Publications by authors named "Dmitry A Bloch"

24 Publications

  • Page 1 of 1

Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase.

Proc Natl Acad Sci U S A 2015 Jun 8;112(25):7695-700. Epub 2015 Jun 8.

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

Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which are widespread among pathogenic bacteria, are the least understood. In particular, the proton-pumping machinery of these Coxs has not yet been elucidated despite the availability of X-ray structure information. Here, we report the discovery of the first (to our knowledge) sodium-pumping Cox (Scox), a cbb3 cytochrome from the extremely alkaliphilic bacterium Thioalkalivibrio versutus. This finding offers clues to the previously unknown structure of the ion-pumping channel in the C-type Coxs and provides insight into the functional properties of this enzyme.
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http://dx.doi.org/10.1073/pnas.1417071112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485098PMC
June 2015

Hidden photoinduced reactivity of the blue fluorescent protein mKalama1.

Phys Chem Chem Phys 2015 May;17(19):12472-85

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.

Understanding the photoinduced dynamics of fluorescent proteins is essential for their applications in bioimaging. Despite numerous studies on the ultrafast dynamics, the delayed response of these proteins, which often results in population of kinetically trapped dark states of various origins, is largely unexplored. Here, by using transient absorption spectroscopy spanning the time scale from picoseconds to seconds, we reveal a hidden reactivity of the bright blue-light emitting protein mKalama1 previously thought to be inert. This protein shows no excited-state proton transfer during its nanosecond excited-state lifetime; however, its tyrosine-based chromophore undergoes deprotonation coupled to non-radiative electronic relaxation. Such deprotonation causes distinct optical absorption changes in the broad UV-to-NIR spectral range (ca. 300-800 nm); the disappearance of the transient absorption signal has a complex nature and spans the whole microsecond-to-second time scale. The mechanisms underlying the relaxation kinetics are disclosed based on the X-ray structural analysis of mKalama1 and the high-level electronic structure calculations of proposed intermediates in the photocycle. We conclude that the non-radiative excited-state decay includes two major branches: internal conversion coupled to intraprotein proton transfer, where a conserved residue E222 serves as the proton acceptor; and ionization induced by two consecutive resonant absorption events, followed by deprotonation of the chromophore radical cation to bulk solvent through a novel water-mediated proton-wire pathway. Our findings open up new perspectives on the dynamics of fluorescent proteins as tracked by its optical transient absorption in the time domain extending up to seconds.
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http://dx.doi.org/10.1039/c5cp00887eDOI Listing
May 2015

Chromophore photoreduction in red fluorescent proteins is responsible for bleaching and phototoxicity.

J Phys Chem B 2014 May 21;118(17):4527-34. Epub 2014 Apr 21.

School of Chemistry and Biochemistry, Georgia Institute of Technology , Atlanta, Georgia 30332-0400, United States.

Red fluorescent proteins (RFPs) are indispensable tools for deep-tissue imaging, fluorescence resonance energy transfer applications, and super-resolution microscopy. Using time-resolved optical spectroscopy this study investigated photoinduced dynamics of three RFPs, KillerRed, mRFP, and DsRed. In all three RFPs, a new transient absorption intermediate was observed, which decays on a microsecond-millisecond time scale. This intermediate is characterized by red-shifted absorption at 1.68-1.72 eV (λmax = 720-740 nm). On the basis of electronic structure calculations, experimental evidence, and published literature, the chemical nature of the intermediate is assigned to an unusual open-shell dianionic chromophore (dianion-radical) formed via photoreduction. A doubly charged state that is not stable in the isolated (gas phase) chromophore is stabilized by the electrostatic field of the protein. Mechanistic implications for photobleaching, blinking, and phototoxicity are discussed.
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http://dx.doi.org/10.1021/jp500919aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010289PMC
May 2014

A single amino acid residue controls ROS production in the respiratory Complex I from Escherichia coli.

