DOI: 10.1002/cbic.201500273

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Involvement of Acidic Amino Acid Residues in Zn2 + Binding to Respiratory Complex I S¦bastien Kriegel,[a, b] Batoul Srour,[a] Stefan Steimle,[c] Thorsten Friedrich,[c] and Petra Hellwig*[a] Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn2 + ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn2 + inhibition of the proton-pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron trans-

fer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm¢1) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn2 + binding. In variant D563NL, which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn2 + binding is changed. Our data support a role for this residue in proton translocation.

Introduction Complex I, the main entry point of electrons into the respiratory chains, catalyzes the oxidation of NADH and the reduction of quinone. The free energy released by the electron transfer reaction is used to translocate four protons across the membrane by an as yet unclear mechanism. Considerable progress has been made in elucidating the structure and functional details of complex I.[1–9] Electron microscopy revealed the twopart structure of the complex: a peripheral arm consisting of the globular subunits and extending in the aqueous environment, and a membrane arm in the phospholipid bilayer.[10–11] Recently, the structures of the bacterial complexes (or parts thereof) from Thermus thermophilus,[1, 12] and Escherichia coli [13] and of the mitochondrial complex from Yarrowia lipolytica[14] and bovine heart[15] was solved. The bacterial complex, consisting of 13 to 15 different subunits, constitutes the catalytic core of the enzyme. Homologues of these subunits with up to 30 additional subunits comprise the mitochondrial complex (~ 1 MDa). Seven of the 14 core

[a] Dr. S. Kriegel, Dr. B. Srour, Prof. P. Hellwig Laboratoire de Bioelectrochimie et Spectroscopie, UMR 7140 Chimie de la MatiÀre Complexe, Universit¦ de Strasbourg, CNRS 1 rue Blaise Pascal, 67070 Strasbourg (France) E-mail: [email protected] [b] Dr. S. Kriegel Universit¦ Paris Diderot, Sorbonne Paris Cit¦ Laboratoire d’Electrochimie Mol¦culaire Unit¦ Mixte de Recherche Universit¦—CNRS No. 7591 B–timent Lavoisier, 15 rue Jean de Baı¨f, 75205 Paris Cedex 13 (France) [c] Dr. S. Steimle, Prof. T. Friedrich Albert-Ludwigs-Universit•t Freiburg, Institut fìr Biochemie Albertstrasse 21, 79104 Freiburg (Germany) Supporting information for this article is available on the WWW under

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subunits are globular proteins that bear the redox groups: one flavin mononucleotide (FMN) and eight to ten iron–sulfur (Fe/ S) clusters. The remaining seven subunits are highly hydrophobic proteins that fold into 64 transmembrane a-helices.[1] Whereas it is well accepted that electrons from NADH are transferred to quinone via FMN and a long chain of Fe/S clusters, the coupling between electron transfer in the peripheral arm and proton translocation through the membrane arm is still under discussion. The inhibition of putative proton pathways is thus an interesting approach to obtain further information on the coupling. Zinc is known to inhibit proton translocation in respiratory complexes I,[16, 17] III,[18, 19] and IV,[20, 21] as well as several other proteins. IC50 for the inhibition of the respiratory complexes was estimated to be in the mid-micromolar range for complexes I and IV and in the mid-nanomolar range for complex III. Under physiological conditions, the levels of free Zn2 + in the mitochondria are close to zero. However, pathologic conditions might have increased Zn2 + concentrations, thus inhibitingoxidative phosphorylation at complex III.[22] It is not clear whether inhibition of complexes I and IV is of physiological relevance. It was shown that zinc inhibits proton translocation in complex IV and causes uncoupling from electron transfer,[23] thus enabling the use of zinc as a tool to investigate proton pumping. Sharpley and Hirst suggested that Zn2 + ions inhibit mitochondrial complex I by blocking protonation of bound ubiquinone or by blocking proton translocation.[16] Binding of zinc to complex I is mechanistically complicated. The IC50 was 10– 50 mm, depending on the pre-incubation time. In addition, the kinetics of the inhibition depended on the state of the enzyme, as during catalysis, zinc only binds slowly and progressively, whereas the resting state is affected faster. No highresolution structure of the membrane domain was available,


