purification. NOC7 (3-[2hydroxy-2-nitrosohydrazino]-N-methyl-1-propanamine) was purchased from Dojin (Kumamoto Japan). Bovine adrenodoxin (Ad), prepared from bovine adrenal glands (56), was kindly provided by Prof. Satoshi Miura (Tokyo University of Science).

The expression plasmids for E. coli SoxR were transformed into E. coli C41(DE3) and co-expressed with the isc operon (57), as described previously (58). SoxR was purified essentially as described previously (58). SoxR protein samples were purified as the oxidized form, and confirmed as >95% homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of SoxR was determined using an extinction coefficient of 12.7 mM-1 cm-1 at 417 nm (59). Fd from spinach was purified using a combination of DE52 and a gel-filtration column (HiLoad 16/60 Superdex 75 pg; GE Healthcare), essentially as described (60). Pulse Radiolysis. Pulse radiolysis experiments were performed using a linear accelerator at the Institute of Scientific and Industrial Research, Osaka University (44, 50, 51, 61). The pulse width and energy were 8 ns and 27 MeV, respectively. The dose was in the range of 15-400 Gy. A 1-kW xenon lamp was used as a light source. After passing through quartz cells with an optical path length of 0.1, 0.3, or 1 cm, the transmitted light intensities were analyzed and monitored by a fast spectrophotometric system composed of a Nikon monochromator, an R-928 photomultiplier, and a Unisoku data analysis system. For time-resolved transient absorption spectral measurements, the monitor light was focused into a quartz optical fiber, which transports the electron pulseinduced transmittance changes to a gated-multichannel spectrometer (Unisoku, TSP-601-02). All experiments were performed at room temperature. SoxR samples for pulse radiolysis were prepared by deoxygenating a solution containing 20-150 µM SoxR, 10 mM sodium phosphate (pH 7.0), 20 mM sodium tartrate, 0.5 M KCl, 0.1 M tert-butyl alcohol (for

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Expression and Purification.

scavenging OH·), and 1-100 mM NaNO2 by repeated evacuation and flushing with argon. The initial concentration of NO generated after the pulse was estimated from the concentration of hydrated electrons (eaq), with the assumption that eaq- reacts quantitatively with NO2-. The eaq- concentration generated by pulse radiolysis was determined based on the absorbance change at 650 nm using an extinction coefficient of 14.1 mM-1 cm-1 (62). The maximum concentration of NO was approximately 110 µM and could be adjusted by varying the dose of the electron beams. Nitrosylation of SoxR and Fd.

the NO donor NOC7 was prepared by dissolving in anaerobic 10 mM NaOH. Aliquots of solutions were rapidly mixed with SoxR or Fd at a molar ratio of 1:2 and incubated for 10-30 min. NO gas was injected into a sample cuvette containing SoxR or Fd after evacuation. Sulfur Determination. Elemental sulfur (S0) was determined by Sörbo’s method (54, 63, 64) using the addition of cyanide ion to generate SCN-, which yields an absorption band at 460 nm upon addition of Fe3+ ions. Fe3+-SCNconcentrations were determined by reference to a calibration curve generated from a solution of sodium thiocyanate. For determination of S0 following NO additions, nitrosylated SoxR and Fd were prepared by injection of NO gas under anaerobic conditions and used immediately. Spectrophotometric Measurements. Optical absorption spectra were measured with a Hitachi U-2900 spectrometer. ESR Measurements. ESR spectra were measured using a JES-RE2X spectrometer (X-band) with 100 kHz field modulation. Nitrosylated SoxR and Fd in ESR tubes were prepared by treatment with NOC7 or NO gas injection under anaerobic conditions, and were frozen at 77 K. Samples for ESR measurements after pulse radiolysis were performed as follows: solutions of SoxR (40100 µM) containing 10 mM potassium phosphate (pH 7.0), 20 mM potassium tartrate, 0.5 M KCl, 0.1 M tertbutyl alcohol, and 50 mM NaNO2 were deoxygenated in ESR tubes by repeated evacuation and flushing with argon. The samples in ESR tubes received one irradiation pulse from the electron linear accelerator, and then