Mol Microbiol 2013 Dec 29;90(6):1190-200. Epub 2013 Oct 29.

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

Reactive oxygen species (ROS) production by respiratory Complex I from Escherichia coli was studied in bacterial membrane fragments and in the isolated and purified enzyme, either solubilized or incorporated in proteoliposomes. We found that the replacement of a single amino acid residue in close proximity to the nicotinamide adenine dinucleotide (NADH)-binding catalytic site (E95 in the NuoF subunit) dramatically increases the reactivity of Complex I towards dioxygen (O2 ). In the E95Q variant short-chain ubiquinones exhibit strong artificial one-electron reduction at the catalytic site, also leading to a stronger increase in ROS production. Two mechanisms can contribute to the observed kinetic effects: (a) a change in the reactivity of flavin mononucleotide (FMN) towards dioxygen at the catalytic site, and (b) a change in the population of the ROS-generating state. We propose the existence of two (closed and open) states of the NAD(+) -bound enzyme as one feature of the substrate-binding site of Complex I. The analysis of the kinetic model of ROS production allowed us to propose that the population of Complex I with reduced FMN is always low in the wild-type enzyme even at low ambient redox potentials, minimizing the rate of reaction with O2 in contrast to E95Q variant.
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http://dx.doi.org/10.1111/mmi.12424DOI Listing
December 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

Energy-converting respiratory Complex I: on the way to the molecular mechanism of the proton pump.

Int J Biochem Cell Biol 2013 Feb 13;45(2):491-511. Epub 2012 Sep 13.

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

In respiring organisms the major energy transduction flux employs the transmembrane electrochemical proton gradient as a physical link between exergonic redox reactions and endergonic ADP phosphorylation. Establishing the gradient involves electrogenic, transmembrane H(+) translocation by the membrane-embedded respiratory complexes. Among others, Complex I (NADH:ubiquinone oxidoreductase) is the most structurally complex and functionally enigmatic respiratory enzyme; its molecular mechanism is as yet unknown. Here we highlight recent progress and discuss the catalytic events during Complex I turnover in relation to their role in energy conversion and to the enzyme structure.
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http://dx.doi.org/10.1016/j.biocel.2012.08.024DOI Listing
February 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

Tightly-bound ubiquinone in the Escherichia coli respiratory complex I.

Biochim Biophys Acta 2012 Sep 1;1817(9):1550-6. Epub 2012 May 1.

University of Helsinki, Finland.

NADH:ubiquinone oxidoreductase (Complex I), the electron input enzyme in the respiratory chain of mitochondria and many bacteria, couples electron transport to proton translocation across the membrane. Complex I is a primary proton pump; although its proton translocation mechanism is yet to be known, it is considered radically different from any other mechanism known for redox-driven proton pumps: no redox centers have been found in its membrane domain where the proton translocation takes place. Here we studied the properties and the catalytic role of the enzyme-bound ubiquinone in the solubilized, purified Complex I from Escherichia coli. The ubiquinone content in the enzyme preparations was 1.3±0.1 per bound FMN residue. Rapid mixing of Complex I with NADH, traced optically, demonstrated that both reduction and re-oxidation kinetics of ubiquinone coincide with the respective kinetics of the majority of Fe-S clusters, indicating kinetic competence of the detected ubiquinone. Optical spectroelectrochemical redox titration of Complex I followed at 270-280nm, where the redox changes of ubiquinone contribute, did not reveal any transition within the redox potential range typical for the membrane pool, or loosely bound ubiquinone (ca. +50-+100mV vs. NHE, pH 6.8). The transition is likely to take place at much lower potentials (E(m) ≤-200mV). Such perturbed redox properties of ubiquinone indicate that it is tightly bound to the enzyme's hydrophobic core. The possibility of two ubiquinone-binding sites in Complex I is discussed.
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http://dx.doi.org/10.1016/j.bbabio.2012.04.013DOI Listing
September 2012

Mechanistic stoichiometry of proton translocation by cytochrome cbb3.