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Full Papers thus no detailed suggestions about the inhibition mechanism were made. Studies with the bacterial complex I from E. coli suggested the presence of at least three Zn2 + -binding sites with different affinities.[17] A low-affinity site (Ki > 700 mm) was shown to interfere with NADH oxidation, and two high-affinity sites (Ki = 55 and 80 mm) compete with protons while not affecting quinone reduction. It was proposed that the high-affinity binding sites are on the cytoplasmic and periplasmic sides of the proposed proton pathways in the membrane arm. Thus, Zn2 + might inhibit complex I mainly by blocking the proton channels.[16, 17] From the pH dependency of Zn2 + inhibition and from FTIR spectroscopy, it was concluded that carboxylic acid residues are involved in Zn2 + binding at these sites.[17] Here we studied the effect of Zn2 + binding by a combined electrochemical and FTIR difference spectroscopic approaches. In addition, investigation of a site-specific variant suggests that position D563L (the superscript denominates the subunit containing the residue) of the horizontal amphipathic helix within the membrane arm is part of one of the proposed proton pathways.

Results and Discussion Binding of Zn2 + to complex I Complex I in H2O and D2O buffer was incubated with Zn2 + , and the evolution of the oxidized-minus-reduced FTIR difference spectra was recorded (Figure 1). For each difference spectrum describing the increasing inhibition, 5–10 difference spectra were averaged to ensure a high S/N ratio. It should be noted that in IR difference spectroscopy (introduced in the early 1980s), spectra are typically obtained by cycling a reaction several times and thus exposing the protein to the reaction over hours.[24–27] Usually identical spectra are seen over time, but here a gradual change was found when Zn2 + ions were present. FTIR difference spectra of complex I in the absence of Zn2 + by the same procedure revealed no change over time (see the Supporting Information), thus revealing no significant difference between the first and the last cycles averaged and confirming that the changes over time were specific to the Zn2 + -treated samples. In the presence of zinc, small but distinct changes in the difference spectra (Figure 1) included changes of signals in the amide I range (1700–1600 cm¢1), decreases of the signals in the amide II region (1580–1520 cm¢1), and modifications to the signatures of protonated residues such as Asp and Glu (see insets). As zinc most likely interacts with residues of the proton translocation pathway, the spectral changes might reflect changes within the proton pathways. By comparing the data at different times, it is evident that particular signals are modified over time and that the intensities of some of the signals changed. This time dependence is in agreement with the time course of the inhibition of the complex by Zn2 + .[17] In a control experiment the his-tagged complex I and the un-tagged (WT) enzyme were compared, as Zn2 + is known to have a strong affinity to polyhistidine moieties (Supporting Information). “Double-difference” spectra were calculated by subtracting the spectra of complex I from those of complex I treated with Zn2 + . No influence of the his-tag was found, as identical signals were obtained in the double-difference spectra of complex I from both purification protocols. As the general shapes of the amide I and amide II bands in Figure 1 were very close to those for the untreated enzyme, incubation with Zn2 + does not influence the redox chemistry of the complex, in accordance with previous kinetic data.[16, 17] Contributions from the backbone

Figure 1. Evolution of oxidized-minus-reduced FTIR spectra (1800– 1200 cm¢1) of Zn2 + -treated complex I over a number of redox cycles in A) H2O or B) D2O buffer. Black line, first cycle; gray lines, intermediate cycles; dark gray line, last cycle. Insets: spectral region 1800 to 1720 cm¢1 (insets to the same scale).

