Publications by authors named "Michael I Verkhovsky"

60 Publications

Oxygen as Acceptor.

EcoSal Plus 2015 ;6(2)

Like most bacteria, Escherichia coli has a flexible and branched respiratory chain that enables the prokaryote to live under a variety of environmental conditions, from highly aerobic to completely anaerobic. In general, the bacterial respiratory chain is composed of dehydrogenases, a quinone pool, and reductases. Substrate-specific dehydrogenases transfer reducing equivalents from various donor substrates (NADH, succinate, glycerophosphate, formate, hydrogen, pyruvate, and lactate) to a quinone pool (menaquinone, ubiquinone, and dimethylmenoquinone). Then electrons from reduced quinones (quinols) are transferred by terminal reductases to different electron acceptors. Under aerobic growth conditions, the terminal electron acceptor is molecular oxygen. A transfer of electrons from quinol to O₂ is served by two major oxidoreductases (oxidases), cytochrome bo₃ encoded by cyoABCDE and cytochrome bd encoded by cydABX. Terminal oxidases of aerobic respiratory chains of bacteria, which use O₂ as the final electron acceptor, can oxidize one of two alternative electron donors, either cytochrome c or quinol. This review compares the effects of different inhibitors on the respiratory activities of cytochrome bo₃ and cytochrome bd in E. coli. It also presents a discussion on the genetics and the prosthetic groups of cytochrome bo₃ and cytochrome bd. The E. coli membrane contains three types of quinones that all have an octaprenyl side chain (C₄₀). It has been proposed that the bo₃ oxidase can have two ubiquinone-binding sites with different affinities. "WHAT'S NEW" IN THE REVISED ARTICLE: The revised article comprises additional information about subunit composition of cytochrome bd and its role in bacterial resistance to nitrosative and oxidative stresses. Also, we present the novel data on the electrogenic function of appBCX-encoded cytochrome bd-II, a second bd-type oxidase that had been thought not to contribute to generation of a proton motive force in E. coli, although its spectral properties closely resemble those of cydABX-encoded cytochrome bd.
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http://dx.doi.org/10.1128/ecosalplus.ESP-0012-2015DOI Listing
July 2016

Accommodation of CO in the di-heme active site of cytochrome bd terminal oxidase from Escherichia coli.

J Inorg Biochem 2013 Jan 24;118:65-7. Epub 2012 Sep 24.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russian Federation.

Catalytic mechanisms of reduction of O(2) to 2H(2)O by respiratory terminal oxidases have been extensively investigated. Tri-heme (b(558), b(595), d) cytochrome bd oxidases presumably utilize a dihemic site composed of high-spin hemes d and b(595). We performed a CO photolysis/recombination study of the purified fully reduced cytochrome bd from Escherichia coli. Spectrum of CO photolysis suggests photodissociation of the ligand from heme d and from part of heme b(595). This is the first clear evidence of interaction of heme b(595) with CO at room temperature. The amount of the heme d-CO species is higher after recombination than before photolysis. In the enzyme population with heme b(595) bound to CO, heme d remains unliganded, hence the dihemic O(2)-reducing pocket in cytochrome bd can bind one rather than two diatomic molecules. Occupancy of the site by one ligand molecule probably blocks access of a second molecule. Thus cytochrome bd exhibits strong negative cooperativity in ligand binding. Immediately after photolysis/recombination CO occupies 100% of the heme d sites, whereas after equilibration, the ligand gets located at heme d in 90-95% and at heme b(595) in 5-10% of the cytochrome. The equilibration process is possibly associated with an exchange of heme d endogenous ligand.
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http://dx.doi.org/10.1016/j.jinorgbio.2012.09.016DOI Listing
January 2013

Urocanate reductase: identification of a novel anaerobic respiratory pathway in Shewanella oneidensis MR-1.

Mol Microbiol 2012 Dec 1;86(6):1452-63. Epub 2012 Nov 1.

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia.

Interpretation of the constantly expanding body of genomic information requires that the function of each gene be established. Here we report the genomic analysis and structural modelling of a previously uncharacterized redox-metabolism protein UrdA (SO_4620) of Shewanella oneidensis MR-1, which led to a discovery of the novel enzymatic activity, urocanate reductase. Further cloning and expression of urdA, as well as purification and biochemical study of the gene's product UrdA and redox titration of its prosthetic groups confirmed that the latter is indeed a flavin-containing enzyme catalysing the unidirectional reaction of two-electron reduction of urocanic acid to deamino-histidine, an activity not reported earlier. UrdA exhibits both high substrate affinity and high turnover rate (K(m)  << 10 μM, k(cat)  = 360 s(-1) ) and strong specificity in favour of urocanic acid. UrdA homologues are present in various bacterial genera, such as Shewanella, Fusobacterium and Clostridium, the latter including the human pathogen Clostridium tetani. The UrdA activity in S. oneidensis is induced by its substrate under anaerobic conditions and it enables anaerobic growth with urocanic acid as a sole terminal electron acceptor. The latter capability can provide the cells of UrdA-containing bacteria with a niche where no other bacteria can compete and survive.
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http://dx.doi.org/10.1111/mmi.12067DOI Listing
December 2012

The fifth electron in the fully reduced caa(3) from Thermus thermophilus is competent in proton pumping.