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SoxR and Fd were nitrosylated by treatment with NOC7 (47) or NO gas injection. A 1 mM stock solution of

the reaction mixture was frozen immediately in liquid N2. ESR spectra were measured at 77 K. Spin concentrations were measured by double integration of the first-derivative ESR spectra and comparison with the corresponding intensities of known standards. RESULTS AND DISCUSSION Nitrosylation of 2Fe-2S Proteins. Nitrosylation of oxidized SoxR (SoxRox) proteins was accomplished by treating with the NO donor NOC7. Addition of the NO-releasing reagent NOC7 to an anaerobic sample of SoxRox was followed by measurement

~450–560 nm, with a smaller increase observed at ~400 nm. Time-course experiments indicated that formation of the DNIC of SoxR was completed within 20 min. Fig. 2-A shows the absorption spectra of SoxRox and the DNIC form of SoxR. The spectrum of the DNIC of SoxR is essentially identical to that reported previously (48). Verification of the formation of DNICs was provided by the presence of an ESR spectrum with a signal at g = 2.03 (Fig. 3). A similar ESR spectrum was obtained on NO gas injection to SoxRox under anaerobic conditions It has been reported that Fd gives no indication of NO-induced cluster degradation, unlike mammalian ferrochelatase (25). In contrast, upon the anaerobic addition of NOC7 to anaerobic sample of Fd, we observed a small change in the absorption of Fd over 30 min (Fig. 2-B). A similar spectral change of Fd was seen on injection of NO gas. The formation of DNICs of Fd was confirmed based on ESR spectra (Fig. 3). Integration of the g = 2.03 signal yielded only 5-8% of the ESR spectrum of the [2Fe-2S] cluster, and correlated with the absorption change accompanying the nitrosylation of Fd (Fig. 2-B). From these results, it is concluded that [2Fe-2S] cluster of SoxR is so readily nitrosylated by low concentration of NO, as compared with that of Fd The amount of elemental sulfur was quantified after reactions of SoxR and Fd with excess NO. Approximately two S0 atoms were detected on completion of the nitrosylation reaction of SoxR (Table 1). In contrast, 0.3 equivalents of S0 were detected after reaction of NO with Fd, reflecting the incomplete nitrosylation of Fd. Reaction of NO with SoxRox by Pulse Radiolysis.

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of UV-visible absorption. Progressive decreases in absorption were observed at 325 nm and in the region

Using pulse radiolysis, we have previously shown that NO is produced by the reaction of eaq- with NO2under deaerated conditions (eq 1) (48, 49).

This was confirmed spectrophotometrically by formation of the NO complex of Fe2+ myoglobin (Mb) (Supplemental Information [SI] Fig. S1 and S2). Here, we report the application of this method to the reaction of NO with the [2Fe-2S] cluster of SoxR. In the absence of NO2-, eaq- rapidly reacted with SoxRox to form the

partially reversed on a time scale of milliseconds (Fig. 4). The recovery process corresponds to reoxidation of [2Fe-2S] cluster of SoxRred by O2-, since the recovery was inhibited by addition of superoxide dismutase (44). In the presence of NO2-, eaq- reacted with both SoxRox and NO2-. The initial rapid decrease in absorption, caused by the reaction of eaq-, decreased with increases in the concentration of NO2-. At an NO2- concentration of 20 mM, the rapid decrease in absorption was completely lost, indicating that all the eaq- generated by pulse radiolysis reacted with NO2- to give NO. Instead, the slower decrease was seen on a time scale of hundreds of microseconds and increased up to an NO2- concentration of 50 mM. Therefore, we conclude that the change in absorption observed in the presence of 50 mM NaNO2 in Fig. 4 is due to the reaction of NO with SoxRox. Under these conditions studied (50 mM NaNO2), eaq- reacts with NO2- nearly quantitatively. When 68 µM NO was generated in a solution of 40 µM SoxRox, the time profile observed at 480 nm was composed of two phases–a fast phase and a slow phase. In contrast, only the faster decrease was observed at 420 nm (Fig. 5). This indicates that at least two distinct reactions are involved under the experimental condition. The effect of SoxRox concentration on the biphasic behavior at 480 nm was investigated at a fixed radiation dose ([NO] = 52 µM). As shown in Fig. 6-A, which depicts absorbance changes normalized to the respective faster phase for each SoxRox concentration, the fraction of the slower phase with respect to the faster phase increased with decreasing SoxRox concentration. In contrast, no second phase was observed at 420 nm (SI Fig. S3). The ratios of the slower phase to the faster absorbance change were plotted against the ratio, [NO]:[SoxRox] (Fig. 6-B). The absorbance changes displayed single-phase at less than ~0.5 equivalents of NO