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The changes induced by zinc binding include negative peaks at positions typical for randomly structured residues (1648 cm¢1) and beta-type structures (hairpins, 1681 cm¢1). The positive signals reflect perturbations of a-helical structures (1656–1665 cm¢1) and b-sheets (1636–1686 cm¢1).[28] Only very small shifts were observed in the spectrum when the buffer was D2O; this indicates that the protein backbone giving rise to the difference signals is not accessible to D2O. It is likely that this protein moiety is part of the membrane arm as suggested for the un-exchangeable fraction of complex I identified


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Full Papers by H/D exchange kinetics of the amide I signal.[29] The same holds for the amide II band (1556(¢) (WT), 1559(¢) cm¢1 (histagged)) and amide II’ band (1488(¢), 1453(¢) cm¢1). These signals barely shifted after H/D exchange. Contributions from protonated acidic residues Very interesting shifts are seen for the n(C=O), nas(COO¢), and ns(COO¢) vibrational modes of protonated and deprotonated Asp and Glu residues.[30, 31] The small signal at 1733 cm¢1, which we previously attributed to the protonation of one or more aspartic or glutamic acid residues[32, 33] in complex I, was found to be perturbed upon zinc binding. The signals at 1756 and 1781 cm¢1 in H2O (Figure 1 A, inset) were associated with the n(C=O) modes of protonated acidic residues in hydrophobic environments. Weaker hydrogen bonds to oxygen atoms of the carboxylic group result in higher frequencies of the C=O vibration.[34] The presence of two peaks is indicative of at least two types of protonated acidic residue, including those that do not share the hydrogen with any other residue. Typically, protonated acidic residues in a hydrophobic environment are observed at around 1740 cm¢1 in proton-translocating proteins.[35–38] In some cases signals at 1761 cm¢1 have been reported, as for example in bacteriorhodopsin[39] and in cytochrome bd,[40] and at 1768 cm¢1 in the aa3 oxidase from Acidianus ambivalens.[41] The signal at 1781 cm¢1, however, has a rather unusual high frequency; this points to a protonated residue in the absence of a hydrogen bond. Both acetic and propanoic acid in the vapor phase show peaks above 1780 cm¢1 representing this particular mode.[41] Ab initio calculations revealed that a n(C=O) vibration with no hydrogen bond is expected at between 1759 and 1776 cm¢1.[34] As acidic residues in the highly hydrophobic environment would not interact with Zn2 + it is most likely that they reside in the inner part of the proposed proton pathways (blocked by Zn2 + ) and are not capable of receiving protons after several reduction/oxidation cycles. Upon H/D exchange the peaks obtained for the inhibited complex I exhibited a 2 cm¢1 downshift and a very slight broadening, typical for protonated acidic residues (Figure 1 and inset). It should be noted that the first two cycles showed slightly weaker S/N ratios, as the H/D exchange was only completed for buried residues after the reducing potential was applied.

the sample containing Zn2 + , together with bands at 1424 (free C¢O) and 1376 cm¢1 (Zn2 + bound C¢O; Figure 1). A signal at 1364 cm¢1 might also be indicative of bidentate Zn2 + binding. For the nas(COO¢) mode around 1560 cm¢1, the assignment is uncertain because of possible overlap with the amide II absorption from the protein backbone and modes from other side chains. Nevertheless, the peak at 1592 cm¢1 is tentatively attributed to the nas(free C¢O) mode of zinc-chelated Asp or Glu residues. Its red-shifted counterpart is attributed to the peak at 1520 cm¢1, but it could also arise from Tyr residues that typically show intense signals at this position.[47] One peak that might include contributions from one of the most common zinc chelator, histidine, is observed at 1601/ 1605 cm¢1. A peak at 1115 cm¢1 (indicative of a His-zinc complex)[48] was not found here, but vibrational modes typical for histidine have been identified for complex I from Y. lipolytica;[49] it cannot be excluded that they are too weak to be seen in our case, as their extinction coefficient is very low. Cys, Tyr, Arg, and Lys residues are also common Zn2 + chelators, and with the exception of Cys they are present in the hydrophilic channels in the membrane arm. Some signals can be tentatively assigned to Tyr residues (Table 1), mainly the peak at 1503 cm¢1, which could represent the n(C=C) mode of zinc-coordinated Tyr. From the multiple signals over 1620 to 1690 cm¢1, chela-

