The Redox-Bohr Group Associated with Iron-Sulfur Cluster N2 of Complex I

Abstract

Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H+/2e−. This finding has significant implications on the discussion about possible proton pumping mechanism for complex I. Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H+/2e−. This finding has significant implications on the discussion about possible proton pumping mechanism for complex I. In mammalian cells mitochondrial complex I (NADH: ubiquinone oxidoreductase) provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme (1Brandt U. Annu. Rev. Biochem. 2006; 75: 69-92Crossref PubMed Scopus (638) Google Scholar) cause numerous human disorders (2Smeitink J. Van den Heuvel L. DiMauro S. Nat. Rev. Genet. 2001; 2: 342-352Crossref PubMed Scopus (542) Google Scholar). Very recently, the x-ray structure of the peripheral arm of complex I from Thermus thermophilus has been solved (3Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (652) Google Scholar). However, it is still unclear how complex I couples electron transfer from NADH to ubiquinone with the vectorial translocation of four protons across the bioenergetic membrane (4Wikström M.K.F. FEBS Lett. 1984; 169: 300-304Crossref PubMed Scopus (207) Google Scholar, 5Galkin A.S. Grivennikova V.G. Vinogradov A.D. FEBS Lett. 1999; 451: 157-161Crossref PubMed Scopus (147) Google Scholar). Numerous hypothetical mechanisms have been proposed for this energy conversion over the years (6Brandt U. Biochim. Biophys. Acta. 1997; 1318: 79-91Crossref PubMed Scopus (196) Google Scholar, 7Ohnishi T. Salerno J.C. FEBS Lett. 2005; 579: 4555-4561Crossref PubMed Scopus (91) Google Scholar, 8Friedrich T. J. Bioenerg. Biomembr. 2001; 33: 169-177Crossref PubMed Scopus (147) Google Scholar, 9Vinogradov A.D. Biochemistry (Mosc.). 2001; 66: 1086-1097Crossref PubMed Scopus (24) Google Scholar). None of the known redox groups of complex I (FMN and 8-9 iron-sulfur clusters) resides in the membrane domain (3Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (652) Google Scholar, 10Hirst J. Carroll J. Fearnley I.M. Shannon R.J. Walker J.E. Biochim. Biophys. Acta. 2003; 1604: 135-150Crossref PubMed Scopus (327) Google Scholar). The Fe4S4 cluster N2 is the last component in a “wire” of seven iron-sulfur clusters connecting FMN, the immediate oxidant for NADH, and substrate ubiquinone (3Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (652) Google Scholar, 11Yano T. Dunham W.R. Ohnishi T. Biochemistry. 2005; 44: 1744-1754Crossref PubMed Scopus (83) Google Scholar). While all other clusters in this chain have a redox midpoint potential of around −250 mV, iron-sulfur cluster N2 has a significantly more positive Em7 of typically −150 mV (12Ingledew W.J. Ohnishi T. Biochem. J. 1980; 186: 111-117Crossref PubMed Scopus (124) Google Scholar, 13Garofano A. Zwicker K. Kerscher S. Okun P. Brandt U. J. Biol. Chem. 2003; 278: 42435-42440Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Moreover, the redox midpoint potential of cluster N2 exhibits pronounced pH dependence between pH 6 and 8 (12Ingledew W.J. Ohnishi T. Biochem. J. 1980; 186: 111-117Crossref PubMed Scopus (124) Google Scholar). Based on this “redox-Bohr” effect (14Chance B. FEBS Lett. 1972; 23: 3-20Crossref PubMed Scopus (97) Google Scholar) and the comparably more positive potential, cluster N2 has been implicated as a key component of the proton pump (6Brandt U. Biochim. Biophys. Acta. 1997; 1318: 79-91Crossref PubMed Scopus (196) Google Scholar, 15Ohnishi T. Biochim. Biophys. Acta. 1998; 1364: 186-206Crossref PubMed Scopus (380) Google Scholar). The structural model for the ubiquinone reducing catalytic core of complex I (1Brandt U. Annu. Rev. Biochem. 2006; 75: 69-92Crossref PubMed Scopus (638) Google Scholar, 16Kashani-Poor N. Zwicker K. Kerscher S. Brandt U. J. Biol. Chem. 2001; 276: 24082-24087Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) that had been deduced from distinct homologies between [NiFe] hydrogenase and complex I (17Sauter M. Böhm R. Böck A. Mol. Microbiol. 