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reduced form of SoxR (SoxRred) in a microsecond time scale. These initial changes in absorption subsequently

to SoxR (SI Fig. S4). The fraction of the slower phase increased with increases in the molar ratio [NO]/[SoxR] and reached a plateau at ~2 equivalents of NO. To further evaluate the relationship between these two phases, the NO dose was decreased from concentrations from 45 µM to 5 µM by reducing the electron beam dose at a fixed SoxRox concentration (30 µM) and plotted the ratios of the slower phase to the faster absorbance change against the ratio [NO]:[SoxRox] (Fig. 6-C). Similar to the results shown in Fig. 6-B, the slower phase fraction under these conditions was absent at less than ~0.5 equivalents of NO to SoxR, and increased with increases in the molar ratio

Accordingly, we propose that the faster phase corresponds to the reaction of 1 NO equivalent with the [2Fe2S] cluster of SoxR, followed by the reaction of 2 NO equivalents. The kinetic difference spectra corresponding the faster and slower phases are shown in Fig. 7. The spectrum at the end of the faster phase (1 ms) exhibited absorption minima at 420, 460, and ~ 550 nm. In contrast, the spectrum of the slower phase at 10 ms had an absorption minimum at 460 nm. A comparison of the species appearing at 10 ms with the DNIC of SoxR revealed that both spectra are similar from 440 to 600 nm, although they are quite distinct in the range of 350 to 440 nm. The spectrum of DNIC has a broad absorption band at ~390 nm (28). The rate constant of the reaction of NO with SoxRox was determined by generating 5-9 µM NO in a solution containing 50-100 µM SoxRox. Under these conditions, the slower absorbance changes at 480 nm were not seen, and the absorption change at 420 nm obeyed pseudo-first-order kinetics, with a rate constant that increased with the concentration of SoxRox (Fig. 8). This indicates that the faster absorbance change is a consequence of a bimolecular reaction of NO with SoxRox. The second-order rate constant of the reaction was calculated to be 1.3 × 108 M-1 s-1. To examine the formation of DNICs after pulse radiolysis of SoxRox in the presence of 50 mM NaNO2, we measured the ESR spectrum of a one-shot irradiated sample. The appearance of the ESR signal at g = 2.03, characteristic of the DNIC of SoxR, was identical to that obtained upon anaerobic introduction of NO gas into a SoxRox solution (Fig. 9). However, quantification of the signal recorded in Fig. 8 yielded a concentration corresponding to only 3-5% of the original cluster. This result reflects the fact that the amount of NO generated

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[NO]/[SoxR]. Beyond ~ 1 equivalent of NO, there was little further increase in the slower phase fraction.

by the pulse is not sufficient for the formation of a DNIC. In addition, only a small cross section of a sample was analyzed in the present experiment and diffusion would occur before the sample was frozen. Reaction of NO with Fd by Pulse Radiolysis. Fig. 10 shows results obtained in similar experiments using Fd. A decrease in absorption at 420 nm was observed in a millisecond time scale (Fig. 10-A). However, the absorbance changes were about 5-times smaller than those of SoxRox. Also in contrast to the results obtained with SoxR, the absorbance changes were not seen at a low dose of irradiation (NO concentrations < 20 µM) (SI Fig. S5). This can be explained by the low