Contributions from deprotonated acidic residues and further Zn2 + chelators In proteins, Zn2 + is typically chelated by three or four side chains in a pyramidal organization;[43] high-affinity binding sites often involve deprotonated Asp/Glu side chains.[44] In IR spectra, the chelation motif of deprotonated acidic residues can be studied. Monodentate chelation of divalent metals, for example, creates two non-equivalent C¢O bonds, splitting both the nas(COO¢) and ns(COO¢) modes into two bands, one at higher and the other at lower wavenumbers.[45, 46] The signal arising from the ns(COO¢) vibration at 1405 cm¢1 was lower in ChemBioChem 2015, 16, 2080 – 2085


Table 1. Tentative assignment of the signals of the oxidized-minus reduced FTIR difference spectra of the his-tag purified complex I from E. coli in presence of Zn2 + , and the variation over time (%: increase of signal intensity, &: decrease of signal intensity, *: no change).

Position [cm¢1] and variation H2O D2 O 1781(+ +) 1756(+ +) +) 1733(+ 1704 1698(+ +) 1681

1779(+ +) 1754(+ +) +) 1750(+ 1733(+ +) 1704 1698(+ +) 1681

1673(+ +) 1673(+ +) 1665(+ +)(sh) 1648 1648 1640 (broad) 1632(¢) 1626 1619 1601 1590 1570/ 1559(¢) 1519(¢) 1503(+ +)

1424 1397(¢) 1376 1364

1640 (broad) 1632(¢) 1619 1605 1590 1570/ 1559(¢) 1515(¢) 1503(+ +) 1460 (broad) 1424 1397(¢) 1376 1364

Tentative assignment

n(C=O)COOH, no H-bond n(C=O)COOH, single H-bond n(C=O)COOH n(C=O)protonated Asp,Glu proton pump n(C=O)Asn,Gln hydrophobic environment n(C=O)Asn,Gln, nas(CN3H5 + )Arg, Amide I b-sheets n(C=O)Asn,Gln, n(C=O) SQ, nas(CN3H5 + )Arg, amide I b-turns & nas(CN3H5 + )Arg & nas(CN3H5 + )Arg, amide I a-helices * n(C=O)Asn,Gln bound to Zn2 + , n(C=O) Q or SQ, amide I random & amide I % % % & * & *

& & * & * %

ns(CN3H5 + )Arg, d(NH3 + )Lys, amide I b-sheets d(NH3 + )Lys, amide I b-sheets d(NH2)Asn,Gln n(C-N and C-C)HisH + nas(COO¢)Asp,Glu bound to Zn2 + , monodentate nas(COO¢)Asp,Glu, amide II

% n(C=C)TyrOH & n(C=C)TyrO¢ bound to Zn2 + , n(C=C)TyrO¢ % d(1H-O-2H) (amide II’) * % * *

ns(COO¢)Asp,Glu bound to Zn2 + , monodentate nas(COO¢)Asp,Glu ns(COO¢)Asp,Glu bound to Zn2 + , monodentate ns(COO¢)Asp,Glu bound to Zn2 + , bidentate

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Full Papers tion to Arg disturbing the nas(CN3H5 + ) and ns(CN3H5 + ) modes can also not be ruled out and would provide a further explanation for the peak pattern in this area.[30, 31] The assignments are summarized in Table 1.

Interaction between Zn2 + and variant D563NL Because the coordination of Zn2 + ions clearly involves acidic residues, we studied variants of the D563L residue. This residue is in the horizontal amphipathic helix aligned to the membrane arm. It is conserved in bacteria and eukaryotes (or replaced by the similar glutamic acid in a few species)[50] and is thus an interesting candidate interaction partner for Zn2 + with complex I. Figure 2 shows the position of D563L. The recently described D563NL variant showed 25 % reduced NADH:ubiquinone oxidoreductase activity (1.2 œ 0.2 mmol min¢1 mg¢1) in proteoliposomes.[50] Compared to the parental protein, the H + /2e¢ stoichiometry was reduced from 4 to 3, thus suggesting that at least one of the proton pathways is perturbed in this mutant. The conservative substitution D563EL led to the same

H + /2e¢ stoichiometry of 3, thus demonstrating that the position of the carboxylic group is also essential and that there is limited flexibility of the protein backbone around this position. The IC50 for Zn2 + with both wild-type and variant D563NL was 40 mm (Figure S3), which is very close to the value reported for the E. coli complex (50 mm).[17] This was as expected, as there are several Zn2 + -binding sites in complex I associated with the four proton pathways. Mutation of one of the possible interaction partners will thus not significantly influence IC50. Figure 3 shows the oxidized-minus-reduced FTIR difference spectra of the native enzyme and variants D563EL and D563NL.