Proc Natl Acad Sci U S A 2012 May 23;109(19):7286-91. Epub 2012 Apr 23.

Helsinki Bioenergetics Group, Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, 00014, Helsinki, Finland.

Cytochrome cbb(3) belongs to the superfamily of respiratory heme-copper oxidases that couple the reduction of molecular oxygen to proton translocation across the bacterial or mitochondrial membrane. The cbb(3)-type enzymes are found only in bacteria, and are both structurally and functionally the most distant from their mitochondrial counterparts. The mechanistic H(+)/e(-) stoichiometry of proton translocation in these cbb(3)-type cytochrome c oxidases has remained controversial. A stoichiometric efficiency of only one-half that of the mitochondrial aa(3)-type enzyme was recently proposed to be related to adaptation of the organism to microaerobic environments. Here, proton translocation by the Rhodobacter sphaeroides enzyme was studied using purified cytochrome cbb(3) reconstituted into liposomes. An H(+)/e(-) stoichiometry of proton translocation close to unity was observed using the oxygen pulse method, but solely in conditions in which the vast majority of the enzyme was fully reduced in the anaerobic state before the O(2) pulse. These data were compared with results using whole cells or spheroplasts, and the discrepancies in the literature data were discussed. Our results suggest that a proton-pumping efficiency of 1 H(+)/e(-) may be achieved using the single-proton uptake pathway identified in the structure of cytochrome cbb(3). The mechanism of proton pumping thus differs from that of the aa(3)-type oxidases of mitochondria and bacteria.
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http://dx.doi.org/10.1073/pnas.1202151109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358842PMC
May 2012

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

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

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

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

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

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

Electrostatic interactions between FeS clusters in NADH:ubiquinone oxidoreductase (Complex I) from Escherichia coli.

Biochemistry 2008 Mar 13;47(10):3185-93. Epub 2008 Feb 13.

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

The redox properties of the cofactors of NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli were studied by following the changes in electron paramagnetic resonance (EPR) and optical spectra upon electrochemical redox titration of the purified protein. At neutral pH, the FMN cofactor had a midpoint redox potential ( E m) approximately -350 mV ( n = 2). Binuclear FeS clusters were well-characterized: N1a was titrated with a single ( n = 1) transition, and E m = -235 mV. In contrast, the titration of N1b can only be fitted with the sum of at least two one-electron Nernstian curves with E m values of -245 and -320 mV. The tetranuclear clusters can also be separated into two groups, either having a single, n = 1, or more complex redox titration curves. The titration curves of the EPR bands attributed to the tetranuclear clusters N2 ( g = 2.045 and g = 1.895) and N6b ( g = 2.089 and g = 1.877) can be presented by the sum of at least two components, each with E m (app) approximately -200/-300 mV and -235/-315 mV, respectively. The titration of the signals at g = 1.956-1.947 (N3 or N7, E m = -315 mV), g = 2.022, and g = 1.932 (Nx, -365 mV) and the low temperature signal at g = 1.929 (N4 or N5, -330 mV) followed Nernstian n = 1 curves. The observed redox titration curves are discussed in terms of intrinsic electrostatic interactions between FeS centers in complex I. A model showing shifts of E m due to the electrostatic interaction between the centers is presented.
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http://dx.doi.org/10.1021/bi702063tDOI Listing
March 2008

Cytochrome bd from Azotobacter vinelandii: evidence for high-affinity oxygen binding.

Biochemistry 2007 Oct 5;46(39):11177-84. Epub 2007 Sep 5.