not observed at least 30 s after pulse radiolysis. Fig. 10-B shows the kinetic difference spectrum at 1 ms after pulse radiolysis of Fd. The spectrum exhibited absorption minima at 420 nm, as was the case for SoxR, whereas the difference spectrum between Fd and the DNIC showed an absorption minimum at 460 nm. In contrast, bovine Ad, which contains a [2Fe-2S] cluster, showed no indication of a reaction with NO after pulse radiolysis (data not shown). Reaction of NO with SoxRred When cells under non-stressed conditions are exposed to NO, NO would predominantly interact with SoxRred because SoxR is maintained in its reduced, inactive form, SoxRred (47) However little is known about the kinetics of the reaction of SoxRred with NO. Here, to follow the reaction of NO with SoxRred, we monitored absorption changes after pulse radiolysis of a solution of SoxRox in the presence of 1 mM NO2-. Under these conditions, eaq- reacts with both NO2- and SoxRox. The concentration of NO generated was approximately 10 µM. The initial absorption decrease at 420 nm was observed. However, further absorbance changes were not observed at least 10 s after pulse radiolysis (data not shown). This may reflect the low affinity of NO for the [2Fe-2S] cluster of SoxRred. It has been reported that treatment of SoxRred with a saturated solution of NO leads to degradation of the iron-cluster to give several species, including reduced Roussin’s red ester (rRRE) and RRE (47). These reactions seem to be complex and occurred more slowly. Reaction Mechanism in Nitrosylation of [2Fe-2S] Proteins The spectrophotometric data presented here suggest the transient formation of 1 and 2 equivalent nitrosylations of the [2Fe-2S] cluster in SoxRox through two successive NO binding steps. This proposal is

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affinity of NO for the [2Fe-2S] cluster of Fd. In addition, it is noteworthy that further absorption changes were

supported by the dependence of the molar ratio ([NO]/[SoxR]) on biphasic absorbance changes (Fig. 6). The observed biphasic character disappeared at less than ~ 0.5 equivalents of NO to SoxR. Under these conditions, a mononitrosyl iron complex would be formed by binding of the first NO equivalent. Binding of the second NO equivalent corresponds to the slower process in Fig. 6. The fraction of the slower phase was increased with increases in the concentration of NO and reached a plateau at ~2 equivalents of NO. On the other hand, the reaction of SoxRox with NO is complete at 4 NO molecules per [2Fe-2S] cluster, yielding two DNICs. In the present method, formation of the DNIC, characterized by an increase in absorption at ~390 nm (27, 29, 34)

portion of [2Fe-2S] clusters formed DNICs, as confirmed by the ESR spectrum of DNICs. The reaction of an additional 2 equivalents of NO may be a slow process. Based on the proposed synthetic model inorganic [2Fe-2S] cluster (36, 37), a possible pathway for the formation of DNIC of SoxR is shown in Scheme I. The reaction of the first NO equivalent with the [2Fe-2S] clusters to form a mononitrosyl iron complex (intermediate I), followed by the reaction of the second NO equivalent to form a dinitrosyl complex (intermediate II). These NO additions in each step result in reductive elimination of 1 mole equivalent of elemental sulfur, respectively. Reactions of another 2 equivalents of NO result in the disassembly of the iron complex to form the corresponding DNIC. In this reaction scheme, Harrop et al (36) proposed that the breakdown of iron-sulfur cluster occurs in tandem with the sequestration of inorganic sulfur. The cysteine residues of SoxR may stabilize the DNIC formed during nitrosylation. When approximately equimolar NO was generated in a solution of SoxRox, two intermediates–a faster and a slower fraction considered to correspond to the mononitrosyl (intermediate I) and the dinitrosyl complexes (intermediate II), respectively–were formed. The most characteristic feature of intermediates I and II are their decreased absorption minima at 420 and 460 nm, respectively. These changes suggest that iron-sulfide and/or iron-cysteine interactions are significantly disrupted during these steps, but they also indicate the reduction of iron and oxidation of S2- to S0. The data in the present study provide experimental support for the formation of two S0 atoms on completion of the nitrosylation reaction of SoxR. The first identified physiological target of NO was soluble guanylate cyclase (sGC). The binding of NO to the ferrous heme in sGC is only limited by the diffusion rate of NO to the heme pocket, with kon ≈ 106-107 M-1