1992; 6: 1523-1532Crossref PubMed Scopus (256) Google Scholar, 18Albracht S.P.J. Biochim. Biophys. Acta. 1993; 1144: 221-224Crossref PubMed Scopus (90) Google Scholar) was now confirmed by the structure of the peripheral arm of complex I (3Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (652) Google Scholar). Like the proximal iron-sulfur cluster of hydrogenase, iron-sulfur cluster N2 is at the interface between two subunits, the PSST subunit and the 49-kDa subunit (Fig. 1). In water-soluble [NiFe] hydrogenases a conserved histidine of the large subunit forms a hydrogen bond to the iron-sulfur cluster ligated to the small subunit (19Volbeda A. Charon M.H. Piras C. Hatchikian E.C. Frey M. Fontecilla-Camps J.C. Nature. 1995; 373: 580-587Crossref PubMed Scopus (1362) Google Scholar). Remarkably, a fully conserved histidine is also found at the corresponding position in the 49-kDa subunit of complex I. Mutating this histidine (His226)to methionine in the 49-kDa subunit of complex I from the strictly aerobic yeast Yarrowia lipolytica shifts the EPR spectrum of reduced iron sulfur cluster N2 to slightly lower field values but does not significantly affect the catalytic activity of complex I (20Grgic L. Zwicker K. Kashani-Poor N. Kerscher S. Brandt U. J. Biol. Chem. 2004; 279: 21193-21199Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Here we show that residue His226 in the 49-kDa subunit of complex I from Y. lipolytica is the redox-Bohr group associated with iron-sulfur cluster N2 but is not involved in the proton pumping mechanism of complex I. Isolation of Mitochondrial Membranes—Mitochondrial membranes were prepared from Y. lipolytica wild-type strain and mutant H226M (20Grgic L. Zwicker K. Kashani-Poor N. Kerscher S. Brandt U. J. Biol. Chem. 2004; 279: 21193-21199Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) as described previously (21Kerscher S. Okun J.G. Brandt U. J. Cell Sci. 1999; 112: 2347-2354Crossref PubMed Google Scholar, 22Kashani-Poor N. Kerscher S. Zickermann V. Brandt U. Biochim. Biophys. Acta. 2001; 1504: 363-370Crossref PubMed Scopus (74) Google Scholar). Isolation of Intact Mitochondria—Intact mitochondria from Y. lipolytica were prepared essentially by the enzymatic digestion method described in Ref. 23Zvyagilskaya R. Parchomenko O. Abramova N. Allard P. Panaretakis T. Pattison-Granberg J. Persson B.L. J. Membr. Biol. 2001; 183: 39-50Crossref PubMed Scopus (28) Google Scholar with minor modifications. Y. lipolytica cells were harvested at early logarithmic stage (OD ∼3-4), washed twice in ice-cold water, resuspended (0.1 g wet cells/ml) at room temperature in 50 mm Tris-HCl buffer (pH 8.6) supplemented with 5 mm dithiothreitol and incubated for 10 min, diluted with water, and washed twice again. After the last centrifugation the weakened cells were resuspended (0.1 g wet cells/ml) in 1.2 m sorbitol and 10 mm Hepes-KOH (pH 7.5) and 3-4 mg/ml zymolyase 20T (from Arthrobacter luteus, ICN Biomedicals) was added to digest the cell wall. The formation of spheroplasts was monitored spectrophotometrically, and usually 10-15 min of incubation at 30 °C was enough to complete the digestion. After that, 0.2 mm Pefablock SC was added, the spheroplast suspension was rapidly cooled and centrifuged at 500-600 × g for 10 min, and the pellet was resuspended and washed twice in the same buffer containing 4 mg/ml fatty acid-free BSA. 3The abbreviations used are: BSA, bovine serum albumin; Mes, 4-morpholineethanesulfonic acid; Mops, 4-morpholinepropanesulfonic acid. The pellet of the last centrifugation was resuspended in grinding buffer (20 mm Tris-HCl (pH 7.3), 0.4 m mannitol, 0.5 mm EDTA, and 4 mg/ml BSA, fatty acid-free). Spheroplasts were disrupted by 20 gentle strokes in a loosely fitting Dounce