Nikolai P. Belevich

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

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 O2 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-μs electron transfer to heme a raises the pKa of a “pump site,” which is loaded by a proton from the inside of the membrane in 150 μs. This loading increases the redox potentials of both hemes a and a3, 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 a3/CuB center, and the electron is transferred to CuB. Finally, in 2.6 ms, the preloaded proton is released from the pump site to the opposite side of the membrane.

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.

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.

PROTON LINKAGE OF CYTOCHROME A OXIDOREDUCTION IN CARBON MONOXIDE-TREATED CYTOCHROME C OXIDASE

Oxidoreduction of the low spin haem a of cytochrome c oxidase was recently reported to be coupled to release/uptake of nearly one proton from/to the enzyme at pH 7.5 in the presence of CO to block oxidoreduction of the binuclear haem a3/CuB centre (N. Capitanio et al., Biochim. Biophys. Acta, 1318 (1997) 255-265). This is difficult to reconcile with earlier findings from several laboratories that the pH-dependence of the Em of haem a is ca. 10 mV/pH unit over a wide pH range in such conditions, which implies redox coupling of only ca. 0.17 H+/e-. In order to resolve this discrepancy, we have performed careful measurements of proton release coupled to oxidation of haem a and CuA in CO-inhibited cytochrome aa3 from bovine heart mitochondria. We find that oxidation of these centres by ferricyanide leads to release of a total of 0.20 protons per enzyme molecule at pH 7.7, increasing to 0.43 protons at pH 6.6, far short of a full 1 H+/e-. Using vesicles reconstituted with cytochrome c oxidase, we also found that all this proton release occurs towards the outside of the vesicles. The observed dependence can be explained by a model in which oxidoreduction of haem a is coupled to uptake and release of ca. 0.17 H+/e-, while oxidoreduction of CuA is linked to a protonatable group which has a pKa of 6.2 when CuA is in the reduced state. In agreement with existing data, this model predicts that the Em of CuA will only be slightly pH dependent in the pH range of these measurements.

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

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 (∼1.5 × 107 m–1 s–1), hydride ion transfer from NADH to FAD (∼3.5 × 103 s–1), and partial electron separation and formation of equivalent fractions of reduced 2Fe-2S cluster and neutral semiquinone of FAD (∼0.97 × 103 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.

Time-resolved single-turnover of caa3 oxidase from Thermus thermophilus. Fifth electron of the fully reduced enzyme converts OH into EH 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.

Chapter 4 Electron Transfer in Respiratory Complexes Resolved by an Ultra-Fast Freeze-Quench Approach

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.

Identification of the coupling step in Na+-translocating NADH:quinone oxidoreductase from real-time kinetics of electron transfer

Bacterial Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) uses a unique set of prosthetic redox groups-two covalently bound FMN residues, a [2Fe-2S] cluster, FAD, riboflavin and a Cys4[Fe] center-to catalyze electron transfer from NADH to ubiquinone in a reaction coupled with Na(+) translocation across the membrane. Here we used an ultra-fast microfluidic stopped-flow instrument to determine rate constants and the difference spectra for the six consecutive reaction steps of Vibrio harveyi Na(+)-NQR reduction by NADH. The instrument, with a dead time of 0.25 ms and optical path length of 1 cm allowed collection of visible spectra in 50-μs intervals. By comparing the spectra of reaction steps with the spectra of known redox transitions of individual enzyme cofactors, we were able to identify the chemical nature of most intermediates and the sequence of electron transfer events. A previously unknown spectral transition was detected and assigned to the Cys4[Fe] center reduction. Electron transfer from the [2Fe-2S] cluster to the Cys4[Fe] center and all subsequent steps were markedly accelerated when Na(+) concentration was increased from 20 μM to 25 mM, suggesting coupling of the former step with tight Na(+) binding to or occlusion by the enzyme. An alternating access mechanism was proposed to explain electron transfer between subunits NqrF and NqrC. According to the proposed mechanism, the Cys4[Fe] center is alternatively exposed to either side of the membrane, allowing the [2Fe-2S] cluster of NqrF and the FMN residue of NqrC to alternatively approach the Cys4[Fe] center from different sides of the membrane.

Real-time optical studies of respiratory Complex I turnover

Reduction of Complex l (NADH:ubiquinone oxidoreductase l) from Escherichia coli by NADH was investigated optically by means of an ultrafast stopped-flow approach. A locally designed microfluidic stopped-flow apparatus with a low volume (0.21Jl) but a long optical path (10 mm) cuvette allowed measurements in the time range from 270 ).IS to seconds. The data acquisition system collected spectra in the visible range every 50 )JS. Analysis of the obtained time-resolved spectral changes upon the reaction of Complex I with NADH revealed three kinetic components with characteristic times of <270 ).IS, 0.45-0.9 ms and 3-6 ms, reflecting reduction of different FeS clusters and FMN. The rate of the major ( T = 0.45-0.9 ms) component was slower than predicted by electron transfer theory for the reduction of all FeS clusters in the intraprotein redox chain. This delay of the reaction was explained by retention of NAD+ in the catalytic site. The fast optical changes in the time range of 0.27- 1.5 ms were not altered significantly in the presence of 1 0-fold excess of NAD+ over NADH. The data obtained on the NuoF E95Q variant of Complex I shows that the single amino acid replacement in the catalytic site caused a strong decrease of NADH binding and/or the hydride transfer from bound NADH to FMN.

Resting state of respiratory Complex I from Escherichia coli

Respiratory Complex I from Escherichia coli may exist in two states, resting (R) and active (A). The conversion from the R‐ to A‐forms occurs spontaneously upon turnover. The fast resting‐to‐active (R/A) transition of membrane‐bound and purified Complex I was studied with the stopped‐flow technique by following NADH oxidation either by absorption decay at 340 nm or using the fluorescent pH indicator, trisodium 8‐hydroxypyrene‐1,3,6‐trisulfonate (pyranine). The R/A transition of Complex I from E. coli occurs upon its turnover in a time interval of ~ 1.5 s. Comparisons between the bacterial Complex I R/A transition and the active/deactive transition of mitochondrial Complex I are discussed.