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was not detected, even following generation of 110 µM NO in a solution of 20-40 µM SoxRox. In fact, only a

s-1 and koff < 10-3 s-1 (65, 66). Such fast and tight binding to heme would guarantee NO signaling even in the presence of O2 in the medium. Similarly, the reaction of NO with SoxRox is very fast, leading to efficient formation of the DNIC. The rate constant (ki) between NO and SoxRox in the first step of Scheme I is 1.3 × 108 M-1 s-1. This rate is at least three orders of magnitude greater than that for the reaction of the [4Fe-4S] cluster of WhiB protein with FNR (~105 M-1 s-1) (53, 54). This difference is a reflection of the protein environment of SoxR, in which the [2Fe-2S] cluster is located on the molecular surface. The upper sulfur atom (S2) and two Fe atoms are fully exposed to solvent (40) (Fig.1). The location of the [2Fe-2S] cluster in a solvent-exposed

multiple signals indicative of possible oxidative stress (67). In Fd and Ad, on the other hand, the [2Fe-2S] cluster is shielded from the solvent by surrounding residues (68, 69). The results presented here provide the following important implications for the NO response in [2Fe-2S] clusters of proteins. The iron-sulfur cluster of Fd was insensitive to NO-induced disassembly, unlike that of SoxR. Moreover, the absorbance changes after pulse radiolysis of Fd were not detected at a low dose of irradiation (NO concentrations < 20 µM). This result suggests that NO binds the [2Fe-2S] cluster of Fd with low affinity. In addition, no slower phase was observed for Fd. The Fd reaction-product spectrum, which exhibited an absorption minimum at 420 nm (Fig. 10), may indicate the reaction of 1 NO equivalent with the Fd cluster, which is analogous to intermediate I of SoxR. NO binding and S2- oxidation occur concomitantly to yield S0 in the model inorganic [2Fe-2S] clusters (36) and SoxR. In Fd and Ad, both sulfur atoms of the [2Fe2S] cluster form similar hydrogen bonds with the main chain. By contrast, the S2 atom of the [2Fe-2S] cluster is fully exposed to the solvent. This may result in an inefficient and slow 2-equivalent NO reaction in Fd. In addition, an unusual CX2CXCX5C motif for the binding of the [2Fe-2S] cluster of SoxR, which has only a single residue (Gly123) between the two internal Cys residues, is different from those of other [2Fe-2S] proteins. Disassembly of the iron-sulfur cluster in SoxRox to form the DNIC is rapid, presumably owing to the high affinity of diferric iron for NO. The mechanisms of NO- and O2--meditated activation differ markedly. The redox regulation with SoxR is mediated by a shift in the redox equilibrium of SoxR (41, 70), with transcription reversibly inhibited by reactions linked to NAD(P)H (71, 72). In contrast, the reaction between NO and SoxRox is irreversible, with

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environment likely enables fast NO binding. SoxR responds not only directly to superoxide (44) but also to

the concomitant formation of a protein-bound dinitrosyl-iron complex (DNIC). Enzymatic pathways, however, may eliminate the DNIC (33). For example, cysteine desulfurase, together with L-cysteine, has been shown to efficiently repair the NO-modified form of the [2Fe-2S] cluster of ferredoxin (73, 74). Conclusions Spectrophotometric analyses revealed the transient formation of 1 and 2 equivalents of nitrosylation products in the [2Fe-2S] cluster of SoxR. The second process was not observed with Fd under the same experimental conditions. These results suggest that the reaction of the second equivalent of NO may be

Acknowledgement We thank members of the Radiation Laboratory of the Institute of Scientific and Industrial Research, Osaka University, for their assistance in operating the accelerator.

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important for the disassembly of a variety of iron-sulfur proteins.

Table 1. Sulfur (S0) Content after Treatment of SoxR and Fd with NO (~1 mM).