Biochim Biophys Acta 2013 Jan 28;1827(1):1-9. Epub 2012 Sep 28.

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation.

The time-resolved kinetics of membrane potential generation coupled to oxidation of the fully reduced (five-electron) caa(3) cytochrome oxidase from Thermus thermophilus by oxygen was studied in a single-turnover regime. In order to calibrate the number of charges that move across the vesicle membrane in the different reaction steps, the reverse electron transfer from heme a(3) to heme a and further to the cytochrome c/Cu(A) has been resolved upon photodissociation of CO from the mixed valence enzyme in the absence of oxygen. The reverse electron transfer from heme a(3) to heme a and further to the cytochrome c/Cu(A) pair is resolved as a single transition with τ~40 μs. In the reaction of the fully reduced cytochrome caa(3) with oxygen, the first electrogenic phase (τ~30 μs) is linked to OO bond cleavage and generation of the P(R) state. The next electrogenic component (τ~50 μs) is associated with the P(R)→F transition and together with the previous reaction step it is coupled to translocation of about two charges across the membrane. The three subsequent electrogenic phases, with time constants of ~0.25 ms, ~1.4 ms and ~4 ms, are linked to the conversion of the binuclear center through the F→O(H)→E(H) transitions, and result in additional transfer of four charges through the membrane dielectric. This indicates that the delivery of the fifth electron from heme c to the binuclear center is coupled to pumping of an additional proton across the membrane.
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http://dx.doi.org/10.1016/j.bbabio.2012.09.013DOI Listing
January 2013

Sodium-dependent movement of covalently bound FMN residue(s) in Na(+)-translocating NADH:quinone oxidoreductase.

Biochemistry 2012 Jul 25;51(27):5414-21. Epub 2012 Jun 25.

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is a component of respiratory electron-transport chain of various bacteria generating redox-driven transmembrane electrochemical Na(+) potential. We found that the change in Na(+) concentration in the reaction medium has no effect on the thermodynamic properties of prosthetic groups of Na(+)-NQR from Vibrio harveyi, as was revealed by the anaerobic equilibrium redox titration of the enzyme's EPR spectra. On the other hand, the change in Na(+) concentration strongly alters the EPR spectral properties of the radical pair formed by the two anionic semiquinones of FMN residues bound to the NqrB and NqrC subunits (FMN(NqrB) and FMN(NqrC)). Using data obtained by pulse X- and Q-band EPR as well as by pulse ENDOR and ELDOR spectroscopy, the interspin distance between FMN(NqrB) and FMN(NqrC) was found to be 15.3 Å in the absence and 20.4 Å in the presence of Na(+), respectively. Thus, the distance between the covalently bound FMN residues can vary by about 5 Å upon changes in Na(+) concentration. Using these results, we propose a scheme of the sodium potential generation by Na(+)-NQR based on the redox- and sodium-dependent conformational changes in the enzyme.
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http://dx.doi.org/10.1021/bi300322nDOI Listing
July 2012

Electrogenic events upon photolysis of CO from fully reduced cytochrome c oxidase.

Biochim Biophys Acta 2012 Feb 18;1817(2):269-75. Epub 2011 Nov 18.

Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

CO photolysis from fully reduced Paracoccus denitrificans aa(3)-type cytochrome c oxidase in the absence of O(2) was studied by time-resolved potential electrometry. Surprisingly, photo dissociation of the uncharged carbon monoxide results in generation of a small-amplitude electric potential with the same sign as the physiological charge separation during activity. The number of electrogenic events after CO photolysis depends on the state of the enzyme. CO photolysis following immediately after activation by an enzymatic turnover, showed a two-component potential development. A fast (~1.5μs) phase was followed by slower potential generation with a time constant varying from 8μs at pH 7 to 250μs at pH 10. The amplitude of the fast phase was independent of the time of incubation after enzyme activation, whereas the slower phase vanished with a time constant of ~25min. CO photolysis from enzyme that had not undergone a prior single turnover showed the fast phase, but the amplitude of the slow phase was reduced to 10-30%. The amplitude of the fast phase corresponds to charge movement of 0.83Å perpendicular to the membrane dielectric, and is independent of the time after enzyme activation. Thus it can be used as an internal ruler for normalization of the electrogenic responses of CcO. The slow phase was absent in the K354M mutant with a blocked proton-conducting K channel. We propose that CO photolysis increases the pK of the K354 residue, which results in its partial protonation, and generation of electric potential.
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http://dx.doi.org/10.1016/j.bbabio.2011.11.005DOI Listing
February 2012

Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis.

Mol Microbiol 2011 Dec 7;82(5):1086-95. Epub 2011 Nov 7.

Helsinki Bioenergetics Group, Institute of Biotechnology, FIN-00014, University of Helsinki, Helsinki, Finland.

The C-terminus of the NuoL subunit of Complex I includes a long amphipathic α-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07883.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274701PMC
December 2011

Aerobic respiratory chain of Escherichia coli is not allowed to work in fully uncoupled mode.