Figure 3. Oxidized-minus-reduced FTIR difference spectra of A) the parent protein, B) variant D563EL, and C) variant D563NL.

Figure 2. Location of one putative zinc binding site in the membrane arm of complex I from E. coli. (PDB ID: 3RKO). A) Structure of the membrane arm lacking NuoH. The position of D563L is indicated by a circle. B) View normal to the membrane plane. The amphipathic helix of NuoL is shown in green and NuoM is in light blue. The side chains of D563L and surrounding polar residues are shown as sticks (oxygen in red, nitrogen in blue, sulfur in yellow). C) View along the membrane plane.

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The spectra of the variants did not show strong differences from that for the parental enzyme, thus confirming that the variants were properly assembled and stable, in accordance with their only mildly changed electron transfer rate. Small shifts in the amide I range (ca. 1664/1623 cm¢1) point to a change in conformation between the oxidized and reduced states.[51] In order to compare the changes that occur in the difference spectra upon Zn2 + addition to the D563NL variant and wildtype, double-difference spectra were calculated, by subtracting the data in the presence and absence of Zn2 + (Figure 4 A, B). Several difference signals were found for wild-type and mutant complexes upon Zn2 + binding, for example at 1704, 1648, 1559 cm¢1, and the signal characteristic for a tyrosine residues at 1519 cm¢1.[47] The complex signal pattern in the amide I range, however, showed small shifts and changes in intensity, 2083

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Full Papers residues are possible zinc-binding sites at the periplasmic side and can be expected to be sensitive to the binding of Zn2 + . These data provide the first step for the identification of the proton pathways, crucial to understand the mechanism of proton translocation by respiratory complex I.

Experimental Section

Figure 4. Double-difference spectra of complex I minus complex I treated with Zn2 + of A) the parent protein, and B) variant D563NL. Inset: region 1800–1700 cm¢1 including the contributions of protonated acidic residues.

and thus reveals changes in the structure of the variant upon electron transfer in the presence of Zn2 + . Furthermore, changes occurred in the important spectral region above 1720 cm¢1, and the signal at 1756 cm¢1 was shifted to 1746 cm¢1 in the D563NL spectrum. The molecular interactions of at least one of the acidic residues in a hydrophobic environment discussed above seem to be influenced in this variant. In addition, the absence of the signal at 1681 cm¢1 is noted together with changes in the amide I and amide II ranges.

Conclusion Structural data suggest four proton pathways in complex I,[1, 14] in accordance with the generally accepted stoichiometry of 4 H + per 2 e¢ .[52] In this study we report that inhibition of the enzyme with zinc produces complicated changes in the vibration spectra, derived from conformational reorganization. These changes are most likely in part of the proton-translocation machinery, as judged from the changes in the infrared signals typical for the protein backbone, acidic amino acid residues buried within the protein, and probably other charged residues. Direct comparison of electrochemically induced FTIR difference spectra between the parental protein and D563NL reveals a change in the spectral signature that arises when Zn2 + binds. This strongly suggests that D563 is indeed one of the zinc-binding sites of complex I. As this variant exhibited reduced proton pumping activity, the participation of this residue in one of the proposed proton pathways is very likely. Thus, our data demonstrate that zinc interferes with proton translocation in complex I as previously suggested.[16, 17] However, this residue is only one of several possible Zn2 + interaction partners. Given the presence of four proton pathways, several ChemBioChem 2015, 16, 2080 – 2085