homogenizer. The suspension was diluted twice with isolation buffer (20 mm Tris-HCl (pH 7.3), 0.6 m mannitol, 0.5 mm EDTA, and 4 mg/ml BSA) and centrifuged at 2000 × g for 10 min. The supernatant was collected and centrifuged once more at 7000 × g for 20 min, and the pellet was resuspended with a smaller volume of isolation buffer and centrifuged again. The mitochondria were resuspended in 500-700 μl of isolation buffer and used immediately. Redox Titration of Iron-Sulfur Cluster N2—Redox titrations of mitochondrial membranes from parental strain and mutant H226M were performed under anaerobic conditions in the presence of redox mediators essentially as described previously (13Garofano A. Zwicker K. Kerscher S. Okun P. Brandt U. J. Biol. Chem. 2003; 278: 42435-42440Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Membranes were diluted in buffer containing sodium acetate, Mes, Mops, Tris, glycine each at 30 mm, 100 mm NaCl, and 1 mm EDTA. The solution was adjusted to the desired pH value at a protein concentration of 25-30 mg/ml. The following redox mediator dyes were added at final concentrations of 50 μm, N,N,N′,N′-tetramethylphenylene diamine, phenazine methosulfate, methylene blue, menadione, resorufin, indigo-trisulfonate, 1,2-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, phenosafranine, benzyl viologen, and methyl viologen. The membrane suspension was poised at appropriate potential values by addition of small amounts of 50 mm dithionite solution. Aliquots were anaerobically transferred into EPR tubes, shock frozen in cold isopentane/methyl cyclohexane (5:1) and stored in liquid nitrogen until measurement. The degree of cluster N2 reduction was monitored by EPR spectroscopy at 12 K. N2 signal intensities were determined after baseline correction and subtraction of high potential components (subtraction of an EPR spectrum obtained at higher redox potential without any detectable N2 signal). Fitting N2 signal intensities to the standard Nernst equation resulted in the given Em values. To check reversibility, after some titrations the membrane suspension was incubated in the presence of oxygen, and the slow autoxidation process was followed by taking several EPR samples. The cluster N2 EPR intensities of these samples at a given potential closely matched those obtained during reductive titration of the solution. Determination of Proton-pumping Stoichiometries—Proton pumping of different segments of the respiratory chain in intact mitochondria from Y. lipolytica was measured at 30 °C as intramitochondrial alkalization using the pH indicator neutral red (80 μm, 529-475 nm). The buffer was as follows: 0.6 m mannitol, 0.5 mm EDTA, 4 mg/ml BSA, 60 mm Tris-HCl (pH 7.6). Consecutively, appropriate substrates, 40 mm KCl, 1 μm valinomycin, and 2 mm cyanide were added, and oxidant pulses were applied by the addition of small amounts of electron acceptors to initiate redox-dependent proton translocation from the matrix. Matrix NADH was generated through reduction by malate dehydrogenase and the pyruvate dehydrogenase complex by adding malate/pyruvate to the mitochondria. The Midpoint Potential of Iron-Sulfur Cluster N2 Is pH-dependent—Similar to complex I from other sources (24Sled V.D. Friedrich T. Leif H. Weiss H. Fukumori Y. Calhoun M.W. Gennis R.B. Ohnishi T. Meinhardt S.W. J. Bioenerg. Biomembr. 