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

Cytochrome bd from Azotobacter vinelandii is a respiratory quinol oxidase that is highly efficient in reducing intracellular oxygen concentration, thus enabling nitrogen fixation under ambient aerobic conditions. Equilibrium measurements of O2 binding to ferrous heme d in the one-electron-reduced form of the A. vinelandii enzyme give Kd(O2) = 0.5 microM, close to the value for the Escherichia coli cytochrome bd (ca. 0.3 microM); thus, both enzymes have similar, high affinity for oxygen. The reaction of the A. vinelandii cytochrome bd in the one-electron-reduced and fully reduced states with O2 is extremely fast approaching the diffusion-controlled limit in water. In the fully reduced state, the rate of O2 binding depends linearly on the oxygen concentration consistently with a simple, single-step process. In contrast, in the one-electron-reduced state the rate of oxygen binding is hyperbolic, implying a more complex binding pattern. Two possible explanations for the saturation kinetics are considered: (A) There is a spectroscopically silent prebinding of oxygen to an unidentified low-affinity saturatable site followed by the oxygen transfer to heme d. (B) Oxygen binding to heme d requires an "activated" state of the enzyme in which an oxygen channel connecting heme d to the bulk is open. This channel is permanently open in the fully reduced enzyme (hence no saturation behavior) but flickers between the open and closed states in the one-electron-reduced enzyme.
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http://dx.doi.org/10.1021/bi700862uDOI Listing
October 2007

Exploring the proton pump mechanism of cytochrome c oxidase in real time.

Proc Natl Acad Sci U S A 2007 Feb 9;104(8):2685-90. Epub 2007 Feb 9.

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

Cytochrome c oxidase catalyzes most of the biological oxygen consumption on Earth, a process responsible for energy supply in aerobic organisms. This remarkable membrane-bound enzyme also converts free energy from O(2) reduction to an electrochemical proton gradient by functioning as a redox-linked proton pump. Although the structures of several oxidases are known, the molecular mechanism of redox-linked proton translocation has remained elusive. Here, correlated internal electron and proton transfer reactions were tracked in real time by spectroscopic and electrometric techniques after laser-activated electron injection into the oxidized enzyme. The observed kinetics establish the long-sought reaction sequence of the proton pump mechanism and describe some of its thermodynamic properties. The 10-micros electron transfer to heme a raises the pK(a) of a "pump site," which is loaded by a proton from the inside of the membrane in 150 micros. This loading increases the redox potentials of both hemes a and a(3), which allows electron equilibration between them at the same rate. Then, in 0.8 ms, another proton is transferred from the inside to the heme a(3)/Cu(B) center, and the electron is transferred to Cu(B). Finally, in 2.6 ms, the preloaded proton is released from the pump site to the opposite side of the membrane.
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http://dx.doi.org/10.1073/pnas.0608794104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1796784PMC
February 2007

Elementary steps of proton translocation in the catalytic cycle of cytochrome oxidase.

Biochim Biophys Acta 2006 May-Jun;1757(5-6):401-7. Epub 2006 May 23.

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

Proton translocation in the catalytic cycle of cytochrome c oxidase (CcO) proceeds sequentially in a four-stroke manner. Every electron donated by cytochrome c drives the enzyme from one of four relatively stable intermediates to another, and each of these transitions is coupled to proton translocation across the membrane, and to uptake of another proton for production of water in the catalytic site. Using cytochrome c oxidase from Paracoccus denitrificans we have studied the kinetics of electron transfer and electric potential generation during several such transitions, two of which are reported here. The extent of electric potential generation during initial electron equilibration between CuA and heme a confirms that this reaction is not kinetically linked to vectorial proton transfer, whereas oxidation of heme a is kinetically coupled to the main proton translocation events during functioning of the proton pump. We find that the rates and amplitudes in multiphase heme a oxidation are different in the OH-->EH and PM-->F steps of the catalytic cycle, and that this is reflected in the kinetics of electric potential generation. We discuss this difference in terms of different driving forces and relate our results, and data from the literature, to proposed mechanisms of proton pumping in cytochrome c oxidase.
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http://dx.doi.org/10.1016/j.bbabio.2006.05.026DOI Listing
September 2006

Thermodynamic properties of the redox centers of Na(+)-translocating NADH:quinone oxidoreductase.