Proc Natl Acad Sci U S A 2011 Oct 10;108(42):17320-4. Epub 2011 Oct 10.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.

Escherichia coli is known to couple aerobic respiratory catabolism to ATP synthesis by virtue of the primary generators of the proton motive force-NADH dehydrogenase I, cytochrome bo(3), and cytochrome bd-I. An E. coli mutant deficient in NADH dehydrogenase I, bo(3) and bd-I can, nevertheless, grow aerobically on nonfermentable substrates, although its sole terminal oxidase cytochrome bd-II has been reported to be nonelectrogenic. In the current work, the ability of cytochrome bd-II to generate a proton motive force is reexamined. Absorption and fluorescence spectroscopy and oxygen pulse methods show that in the steady-state, cytochrome bd-II does generate a proton motive force with a H(+)/e(-) ratio of 0.94 ± 0.18. This proton motive force is sufficient to drive ATP synthesis and transport of nutrients. Microsecond time-resolved, single-turnover electrometry shows that the molecular mechanism of generating the proton motive force is identical to that in cytochrome bd-I. The ability to induce cytochrome bd-II biosynthesis allows E. coli to remain energetically competent under a variety of environmental conditions.
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http://dx.doi.org/10.1073/pnas.1108217108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198357PMC
October 2011

The cytochrome bd respiratory oxygen reductases.

Biochim Biophys Acta 2011 Nov 1;1807(11):1398-413. Epub 2011 Jul 1.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russian Federation.

Cytochrome bd is a respiratory quinol: O₂ oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O₂ and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O₂-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O₂, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated.
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http://dx.doi.org/10.1016/j.bbabio.2011.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3171616PMC
November 2011

The D-channel of cytochrome oxidase: an alternative view.

Biochim Biophys Acta 2011 Oct 18;1807(10):1273-8. Epub 2011 May 18.

University of Helsinki, Helsinki, Finland.

The D-pathway in A-type cytochrome c oxidases conducts protons from a conserved aspartate on the negatively charged N-side of the membrane to a conserved glutamic acid at about the middle of the membrane dielectric. Extensive work in the past has indicated that all four protons pumped across the membrane on reduction of O(2) to water are transferred via the D-pathway, and that it is also responsible for transfer of two out of the four "chemical protons" from the N-side to the binuclear oxygen reduction site to form product water. The function of the D-pathway has been discussed in terms of an apparent pK(a) of the glutamic acid. After reacting fully reduced enzyme with O(2), the rate of formation of the F state of the binuclear heme-copper active site was found to be independent of pH up to pH~9, but to drop off at higher pH with an apparent pK(a) of 9.4, which was attributed to the glutamic acid. Here, we present an alternative view, according to which the pH-dependence is controlled by proton transfer into the aspartate residue at the N-side orifice of the D-pathway. We summarise experimental evidence that favours a proton pump mechanism in which the proton to be pumped is transferred from the glutamic acid to a proton-loading site prior to proton transfer for completion of oxygen reduction chemistry. The mechanism is discussed by which the proton-pumping activity is decoupled from electron transfer by structural alterations of the D-pathway. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.
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http://dx.doi.org/10.1016/j.bbabio.2011.05.013DOI Listing
October 2011

Time-resolved single-turnover of caa(3) oxidase from Thermus thermophilus. Fifth electron of the fully reduced enzyme converts O(H) into E(H) state.

Biochim Biophys Acta 2011 Sep 14;1807(9):1162-9. Epub 2011 May 14.

Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Russian Federation.

The oxidative part of the catalytic cycle of the caa(3)-type cytochrome c oxidase from Thermus thermophilus was followed by time-resolved optical spectroscopy. Rate constants, chemical nature and the spectral properties of the catalytic cycle intermediates (Compounds A, P, F) reproduce generally the features typical for the aa(3)-type oxidases with some distinctive peculiarities caused by the presence of an additional 5-th redox-center-a heme center of the covalently bound cytochrome c. Compound A was formed with significantly smaller yield compared to aa(3) oxidases in general and to ba(3) oxidase from the same organism. Two electrons, equilibrated between three input redox-centers: heme a, Cu(A) and heme c are transferred in a single transition to the binuclear center during reduction of the compound F, converting the binuclear center through the highly reactive O(H) state into the final product of the reaction-E(H) (one-electron reduced) state of the catalytic site. In contrast to previous works on the caa(3)-type enzymes, we concluded that the finally produced E(H) state of caa(3) oxidase is characterized by the localization of the fifth electron in the binuclear center, similar to the O(H)→E(H) transition of the aa(3)-type oxidases. So, the fully-reduced caa(3) oxidase is competent in rapid electron transfer from the input redox-centers into the catalytic heme-copper site.
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http://dx.doi.org/10.1016/j.bbabio.2011.05.006DOI Listing
September 2011

A combined quantum chemical and crystallographic study on the oxidized binuclear center of cytochrome c oxidase.

Biochim Biophys Acta 2011 Jul 4;1807(7):769-78. Epub 2011 Jan 4.

Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland.