Sample preparation: His-tagged complex I and variants were prepared as previously described.[50, 53] Briefly, membrane proteins were extracted with n-dodecyl-b-d-maltopyranoside (DDM; AppliChem, Darmstadt, Germany) and purified by anion exchange chromatography on Fractogel EMD TMAE Hicap (M) exchanger (Merck Millipore). Fractions with NADH/ferricyanide oxidoreductase activity were combined and applied to affinity chromatography on ProBond Ni-IDA resin (Life Technologies). Bound proteins were eluted with an imidazole step gradient. Fractions with NADH/ferricyanide oxidoreductase activity were combined and concentrated (Amicon Ultra-15, Millipore, 100 kDa MWCO). Complex I lacking the hexahistidine tag (introduced for purification purposes) was prepared as previously described.[54] Briefly, membrane proteins were extracted by DDM as above. Complex I was purified by anion exchange chromatography on Fractogel EMD TMAE Hicap (M), size-exclusion chromatography on Sephacryl S-300 HR (GE Healthcare), and a second anion exchange chromatography on Source 15Q (GE Healthcare). For IR spectroscopy, samples were in MES/NaOH (50 mm, pH 6.3) with NaCl (50 mm) and DDM (0.01 %). For Zn2 + inhibition, ZnSO4·H2O (1 mL; Sigma–Aldrich) in the same buffer was added to complex I (7 mL) to achieve a final Zn2 + /complex I molar ratio of 100:1 (complex I: 80 mg mL¢1, ZnSO4 15 mm). The sample was mixed in a Thermomixer (Eppendorf) for 3 h at 4 8C To exchange the buffer from H2O to D2O, complex I samples were repeatedly washed with D2O buffer by ultrafiltration for at least 3 h at 4 8C prior to the experiments. This time is necessary to ensure that the solvent-accessible, exchangeable water molecules bound to complex I[29] are fully exchanged. NADH:ubiquinone oxidoreductase activity: NADH:decylubiquinone oxidoreductase activity was determined by monitoring the decrease in NADH concentration (e340 = 6.3 mm¢1 cm¢1) in a TIDAS II spectrometer (J&M Analytik, Essingen, Germany).[54] Complex I (5 mg) was mixed (1:1, w/w) with E. coli polar lipids (Avanti) and added to the assay buffer (MES/NaOH (50 mm, pH 6.0) with NaCl (50 mm)) at 25 8C. Decylubiquinone (60 mm; Sigma–Aldrich) was added and incubated for 1 min before the reaction was started by the addition of NADH (150 mm; Carl Roth). To measure Zn2 + inhibition, ZnSO4 (0–500 mm) was added 1 min before the addition of decylubiquinone. FTIR: An homebuilt ultra-thin-layer spectroelectrochemical cell was used as described previously.[32] To avoid irreversible protein adhesion, the gold grid working electrode was modified by treatment with cysteamine and mercaptopropionic acid (2 mm each) for 1 h and then washed with deionized water. In order to accelerate the redox reaction, a mixture of 19 mediators[32] was added in a substoichiometric concentration (40 mm each). The assay solution (7–8 mL) was placed in the electrochemical cell (path length < 10 mm, as determined at the beginning of each experiment). All experiments were performed at 278 K. Control experiments (buffer and mediators only) led to only small signals below 1300 cm¢1 (Figure S5).


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Full Papers FTIR spectra were recorded as a function of the applied potential using a setup combining an IR beam from a Vertex 70 interferometer (Bruker) for the 4000–1000 cm¢1 range. First, the protein was equilibrated at an initial electrode potential, and a single-beam spectrum was recorded. Then the final potential was applied, and a single-beam spectrum was again recorded after equilibration. Equilibration generally took less than 4 min for the full potential step from 0.2 to ¢0.7 V (vs. Ag/AgCl). Difference spectra were calculated from two single-beam spectra, with the initial spectrum taken as a reference. Typically, 2 Õ 256 interferograms at 4 cm¢1 resolution were added for each single-beam spectrum, and Fourier transformed with triangular apodization and a zero filling factor of 2. Except for the experiment in which the evolution of the ox-red spectra was followed, at least 40 difference spectra were averaged. It is noted that averaging several difference spectra was necessary to obtain a good S/N ratio.