Biochemistry 2006 Mar;45(10):3421-8

Department of Molecular Energetics of Microorganisms, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.

Redox titration of all optically detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) at pH 7.5 showed that the functionally active enzyme possesses only three titratable flavin cofactors, one noncovalently bound FAD and two covalently bound FMN residues. All three flavins undergo different redox transitions during the function of the enzyme. The noncovalently bound FAD works as a "classical" two-electron carrier with a midpoint potential (E(m)) of -200 mV. Each of the FMN residues is capable of only one-electron reduction: one from neutral flavosemiquinone to fully reduced flavin (E(m) = 20 mV) and the other from oxidized flavin to flavosemiquinone anion (E(m) = -150 mV). The lacking second half of the redox transitions for the FMNs cannot be reached under our experimental conditions and is most likely not employed in the catalytic cycle. Besides the flavins, a [2Fe-2S] cluster was shown to function in the enzyme as a one-electron carrier with an E(m) of -270 mV. The midpoint potentials of all the redox transitions determined in the enzyme were found to be independent of Na(+) concentration. Even the components that exhibit very strong retardation in the rate of their reduction by NADH at low sodium concentrations experienced no change in the E(m) values when the concentration of the coupling ion was changed 1000 times. On the basis of these data, plausible mechanisms for the translocation of transmembrane sodium ions by Na(+)-NQR are discussed.
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http://dx.doi.org/10.1021/bi052422xDOI Listing
March 2006

An elementary reaction step of the proton pump is revealed by mutation of tryptophan-164 to phenylalanine in cytochrome c oxidase from Paracoccus denitrificans.

Biochemistry 2005 Dec;44(50):16502-12

Helsinki Bioenergetics Group, Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, PB 65 (Viikinkaari 1), FIN-00014, Helsinki, Finland.

Cytochrome c oxidase couples reduction of dioxygen to water to translocation of protons over the inner mitochondrial or bacterial membrane. A likely proton acceptor for pumped protons is the Delta-propionate of heme a(3), which may receive the proton via water molecules from a conserved glutamic acid (E278 in subunit I of the Paracoccus denitrificans enzyme) and which receives a hydrogen bond from a conserved tryptophan, W164. Here, W164 was mutated to phenylalanine (W164F) to further explore the role of the heme a(3) Delta-propionate in proton translocation. FTIR spectroscopy showed changes in vibrations possibly attributable to heme propionates, and the midpoint redox potential of heme a(3) decreased by approximately 50 mV. The reaction of the oxidized W164F enzyme with hydrogen peroxide yielded substantial amounts of the intermediate F' even at high pH, which suggests that the mutation rearranges the local electric field in the binuclear center that controls the peroxide reaction. The steady-state proton translocation stoichiometry of the W164F enzyme dropped to approximately 0.5 H(+)/e(-) in cells and reconstituted proteoliposomes. Time-resolved electrometric measurements showed that when the fully reduced W164F enzyme reacted with O(2), the membrane potential generated in the fast phase of this reaction was far too small to account either for full proton pumping or uptake of a substrate proton from the inside of the proteoliposomes. Time-resolved optical spectroscopy showed that this fast electrometric phase occurred with kinetics corresponding to the transition from state A to P(R), whereas the subsequent transition to the F state was strongly delayed. This is due to a delay of reprotonation of E278 via the D-pathway, which was confirmed by observation of a slowed rate of Cu(A) oxidation and which explains the small amplitude of the fast charge transfer phase. Surprisingly, the W164F mutation thus mimics a weak block of the D-pathway, which is interpreted as an effect on the side chain isomerization of E278. The fast charge translocation may be due to transfer of a proton from E278 to a "pump site" above the heme groups and is likely to occur also in wild-type enzyme, though not distinguished earlier due to the high-amplitude membrane potential formation during the P(R)--> F transition.
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http://dx.doi.org/10.1021/bi0511336DOI Listing
December 2005
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