Marina L. Verkhovskaya

Proton translocation by cytochrome c oxidase.

Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane,,. The enzymes catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c, and an oxidative phase, in which the reduced enzyme is oxidized by O2. Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase, but this has been challenged. Here, with the purified enzyme inlaid in liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase. pH measurements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase. We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O2. One half of it is utilized for proton-pumping during oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.

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 μs 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 ≈90 μs, 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, ≈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.

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

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.

Translocation of electrical charge during a single turnover of cytochrome-c oxidase

Abstract In cell respiration, cytochrome-c oxidase utilizes electrons from catabolism to reduce O2 to water. Energy is conserved as an electrochemical proton gradient across the mitochondrial membrane, which drives the synthesis of ATP. Electrical charge translocation during the reaction of the reduced enzyme with O2 takes place in two phases of identical amplitude. The first phase (τ1=0.2 ms) occurs after an initial lag, and appears to correspond to the transition from a peroxy to a ferryl intermediate in the oxygen chemistry. The second phase (τ2=2.6 ms) matches the transition from the ferryl intermediate to the oxidised enzyme. These findings define the kinetic linkage between the chemistry and the major events of proton pumping by the enzyme.

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 bo3, and cytochrome bd-I. An E. coli mutant deficient in NADH dehydrogenase I, bo3 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.

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.

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

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.

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

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.

Heme-copper oxidases with modified D- and K-pathways are yet efficient proton pumps

The cytochrome aa 3‐type quinol oxidase from the archaeon Acidianus ambivalens and the ba3 ‐type cytochrome c oxidase from Thermus thermophilus are divergent members of the heme‐copper oxidase superfamily of enzymes. In particular they lack most of the key residues involved in the proposed proton transfer pathways. The pumping capability of the A. ambivalens enzyme was investigated and found to occur with the same efficiency as the canonical enzymes. This is the first demonstration of pumping of 1 H+/electron in a heme‐copper oxidase that lacks most residues of the K‐ and D‐channels. Also, the structure of the ba3 oxidase from T. thermophilus was simulated by mutating Phe274 to threonine and Glu278 to isoleucine in the D‐pathway of the Paracoccus denitrificans cytochrome c oxidase. This modification resulted in full efficiency of proton translocation albeit with a substantially lowered turnover. Together, these findings show that multiple structural solutions for efficient proton conduction arose during evolution of the respiratory oxidases, and that very few residues remain invariant among these enzymes to function in a common proton‐pumping mechanism.

The caa(3) terminal oxidase of Rhodothermus marinus lacking the key glutamate of the D-channel is a proton pump.

The thermohalophilic bacterium Rhodothermus marinus expresses a caa(3)-type dioxygen reductase as one of its terminal oxidases. The subunit I amino acid sequence shows the presence of all the essential residues of the D- and K-proton channels, defined in most heme-copper oxidases, with the exception of the key glutamate residue located in the middle of the membrane dielectric (E278 in Paracoccus denitrificans). On the basis of homology modeling studies, a tyrosine residue (Y256, R. marinus numbering) has been proposed to act as a functional substitute [Pereira, M. M., Santana, M., Soares, C. M., Mendes, J., Carita, J. N., Fernandes, A. S., Saraste, M., Carrondo, M. A., and Teixeira, M. (1999) Biochim. Biophys. Acta 1413, 1-13]. Here, R. marinus caa(3) oxidase was reconstituted in liposomes and shown to operate as a proton pump, translocating protons from the cytoplasmic side of the bacterial inner membrane to the periplasmatic space with a stoichiometry of 1H(+)/e(-), as in the case in heme-copper oxidases that contain the glutamate residue. Possible mechanisms of proton transfer in the D-channel with the participation of the tyrosine residue are discussed. The observation that the tyrosine residue is conserved in several other members of the heme-copper oxidase superfamily suggests a common alternative mode of action for the D-channel.