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain. By reducing oxygen to water, it generates a proton gradient across the mitochondrial or bacterial membrane. Recently, two independent X-ray crystallographic studies ((Aoyama et al. Proc. Natl. Acad. Sci. USA 106 (2009) 2165-2169) and (Koepke et al. Biochim. Biophys. Acta 1787 (2009) 635-645)), suggested that a peroxide dianion might be bound to the active site of oxidized CcO. We have investigated this hypothesis by combining quantum chemical calculations with a re-refinement of the X-ray crystallographic data and optical spectroscopic measurements. Our data suggest that dianionic peroxide, superoxide, and dioxygen all form a similar superoxide species when inserted into a fully oxidized ferric/cupric binuclear site (BNC). We argue that stable peroxides are unlikely to be confined within the oxidized BNC since that would be expected to lead to bond splitting and formation of the catalytic P intermediate. Somewhat surprisingly, we find that binding of dioxygen to the oxidized binuclear site is weakly exergonic, and hence, the observed structure might have resulted from dioxygen itself or from superoxide generated from O(2) by the X-ray beam. We show that the presence of O(2) is consistent with the X-ray data. We also discuss how other structures, such as a mixture of the aqueous species (H(2)O+OH(-) and H(2)O) and chloride fit the experimental data.
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http://dx.doi.org/10.1016/j.bbabio.2010.12.016DOI Listing
July 2011

Proton-coupled electron transfer in cytochrome oxidase.

Chem Rev 2010 Dec 5;110(12):7062-81. Epub 2010 Nov 5.

Helsinki Bioenergetics Group, Structural Biology and Biophysics Program, Institute of Biotechnology, University of Helsinki, P.O. Box 65, FI-00014 Helsinki, Finland.

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http://dx.doi.org/10.1021/cr1002003DOI Listing
December 2010

Initiation of the proton pump of cytochrome c oxidase.

Proc Natl Acad Sci U S A 2010 Oct 11;107(43):18469-74. Epub 2010 Oct 11.

Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, PB 65 (Viikinkaari 1), FI-00014, Helsinki, Finland.

Cytochrome c oxidase is the terminal enzyme of the respiratory chain that is responsible for biological energy conversion in mitochondria and aerobic bacteria. The membrane-bound enzyme converts free energy from oxygen reduction to an electrochemical proton gradient by functioning as a redox-coupled proton pump. Although the 3D structure and functional studies have revealed proton conducting pathways in the enzyme interior, the location of proton donor and acceptor groups are not fully identified. We show here by time-resolved optical and FTIR spectroscopy combined with time-resolved electrometry that some mutant enzymes incapable of proton pumping nevertheless initiate catalysis by proton transfer to a proton-loading site. A conserved tyrosine in the so-called D-channel is identified as a potential proton donor that determines the efficiency of this reaction.
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http://dx.doi.org/10.1073/pnas.1010974107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2972982PMC
October 2010

Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump.

Biochim Biophys Acta 2010 Jun-Jul;1797(6-7):738-46. Epub 2010 Jan 4.

Helsinki Bioenergetics Group, Institute of Biotechnology, P.O. Box 65, (Viikinkaari 1), 00014, University of Helsinki, Helsinki, Finland.

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) is a component of the respiratory chain of various bacteria. This enzyme is an analogous but not homologous counterpart of mitochondrial Complex I. Na+-NQR drives the same chemistry and also uses released energy to translocate ions across the membrane, but it pumps Na+ instead of H+. Most likely the mechanism of sodium pumping is quite different from that of proton pumping (for example, it could not accommodate the Grotthuss mechanism of ion movement); this is why the enzyme structure, subunits and prosthetic groups are completely special. This review summarizes modern knowledge on the structural and catalytic properties of bacterial Na+-translocating NADH:quinone oxidoreductases. The sequence of electron transfer through the enzyme cofactors and thermodynamic properties of those cofactors is discussed. The resolution of the intermediates of the catalytic cycle and localization of sodium-dependent steps are combined in a possible molecular mechanism of sodium transfer by the enzyme.
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http://dx.doi.org/10.1016/j.bbabio.2009.12.020DOI Listing
January 2011

Oxygen as Acceptor.

EcoSal Plus 2009 Aug;3(2)

Like most bacteria, Escherichia coli has a flexible and branched respiratory chain that enables the prokaryote to live under a variety of environmental conditions, from highly aerobic to completely anaerobic. In general, the bacterial respiratory chain is composed of dehydrogenases, a quinone pool, and reductases. Substrate specific dehydrogenases transfer reducing equivalents from various donor substrates (NADH, succinate, glycerophoshate, formate, hydrogen, pyruvate, and lactate) to a quinone pool (menaquinone, ubiquinone, and demethylmenoquinone). Then electrons from reduced quinones (quinols) are transferred by terminal reductases to different electron acceptors. Under aerobic growth conditions, the terminal electron acceptor is molecular oxygen. A transfer of electrons from quinol to O2 is served by two major oxidoreductases (oxidases), cytochrome bo3 and cytochrome bd. Terminal oxidases of aerobic respiratory chains of bacteria, which use O2 as the final electron acceptor, can oxidize one of two alternative electron donors, either cytochrome c or quinol. This review compares the effects of different inhibitors on the respiratory activities of cytochrome bo3 and cytochrome bd in E. coli. It also presents a discussion on the genetics and the prosthetic groups of cytochrome bo3 and cytochrome bd. The E. coli membrane contains three types of quinones which all have an octaprenyl side chain (C40). It has been proposed that the bo3 oxidase can have two ubiquinone-binding sites with different affinities. The spectral properties of cytochrome bd-II closely resemble those of cydAB-encoded cytochrome bd.
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http://dx.doi.org/10.1128/ecosalplus.3.2.7DOI Listing
August 2009

Redox properties of the prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase. 2. Study of the enzyme by optical spectroscopy.