Acknowledgements We are grateful to the r¦gion Alsace, the Centre International de Recherches aux FrontiÀres de la Chimie (FRC), the Centre National de Recherche Scientifique (CNRS), the Institut Universitaire de France (IUF) and the Deutsche Forschungsgemeinschaft (DFG) Keywords: complex I · infrared difference spectroscopy · inhibitors · IR spectroscopy · proton pumping · Zn2 + [1] R. Baradaran, J. M. Berrisford, G. S. Minhas, L. A. Sazanov, Nature 2013, 494, 443 – 448. [2] U. Brandt, Annu. Rev. Biochem. 2006, 75, 69 – 92. [3] J. Hirst, Annu. Rev. Biochem. 2013, 82, 551 – 575. [4] M. Verkhovskaya, D. A. Bloch, Int. J. Biochem. Cell Biol. 2013, 45, 491 – 511. [5] J. E. Walker, Quart. Rev. Biophys. 1992, 25, 253 – 324. [6] T. Friedrich, J. Bioenerg. Biomembr. 2014, 46, 255 – 268. [7] T. Ohnishi, J. C. Salerno, FEBS Lett. 2005, 579, 4555 – 4561. [8] U. Brandt, S. Kerscher, S. Drçse, K. Zwicker, V. Zickermann, FEBS Lett. 2003, 545, 9 – 17. [9] T. Yagi, A. Matsuno-Yagi, Biochemistry 2003, 42, 2266 – 2274. [10] N. Grigorieff, Curr. Opin. Struct. Biol. 1999, 9, 476 – 483. [11] V. Gu¦nebaut, A. Schlitt, H. Weiss, K. Leonard, T. Friedrich, J. Mol. Biol. 1998, 276, 105 – 112. [12] L. A. Sazanov, P. Hinchliffe, Science 2006, 311, 1430 – 1436. [13] R. G. Efremov, R. Baradaran, L. A. Sazanov, Nature 2010, 465, 441 – 445. [14] V. Zickermann, C. Wirth, H. Nasiri, K. Siegmund, H. Schwalbe, C. Hunte, U. Brandt, Science 2015, 347, 44 – 49. [15] K. R. Vinothkumar, J. Zhu, J. Hirst, Nature 2014, 515, 80 – 84. [16] M. S. Sharpley, J. Hirst, J. Biol. Chem. 2006, 281, 34803 – 34809. [17] M. Schulte, D. Mattay, S. Kriegel, P. Hellwig, T. Friedrich, Biochemistry 2014, 53, 6332 – 6339. [18] D.-W. Lee, Y. El Khoury, F. Francia, B. Zambelli, S. Ciurli, G. Venturoli, P. Hellwig, F. Daldal, Biochemistry 2011, 50, 4263 – 4272. [19] T. A. Link, G. von Jagow, J. Biol. Chem. 1995, 270, 25001 – 25006. [20] P. L. Martino, G. Capitanio, N. Capitanio, S. Papa, Biochim. Biophys. Acta Bioenergetics 2011, 1807, 1075 – 1082.

