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

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.

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

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.

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

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.

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

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.

Initiation of the proton pump of cytochrome c oxidase.

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.

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

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.

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

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.

The cytochrome bd respiratory oxygen reductases.

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.

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.

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.

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

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.

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

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.

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase.

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.

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

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.

Glutamic acid 242 is a valve in the proton pump of cytochrome c oxidase.

Aerobic life is based on a molecular machinery that utilizes oxygen as a terminal electron sink. The membrane-bound cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water in mitochondria and many bacteria. The energy released in this reaction is conserved by pumping protons across the mitochondrial or bacterial membrane, creating an electrochemical proton gradient that drives production of ATP. A crucial question is how the protons pumped by CcO are prevented from flowing backwards during the process. Here, we show by molecular dynamics simulations that the conserved glutamic acid 242 near the active site of CcO undergoes a protonation state-dependent conformational change, which provides a valve in the pumping mechanism. The valve ensures that at any point in time, the proton pathway across the membrane is effectively discontinuous, thereby preventing thermodynamically favorable proton back-leakage while maintaining an overall high efficiency of proton translocation. Suppression of proton leakage is particularly important in mitochondria under physiological conditions, where production of ATP takes place in the presence of a high electrochemical proton gradient.

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

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.

Real-time electron transfer in respiratory complex I.

Electron transfer in complex I from Escherichia coli was investigated by an ultrafast freeze-quench approach. The reaction of complex I with NADH was stopped in the time domain from 90 mus to 8 ms and analyzed by electron paramagnetic resonance (EPR) spectroscopy at low temperatures. The data show that after binding of the first molecule of NADH, two electrons move via the FMN cofactor to the iron-sulfur (Fe/S) centers N1a and N2 with an apparent time constant of approximately 90 mus, implying that these two centers should have the highest redox potential in the enzyme. The rate of reduction of center N2 (the last center in the electron transfer sequence) is close to that predicted by electron transfer theory, which argues for the absence of coupled proton transfer or conformational changes during electron transfer from FMN to N2. After fast reduction of N1a and N2, we observe a slow, approximately 1-ms component of reduction of other Fe/S clusters. Because all elementary electron transfer rates between clusters are several orders of magnitude higher than this observed rate, we conclude that the millisecond component is limited by a single process corresponding to dissociation of the oxidized NAD(+) molecule from its binding site, where it prevents entry of the next NADH molecule. Despite the presence of approximately one ubiquinone per enzyme molecule, no transient semiquinone formation was observed, which has mechanistic implications, suggesting a high thermodynamic barrier for ubiquinone reduction to the semiquinone radical. Possible consequences of these findings for the proton translocation mechanism are discussed.

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

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.

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

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.

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

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.

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

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.