Biochemistry 2009 Jul;48(27):6299-304

Department of Molecular Energetics of Microorganisms, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia.

Redox titration of the electronic spectra of the prosthetic groups of the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio harveyi at different pH values showed five redox transitions corresponding to the four flavin cofactors of the enzyme and one additional transition reflecting oxidoreduction of the [2Fe-2S] cluster. The pH dependence of the measured midpoint redox potentials showed that the two-electron reduction of the FAD located in the NqrF subunit was coupled with the uptake of only one H(+). The one-electron reduction of neutral semiquinone of riboflavin and the formation of anion flavosemiquinone from the oxidized FMN bound to the NqrB subunit were not coupled to any proton uptake. The two sequential one-electron reductions of the FMN residue bound to the NqrC subunit showed pH-independent formation of anion radical in the first step and the formation of fully reduced flavin coupled to the uptake of one H(+) in the second step. All four flavins stayed in the anionic form in the fully reduced enzyme. None of the six redox transitions in Na(+)-NQR showed dependence of its midpoint redox potential on the concentration of sodium ions. A model of the sequence of electron transfer steps in the enzyme is suggested.
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http://dx.doi.org/10.1021/bi900525vDOI Listing
July 2009

Redox properties of the prosthetic groups of Na(+)-translocating nadh:quinone oxidoreductase. 1. Electron paramagnetic resonance study of the enzyme.

Biochemistry 2009 Jul;48(27):6291-8

Department of Molecular Energetics of Microorganisms, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia.

Redox properties of all EPR-detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio harveyi were studied at pH 7.5 using cryo-EPR spectroelectrochemistry. Titration shows five redox transitions. One with E(m) = -275 mV belongs to the reduction of the [2Fe-2S] cluster, and the four others reflect redox transitions of flavin cofactors. Two transitions (E(m)(1) = -190 mV and E(m)(2) = -275 mV) originate from the formation of FMN anion radical, covalently bound to the NqrC subunit, and its subsequent reduction. The remaining two transitions arise from the two other flavin cofactors. A high potential (E(m) = -10 mV) transition corresponds to the reduction of riboflavin neutral radical, which is stable at rather high redox potentials. An E(m) = -130 mV transition reflects the formation of FMN anion radical from a flavin covalently bound to the NqrB subunit, which stays as a radical down to very low potentials. Taking into account the EPR-silent, two-electron transition of noncovalently bound FAD located in the NqrF subunit, there are four flavins in Na(+)-NQR all together. Defined by dipole-dipole magnetic interaction measurements, the interspin distance between the [2Fe-2S](+) cluster and the NqrB subunit-bound FMN anion radical is found to be 22.5 +/- 1.5 A, which means that for the functional electron transfer between these two centers another cofactor, most likely FMN bound to the NqrC subunit, should be located.
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http://dx.doi.org/10.1021/bi900524mDOI Listing
July 2009

Elevated proton leak of the intermediate OH in cytochrome c oxidase.

Biophys J 2009 Jun;96(11):4733-42

Institute of Biotechnology, 00014 University of Helsinki, Helsinki, Finland.

The kinetics of the formation and relaxation of transmembrane electric potential (Deltapsi) during the complete single turnover of CcO was studied in the bovine heart mitochondrial and the aa(3)-type Paracoccus denitrificans enzymes incorporated into proteoliposome membrane. The real-time Deltapsi kinetics was followed by the direct electrometry technique. The prompt oxidation of CcO and formation of the activated, oxidized (O(H)) state of the enzyme leaves the enzyme trapped in the open state that provides an internal leak for protons and thus facilitates dissipation of Deltapsi (tau(app) < or = 0.5-0.8 s). By contrast, when the enzyme in the O(H) state is rapidly re-reduced by sequential electron delivery, Deltapsi dissipates much slower (tau(app) > 3 s). In P. denitrificans CcO proteoliposomes the accelerated Deltapsi dissipation is slowed down by a mutational block of the proton conductance through the D-, but not K-channel. We concluded that in contrast to the other intermediates the O(H) state of CcO is vulnerable to the elevated internal proton leak that proceeds via the D-channel.
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http://dx.doi.org/10.1016/j.bpj.2009.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711495PMC
June 2009

Heme/heme redox interaction and resolution of individual optical absorption spectra of the hemes in cytochrome bd from Escherichia coli.

Biochim Biophys Acta 2009 Oct 18;1787(10):1246-53. Epub 2009 May 18.

Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

Cytochrome bd is a terminal component of the respiratory chain of Escherichia coli catalyzing reduction of molecular oxygen to water. It contains three hemes, b(558), b(595), and d. The detailed spectroelectrochemical redox titration and numerical modeling of the data reveal significant redox interaction between the low-spin heme b(558) and high-spin heme b(595), whereas the interaction between heme d and either hemes b appears to be rather weak. However, the presence of heme d itself decreases much larger interaction between the two hemes b. Fitting the titration data with a model where redox interaction between the hemes is explicitly included makes it possible to extract individual absorption spectra of all hemes. The alpha- and beta-band reduced-minus-oxidized difference spectra agree with the data published earlier ([22] J.G. Koland, M.J. Miller, R.B. Gennis, Potentiometric analysis of the purified cytochrome d terminal oxidase complex from Escherichia coli, Biochemistry 23 (1984) 1051-1056., and [23] R.M. Lorence, J.G. Koland, R.B. Gennis, Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of "cytochrome a(1)" as cytochrome b(595), Biochemistry 25 (1986) 2314-2321.). The Soret band spectra show lambda(max)=429.5 nm, lambda(min) approximately 413 nm (heme b(558)), lambda(max)=439 nm, lambda(min) approximately 400+/-1 nm (heme b(595)), and lambda(max)=430 nm, lambda(min)=405 nm (heme d). The spectral contribution of heme d to the complex Soret band is much smaller than those of either hemes b; the Soret/alpha (DeltaA(430):DeltaA(629)) ratio for heme d is 1.6.
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http://dx.doi.org/10.1016/j.bbabio.2009.05.003DOI Listing
October 2009

Mechanism and energetics by which glutamic acid 242 prevents leaks in cytochrome c oxidase.

Biochim Biophys Acta 2009 Oct 3;1787(10):1205-14. Epub 2009 May 3.

Helsinki Bioenergetics Group, Structural Biology and Biophysics Program, Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

Cytochrome c oxidase (CcO) is the terminal enzyme of aerobic respiration. The energy released from the reduction of molecular oxygen to water is used to pump protons across the mitochondrial or bacterial membrane. The pump function introduces a mechanistic requirement of a valve that prevents protons from flowing backwards during the process. It was recently found that Glu-242, a key amino acid in transferring protons to be pumped across the membrane and to the site of oxygen reduction, fulfils the function of such a valve by preventing simultaneous contact to the pump site and to the proton-conducting D-channel (Kaila V.R.I. et al. Proc. Natl. Acad. Sci. USA 105, 2008). Here we have incorporated the valve model into the framework of the reaction mechanism. The function of the Glu valve is studied by exploring how the redox state of the surrounding metal centers, dielectric effects, and membrane potential, affects the energetics and leaks of this valve. Parallels are drawn between the dynamics of Glu-242 and the long-standing obscure difference between the metastable O(H) and stable O states of the binuclear center. Our model provides a suggestion for why reduction of the former state is coupled to proton translocation while reduction of the latter is not.
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http://dx.doi.org/10.1016/j.bbabio.2009.04.008DOI Listing
October 2009

Time-resolved OH-->EH transition of the aberrant ba3 oxidase from Thermus thermophilus.

Biochim Biophys Acta 2009 Mar;1787(3):201-5

Department of Molecular Energetics of Microorganisms, Lomonosov Moscow State University, Moscow, Russian Federation.

The kinetics of single-electron injection into the oxidized nonrelaxed state (OH --> EH transition) of the aberrant ba3 cytochrome oxidase from Thermus thermophilus, noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component (tau approximately 17 mus) reflects oxidation of CuA and reduction of the heme groups (low-spin heme b and high-spin heme a3 in a ratio close to 50:50). The subsequent component (tau 420 mus) includes reoxidation of both hemes by CuB. This is in significant contrast to the OH--> EH transition of the aa3-type cytochrome oxidase from Paracoccus denitrificans, where the fastest phase is exclusively due to transient reduction of the low-spin heme a, without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba3 oxidase was similar to that in aa3 oxidase and only included rapid electron transfer from CuA to the low-spin heme b. This indicates a functional difference between the relaxed O and the pulsed OH forms also in the ba3 oxidase from T. thermophilus.
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http://dx.doi.org/10.1016/j.bbabio.2008.12.020DOI Listing
March 2009

Chapter 4 Electron transfer in respiratory complexes resolved by an ultra-fast freeze-quench approach.

Methods Enzymol 2009 ;456:75-93

Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

The investigation of the molecular mechanism of the respiratory chain complexes requires determination of the time-dependent evolution of the catalytic cycle intermediates. The ultra-fast freeze-quench approach makes possible trapping such intermediates with consequent analysis of their chemical structure by means of different physical spectroscopic methods (e.g., EPR, optic, and Mössbauer spectroscopies). This chapter presents the description of a setup that allows stopping the enzymatic reaction in the time range from 100 microsec to tens of msec. The construction and production technology of the mixer head, ultra-fast freezing device, and accessories required for collecting a sample are described. Ways of solving a number of problems emerging on freezing of the reaction mixture and preparing the samples for EPR spectroscopy are proposed. The kinetics of electron transfer reaction in the first enzyme of the respiratory chain, Complex I (NADH: ubiquinone oxidoreductase), is presented as an illustration of the freeze-quench approach. Time-resolved EPR spectra indicating the redox state of FeS clusters of the wild-type and mutant (R274A in subunit NuoCD) Complex I from Escherichia coli are shown.
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http://dx.doi.org/10.1016/S0076-6879(08)04404-2DOI Listing
June 2009

Active site of cytochrome cbb3.