1993; 25: 347-356Crossref PubMed Scopus (84) Google Scholar) the N2 midpoint potential of Y. lipolytica complex I at pH 7 is −140 mV (Fig. 2; Ref. 13Garofano A. Zwicker K. Kerscher S. Okun P. Brandt U. J. Biol. Chem. 2003; 278: 42435-42440Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To determine the redox-Bohr effect of iron-sulfur cluster N2 in Y. lipolytica mitochondrial membranes we measured its midpoint potential in the range between pH 5 and 9 (Fig. 2B). Due to some instability of the cluster at extreme pH values, it was difficult to reliably quantify the degree of reduction in particular at more positive redox potentials. Therefore, the Em values determined at pH 5 and 9 were not used for calculating the pH profile of iron-sulfur cluster N2. The pH dependence of the midpoint potential of iron-sulfur cluster N2 could be described by assuming a single protonated group. The fitted curve had a maximal slope of −36 mV/pH, and pK values of 5.7 for the oxidized state and 7.3 for the reduced state were calculated (Fig. 2B). These pK values fit well to a histidine side chain as the protonated group involved. His226 Is the Redox-Bohr Group Associated with Iron-Sulfur Cluster N2—We have shown previously that the mutation of histidine 226 in the 49-kDa subunit to methionine has little effect on steady-state activity and inhibitor sensitivity of complex I (20Grgic L. Zwicker K. Kashani-Poor N. Kerscher S. Brandt U. J. Biol. Chem. 2004; 279: 21193-21199Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, when we determined the redox midpoint potential of complex I from mutant H226M at pH 7, we found that it was shifted to the negative from −140 mV to −220 mV (Fig. 2A). More importantly, the Em value did not change significantly from pH 6 to 8, indicating that the mutation had completely abolished the pH dependence of this redox group (Fig. 2B). This indicated that the redox-Bohr group associated with iron-sulfur cluster N2 of complex I is the conserved histidine at position 226 of the 49-kDa subunit. It should be noted that complex I from the mutant was somewhat more sensitive to extreme pH values limiting the range where the redox-midpoint potential could be determined. His226 Is Not Part of the Proton Pump—If the redox-Bohr effect of cluster N2 was part of the proton-translocating machinery of complex I, abolishing the pH dependence of the midpoint potential should impair proton pumping. Therefore, we determined the H+/e− stoichiometry for complex I in intact mitochondria from the parental strain and from mutant H226M using the oxidant pulse technique (25Mitchell P. Moyle J. Mitchell R.M. Methods Enzymol. 1979; 55: 627-640Crossref PubMed Scopus (28) Google Scholar): pulses of oxidant were added to coupled mitochondria in the presence of excess reductant. Proton extrusion was monitored using the pH indicator neutral red. By choosing appropriate reductants and oxidants, the proton pumping activity of individual complexes or a larger segment of the respiratory chain could be monitored (Fig. 3). The response was calibrated internally (4Wikström M.K.F. FEBS Lett. 1984; 169: 300-304Crossref PubMed Scopus (207) Google Scholar) by measuring proton pumping of respiratory complex III (cytochrome bc1 complex) that has a well established pumping stoichiometry of 1 H+/e−. When we probed the segment comprising complexes I and III in the parental strain by malate/pyruvate and ferricyanide, proton pumping per added oxidation equivalent was about three times higher than for complex III alone that was probed separately using ubiquinol and ferrycyanide (Fig. 3A). With five different batches of mitochondria an overall stoichiometry of 2.8 ± 0.1 H+/e− for complexes I + III was determined. When complex I alone was probed with malate/pyruvate and ubiquinone the measured stoichiometry was 1.7 ± 0.1 H+/e− (n = 3). This indicated that, like its counterpart from bovine heart (5Galkin A.S. Grivennikova V.G. Vinogradov A.D. FEBS Lett. 1999; 451: 157-161Crossref PubMed Scopus (147) Google Scholar), complex I from Y. lipolytica pumped four H+ per NADH oxidized (2 e−) across the inner mitochondrial membrane. With mutant H226M we determined stoichiometries of 2.8 ± 0.1 H+/e− (n = 5) for complexes I + III and 1.9 ± 0.1 H+/e− (n = 2) for complex I alone (Fig. 3B). Moreover, mitochondria from parental and mutant strain exhibited almost identical respiration rates and synthesized the same amount of ATP per oxygen consumed. This also indicated