[2Fe-2S] (µM)

S0 (µM)

S0/[2Fe-2S]

SoxR1

30

54.8

1.82

SoxR2

40

88.5

2.21

SoxR3

50

86.0

1.72

Fd

80

25.2

0.32

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Sample

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Scheme 1. Possible reaction pathways for [2Fe-2S] clusters with NO, based on the proposed synthetic model inorganic [2Fe-2S] cluster [36, 37].

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Figure Legends

Fig. 2. (A) Changes in UV/vis absorption spectra upon the rapid mixing of SoxRox (30 µM) with NOC7 (85 µM) at pH 7.6 in a 20 mM Mops buffer containing 10 mM sodium tartrate. Spectra were recorded µ before (solid line) and 30 min after (dotted line) treatment with NOC7. (B) Changes in UV/vis absorption spectra upon the rapid mixing of oxidized Fd (42 µM) with NOC7 (105 µM) at pH 7.0 (10 mM phosphate buffer). Spectra were recorded before (solid line) and 60 min after (dotted line).

Fig. 3. ESR spectra (77 K) of the DNIC species formed reactions of SoxR (dotted line) and spinach Fd (solid line) 30 min after addition of 2 equivalents of NOC 7 at room temperature.

Fig. 4. Absorption changes after pulse radiolysis of SoxRox in the presence of NO2- measured at 420 nm. The samples contained 40 µM SoxRox, 10 mM sodium phosphate buffer (pH 7.0), 20 mM sodium tartrate, and 0.1 M tert-butyl alcohol.

Fig. 5. Absorption changes after pulse radiolysis of SoxRox, measured at 420 and 480 nm in the presence of 50 mM NaNO2. Samples contained 40 µM SoxRox and 68 µM NO.

Fig. 6. (A) SoxR-concentration dependence of absorption changes monitored at 480 nm after pulse radiolysis in the presence of 50 mM NaNO2. Samples contained 52 µM NO. Absorbance changes of the slower phase were normalized to the respective faster phase for each SoxRox concentration (B) The SoxRconcentration dependence of the slow phase fraction. The ratios ([B]/[A]) are plotted against the concentration of SoxR, where A and B are the absorbance changes of faster and slower absorbance changes, respectively. (C) The NO concentration dependence of the slower phase-fraction. Samples contained 30 µM SoxRox.

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Fig. 1. Structure of the SoxR-soxS promoter complex. A close-up view of the [2Fe-2S] cluster is shown. These structures were reproduced with PyMol using a structure from the Protein Data Bank (code 2ZHG).

Fig. 7 Comparison of the kinetic difference spectra obtained 1 ms and 10 ms after pulse radiolysis with DNICs minus the oxidized difference spectrum (dashed line). Samples contained 70 µM SoxRox and 75 µM NO.

Fig. 8. SoxR concentration-dependence of apparent rate constants of the absorption decrease at 420 nm.

Fig. 9. ESR spectra of SoxRox treated with a saturated solution of NO and SoxRox after pulse radiolysis in the presence of NaNO2 at 77 K.

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Fig. 10. Absorption change at 420 nm after pulse radiolysis of spinach Fd, measured in the presence of 50 mM NaNO2. Samples contained 80 µM Fd, 10 mM sodium phosphate buffer (pH 7.0), 0.1 M tert-butyl alcohol. 39 µM NO was generated. (B) Comparison of the kinetic difference spectrum obtained 1 ms (●) with that of NOC7-treated Fd minus oxidized Fd (solid line).

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Fig. 1. Structure of the SoxR-soxS promoter complex. A close-up view of the [2Fe-2S] cluster is shown. These structures were reproduced with PyMol using a structure from the Protein Data Bank (code 2ZHG). 254x190mm (96 x 96 DPI)

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Mechanistic studies on formation of the dinitrosyl iron complex of the [2Fe-2S] cluster of SoxR protein.

The transcriptional activation of SoxR in Escherichia coli occurs through direct modification of the [2Fe-2S] by nitric oxide (NO) to form a dinitrosy...
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