J Biol Chem 2009 Apr 28;284(17):11301-8. Epub 2009 Feb 28.

Helsinki Bioenergetics Group, Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P. O. Box 65 (Viikinkaari 1), 00014 Helsinki, Finland.

Cytochrome cbb(3) is the most distant member of the heme-copper oxidase family still retaining the following major feature typical of these enzymes: reduction of molecular oxygen to water coupled to proton translocation across the membrane. The thermodynamic properties of the six redox centers, five hemes and a copper ion, in cytochrome cbb(3) from Rhodobacter sphaeroides were studied using optical and EPR spectroscopy. The low spin heme b in the catalytic subunit was shown to have the highest midpoint redox potential (E(m)(,7) +418 mV), whereas the three hemes c in the two other subunits titrated with apparent midpoint redox potentials of +351, +320, and +234 mV. The active site high spin heme b(3) has a very low potential (E(m)(,7) -59 mV) as opposed to the copper center (Cu(B)), which has a high potential (E(m)(,7) +330 mV). The EPR spectrum of the ferric heme b(3) has rhombic symmetry. To explain the origins of the rhombicity, the Glu-383 residue located on the proximal side of heme b(3) was mutated to aspartate and to glutamine. The latter mutation caused a 10 nm blue shift in the optical reduced minus oxidized heme b(3) spectrum, and a dramatic change of the EPR signal toward more axial symmetry, whereas mutation to aspartate had far less severe consequences. These results strongly suggest that Glu-383 is involved in hydrogen bonding to the proximal His-405 ligand of heme b(3), a unique interaction among heme-copper oxidases.
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http://dx.doi.org/10.1074/jbc.M808839200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2670135PMC
April 2009

Primary steps of the Na+-translocating NADH:ubiquinone oxidoreductase catalytic cycle resolved by the ultrafast freeze-quench approach.

J Biol Chem 2009 Feb 30;284(9):5533-8. Epub 2008 Dec 30.

Department of Molecular Energetics of Microorganisms, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia.

The Na(+)-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR) is a component of respiratory chain of various bacteria, and it generates a redox-driven transmembrane electrochemical Na(+) potential. Primary steps of the catalytic cycle of Na(+)-NQR from Vibrio harveyi were followed by the ultrafast freeze-quench approach in combination with conventional stopped-flow technique. The obtained sequence of events includes NADH binding ( approximately 1.5 x 10(7) m(-1) s(-1)), hydride ion transfer from NADH to FAD ( approximately 3.5 x 10(3) s(-1)), and partial electron separation and formation of equivalent fractions of reduced 2Fe-2S cluster and neutral semiquinone of FAD ( approximately 0.97 x 10(3) s(-1)). In the last step, a quasi-equilibrium is approached between the two states of FAD: two-electron reduced (50%) and one-electron reduced (the other 50%) species. The latter, neutral semiquinone of FAD, shares the second electron with the 2Fe-2S center. The transient midpoint redox potentials for the cofactors obtained during the fast kinetics measurements are very different from ones achieved during equilibrium redox titration and show that the functional states of the enzyme realized during its turning over cannot be modeled by the equilibrium approach.
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http://dx.doi.org/10.1074/jbc.M808984200DOI Listing
February 2009

The role of the invariant glutamate 95 in the catalytic site of Complex I from Escherichia coli.

Biochim Biophys Acta 2009 Jan 13;1787(1):68-73. Epub 2008 Nov 13.

Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, Finland.

Replacement of glutamate 95 for glutamine in the NADH- and FMN-binding NuoF subunit of E. coli Complex I decreased NADH oxidation activity 2.5-4.8 times depending on the used electron acceptor. The apparent K(m) for NADH was 5.2 and 10.4 microM for the mutant and wild type, respectively. Analysis of the inhibitory effect of NAD(+) on activity showed that the E95Q mutation caused a 2.4-fold decrease of K(i)(NAD+) in comparison to the wild type enzyme. ADP-ribose, which differs from NAD(+) by the absence of the positively charged nicotinamide moiety, is also a competitive inhibitor of NADH binding. The mutation caused a 7.5-fold decrease of K(i)(ADP-ribose) relative to wild type enzyme. Based on these findings we propose that the negative charge of Glu95 accelerates turnover of Complex I by electrostatic interaction with the negatively charged phosphate groups of the substrate nucleotide during operation, which facilitates release of the product NAD(+). The E95Q mutation was also found to cause a positive shift of the midpoint redox potential of the FMN, from -350 mV to -310 mV, which suggests that the negative charge of Glu95 is also involved in decreasing the midpoint potential of the primary electron acceptor of Complex I.
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http://dx.doi.org/10.1016/j.bbabio.2008.11.002DOI Listing
January 2009

The protonation state of the cross-linked tyrosine during the catalytic cycle of cytochrome c oxidase.