that proton pumping was not at all affected by the mutation. Our data indicate that residue His226 of the 49-kDa subunit of Y. lipolytica complex I is the protonated group that is responsible for the pH dependence of the redox midpoint potential of iron-sulfur cluster N2. This is consistent with the fact that in the structure of the peripheral arm of T. thermophilus complex I the corresponding histidine is found within 4 Å of iron-sulfur cluster N2 (3Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (652) Google Scholar). In earlier studies, we had changed this histidine to alanine, glutamine, or cysteine. These mutations also had only moderate effects on the catalytic activity of complex I, but the EPR signal of iron-sulfur cluster N2 was markedly reduced (16Kashani-Poor N. Zwicker K. Kerscher S. Brandt U. J. Biol. Chem. 2001; 276: 24082-24087Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 20Grgic L. Zwicker K. Kashani-Poor N. Kerscher S. Brandt U. J. Biol. Chem. 2004; 279: 21193-21199Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). It is quite remarkable that removing the redox-Bohr group of iron-sulfur cluster N2 and thereby abolishing the pH dependence of its redox midpoint potential and shifting Em7 by −80 mV had no effect on the proton pumping capacity of complex I. Because such dramatic changes in the physicochemical properties of iron-sulfur cluster N2 had no functional consequences, one can effectively rule out any specific role for this redox center within the energy converting mechanism of complex I other than serving as the electron donor for ubiquinone. In mutant H226M, electrons that are fed into complex I from NADH stay on a very negative potential until they reach ubiquinone. The unexpected observation that this does not affect proton pumping by complex I strongly suggests that the energy conversion driving proton translocation across the membrane is immediately linked to the redox chemistry of ubiquinone but is not associated to one of the electron transfers upstream. As implicated in several proposed mechanisms for complex I (6Brandt U. Biochim. Biophys. Acta. 1997; 1318: 79-91Crossref PubMed Scopus (196) Google Scholar, 7Ohnishi T. Salerno J.C. FEBS Lett. 2005; 579: 4555-4561Crossref PubMed Scopus (91) Google Scholar, 26Brandt U. Biofactors. 1999; 9: 95-101Crossref PubMed Scopus (45) Google Scholar), redox-linked protonations and deprotonations of ubiquinone intermediates could be directly employed to drive vectorial proton translocation. In more recent models combinations of direct and conformational coupling mechanisms have been suggested to account for the observed pumping stoichimetry of complex I (7Ohnishi T. Salerno J.C. FEBS Lett. 2005; 579: 4555-4561Crossref PubMed Scopus (91) Google Scholar, 8Friedrich T. J. Bioenerg. Biomembr. 2001; 33: 169-177Crossref PubMed Scopus (147) Google Scholar). In Y. Biochemistry. 33: PubMed Scopus Google Scholar) and structural Carroll J. L.A. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) for redox-linked conformational changes of complex I has been However, the of all redox from the membrane domain (3Sazanov L.A. Hinchliffe P. Science. 2006; 311: 1430-1436Crossref PubMed Scopus (652) Google Scholar) to any direct between redox chemistry and vectorial proton translocation as by P. Biol. Rev. PubMed Google Scholar). coupling by range conformational changes Therefore, we that, the redox of ubiquinone the driving force for proton protonation of the substrate reduction is a the protons involved in chemistry are not those that are across the membrane. we that reduction of ubiquinone at the interface between the 49-kDa and PSST subunit conformational changes that are to of the membrane arm as the The most for this are the and they to C. C. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, I.M. Walker J.E. Biochim. Biophys. Acta. 1992; PubMed Scopus Google Scholar). Zickermann for