ChemBioChem 2015, 16, 2080 – 2085

[21] D. A. Mills, B. Schmidt, C. Hiser, E. Westley, S. Ferguson-Miller, J. Biol. Chem. 2002, 277, 14894 – 14901. [22] B. Florian´czyk, Folia Neuropathol. 2009, 47, 234 – 239. [23] A. Kannt, T. Ostermann, H. Mìller, M. Ruitenberg, FEBS Lett. 2001, 503, 142 – 146. [24] R. B. Gennis, FEBS Lett. 2003, 555, 2 – 7. [25] R. Vogel, F. Siebert, Curr. Opin. Chem. Biol. 2000, 4, 518 – 523. [26] K. Gerwert, Biol. Chem. 1999, 380, 931 – 935. [27] A. Barth, Biochim. Biophys. Acta Bioenergetics 2007, 1767, 1073 – 1101. [28] E. Goormaghtigh, V. Cabiaux, J. M. Ruysschaert, Sub-Cell. Biochem. 1994, 23, 405 – 450. [29] R. Hielscher, T. Friedrich, P. Hellwig, ChemPhysChem 2011, 12, 217 – 224. [30] M. Wolpert, P. Hellwig, Spectrochim. Acta Part A 2006, 64, 987 – 1001. [31] A. Barth, Prog. Biophys. Mol. Biol. 2000, 74, 141 – 173. [32] P. Hellwig, D. Scheide, S. Bungert, W. M•ntele, T. Friedrich, Biochemistry 2000, 39, 10884 – 10891. [33] D. Flemming, P. Hellwig, S. Lepper, D. P. Kloer, T. Friedrich, J. Biol. Chem. 2006, 281, 24781 – 24789. [34] B. Nie, J. Stutzman, A. Xie, Biophys. J. 2005, 88, 2833 – 2847. [35] F. Siebert, W. M•ntele, K. Gerwert, Eur. J. Biochem. 1983, 136, 119 – 127. [36] F. Siebert, W. M•ntele, Eur. J. Biochem. 1983, 130, 565 – 573. [37] K. J. Rothschild, W. A. Cantore, H. Marrero, Science 1983, 219, 1333 – 1335. [38] P. R. Rich, A. Mar¦chal, Biochim. Biophys. Acta Bioenergetics 2008, 1777, 912 – 918. [39] K. Fahmy, O. Weidlich, M. Engelhard, H. Sigrist, F. Siebert, Biochemistry 1993, 32, 5862 – 5869. [40] J. Zhang, W. Oettmeier, R. B. Gennis, P. Hellwig, Biochemistry 2002, 41, 4612 – 4617. [41] P. Hellwig, C. M. Gomes, M. Teixeira, Biochemistry 2003, 42, 6179 – 6184. [42] National Institute of Standards and Technology: IR Spectra of Acetic and Propanoic Acid in Vapor Phase, 2013: cbook.cgi?ID = C64197&Type = IR-SPEC&Index = 1#IR-SPEC (acetic acid) and = C79094&Units = SI&Mask = 80#IR-Spec (propanoic acid). [43] K. A. McCall, C.-c. Huang, C. A. Fierke, J. Nutr. 2000, 130, 1437S – 1446S. [44] L. Giachini, F. Francia, A. Mallardi, G. Palazzo, E. CarpenÀ, F. Boscherini, G. Venturoli, Biophys. J. 2005, 88, 2038 – 2046. [45] M. Nara, M. Tasumi, M. Tanokura, T. Hiraoki, M. Yazawa, A. Tsutsumi, FEBS Lett. 1994, 349, 84 – 88. [46] Y. Neehaul, O. Ju‚rez, B. Barquera, P. Hellwig, Biochemistry 2013, 52, 3085 – 3093. [47] D. Flemming, P. Hellwig, T. Friedrich, J. Biol. Chem. 2003, 278, 3055 – 3062. [48] S. Gourion-Arsiquaud, S. Chevance, P. Bouyer, L. Garnier, J.-L. Montillet, A. Bondon, C. Berthomieu, Biochemistry 2005, 44, 8652 – 8663. [49] D. Marshall, N. Fisher, L. Grigic, V. Zickermann, U. Brandt, R. J. Shannon, J. Hirst, R. Lawrence, P. R. Rich, Biochemistry 2006, 45, 5458 – 5467. [50] S. Steimle, M. Willistein, P. Hegger, M. Janoschke, H. Erhardt, T. Friedrich, FEBS Lett. 2012, 586, 699 – 704. [51] T. Friedrich, P. Hellwig, Biochim. Biophys. Acta Bioenergetics 2010, 1797, 659 – 663. [52] M. O. Ripple, N. Kim, R. Springett, J. Biol. Chem. 2013, 288, 5374 – 5380. [53] T. Pohl, M. Uhlmann, M. Kaufenstein, T. Friedrich, Biochemistry 2007, 46, 10694 – 10702. [54] S. Stolpe, T. Friedrich, J. Biol. Chem. 2004, 279, 18377 – 18383. Manuscript received: June 1, 2015 Accepted article published: July 6, 2015 Final article published: July 31, 2015


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Involvement of Acidic Amino Acid Residues in Zn(2+) Binding to Respiratory Complex I.

Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn(2+) ions are capable of binding to acidic residu...
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