J Biol Chem 2008 Dec 17;283(50):34907-12. Epub 2008 Oct 17.

Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, P. O. Box 65, Viikinkaari 1, FI-00014 Helsinki, Finland.

Cytochrome c oxidase is the terminal complex of the respiratory chain in mitochondria and some aerobic bacteria and is responsible for most of the O(2) consumption in biology. The key reaction in the catalysis of O(2) reduction is O-O bond scission that requires four electrons and a proton. In our recent work (Gorbikova, E. A., Belevich, I., Wikstrom, M., and Verkhovsky, M. I. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 10733-10737), it was shown that the cross-linked Tyr-280 (Paracoccus denitrificans numbering) provides the proton for O-O bond cleavage. The deprotonated Tyr-280 must be reprotonated later on in the catalytic cycle to serve as a proton donor for the next oxygen reduction event. To find the reaction step at which the cross-linked Tyr-280 becomes reprotonated, all further steps of the catalytic cycle after O-O bond cleavage were followed by infrared spectroscopy. We found that complete reprotonation of the tyrosine is linked to the formation of the one-electron reduced state coupled to reduction of the Cu(B) site.
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http://dx.doi.org/10.1074/jbc.M803511200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3259884PMC
December 2008

The proton donor for O-O bond scission by cytochrome c oxidase.

Proc Natl Acad Sci U S A 2008 Aug 29;105(31):10733-7. Epub 2008 Jul 29.

Helsinki Bioenergetics Group, Institute of Biotechnology, University of Helsinki, P.O. Box 65 Viikinkaari 1, FI-00014, Helsinki, Finland.

Cytochrome c oxidase is the main catalyst of oxygen consumption in mitochondria and many aerobic bacteria. The key step in oxygen reduction is scission of the O-O bond and formation of an intermediate P(R) of the binuclear active site composed of heme a(3) and Cu(B). The donor of the proton required for this reaction has been suggested to be a unique tyrosine residue (Tyr-280) covalently cross-linked to one of the histidine ligands of Cu(B). To test this idea we used the Glu-278-Gln mutant enzyme from Paracoccus denitrificans, in which the reaction with oxygen stops at the P(R) intermediate. Three different time-resolved techniques were used. Optical spectroscopy showed fast (approximately 60 micros) appearance of the P(R) species along with full oxidation of heme a, and FTIR spectroscopy revealed a band at 1,308 cm(-1), which is characteristic for the deprotonated form of the cross-linked Tyr-280. The development of electric potential during formation of the P(R) species suggests transfer of a proton over a distance of approximately 4 A perpendicular to the membrane plane, which is close to the distance between the oxygen atom of the hydroxyl group of Tyr-280 and the bound oxygen. These results strongly support the hypothesis that the cross-linked tyrosine is the proton donor for O-O bond cleavage by cytochrome c oxidase and strengthens the view that this tyrosine also provides the fourth electron in O(2) reduction in conditions where heme a is oxidized.
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http://dx.doi.org/10.1073/pnas.0802512105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504829PMC
August 2008

Glutamate 107 in subunit I of cytochrome bd from Escherichia coli is part of a transmembrane intraprotein pathway conducting protons from the cytoplasm to the heme b595/heme d active site.

Biochemistry 2008 Jul 3;47(30):7907-14. Epub 2008 Jul 3.

Department of Molecular Energetics of Microorganisms, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russian Federation.

Cytochrome bd is a terminal quinol:O 2 oxidoreductase of the respiratory chain of Escherichia coli. The enzyme generates protonmotive force without proton pumping and contains three hemes, b 558, b 595, and d. A highly conserved glutamic acid residue of transmembrane helix III in subunit I, E107, was suggested to be part of a transmembrane pathway delivering protons from the cytoplasm to the oxygen-reducing site. When E107 is replaced with leucine, the hemes are retained but the ubiquinol-1-oxidase activity is lost. We compared wild-type and E107L mutant enzymes during single turnover using absorption and electrometric techniques with a microsecond time resolution. Both wild-type and E107L mutant cytochromes bd in the fully reduced state bind O 2 rapidly, but the formation of the oxoferryl species in the mutant is dramatically retarded as compared to the wild type. Intraprotein electron redistribution induced by the photolysis of CO bound to ferrous heme d in the one-electron-reduced wild-type enzyme is coupled to the membrane potential generation, whereas the mutant cytochrome bd shows no such potential generation. The E107L mutation also causes decrease of midpoint redox potentials of hemes b 595 and d by 25-30 mV and heme b 558 by approximately 70 mV. There are two protonatable groups redox-linked to hemes b 595 and d in the active site, one of which has been recently identified as E445, whereas the second group remains unknown. Here we propose that E107 is either the second group or a key residue of a proposed proton delivery pathway leading from the cytoplasm toward this second group.
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http://dx.doi.org/10.1021/bi800435aDOI Listing
July 2008

Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I).

Biochim Biophys Acta 2008 Sep 9;1777(9):1166-72. Epub 2008 Jun 9.

Helsinki Bioenergetics Group, Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Finland.

Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.
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http://dx.doi.org/10.1016/j.bbabio.2008.06.001DOI Listing
September 2008
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