Activation of Proton Translocation by Respiratory Complex I.

Activation of proton pumping by reconstituted and native membrane-bound Complex I was studied using optical electric potential- and pH-sensitive probes. We find that reconstituted Complex I has a delay in proton translocation, which is significantly longer than the delay in quinone reductase activity, indicating an initially decoupled state of Complex I. Studies of the amount of NADH required for the activation of pumping indicate the prerequisite of multiple turnovers. Proton pumping by Complex I was also activated by NADPH, excluding significant reduction of Complex I and a preexisting Δψ as activation factors. Co-reconstitution of Complex I and ATPase did not indicate an increased membrane permeability for protons in the uncoupled Complex I state. The delay in Complex I proton pumping activation was also observed in subbacterial vesicles. While it is negligible at room temperature, it strongly increases at a lower temperature. We conclude that Complex I undergoes a conversion from a decoupled state to a coupled state upon activation. The possible origins and importance of the observed phenomenon are discussed.

Time-resolved generation of membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus coupled to single electron injection into the O and OH states.

Two electrogenic phases with characteristic times of ~14µs and ~290µs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized "relaxed" O state of ba3 oxidase from T. thermophilus (O→E transition). The rapid phase reflects electron redistribution between CuA and heme b. The slow phase includes electron redistribution from both CuA and heme b to heme a3, and electrogenic proton transfer coupled to reduction of heme a3. The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a3 is reduced, but there is no proton pumping and no reduction of CuB. Single-electron reduction of the oxidized "unrelaxed" state (OH→EH transition) is accompanied by electrogenic reduction of the heme b/heme a3 pair by CuA in a "fast" phase (~22µs) and transfer of protons in "middle" and "slow" electrogenic phases (~0.185ms and ~0.78ms) coupled to electron redistribution from the heme b/heme a3 pair to the CuB site. The "middle" and "slow" electrogenic phases seem to be associated with transfer of protons to the proton-loading site (PLS) of the proton pump, but when all injected electrons reach CuB the electronic charge appears to be compensated by back-leakage of the protons from the PLS into the binuclear site. Thus proton pumping occurs only to the extent of ~0.1 H+/e-, probably due to the formed membrane potential in the experiment.

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.

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.

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.

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.

Fluorescent signals associated with respiratory Complex I revealed conformational changes in the catalytic site.

Fluorescent signals associated with Complex I (NADH:ubiquinone oxidoreductase type I) upon its reduction by NADH without added acceptors and upon NADH:ubiquinone oxidoreduction were studied. Two Complex I-associated redox-dependent signals were observed: with maximum emission at 400 nm (λex = 320 nm) and 526 nm (λex = 450 nm). The 400 nm signal derived from ubiquinol accumulated in Complex I/DDM (n-dodecyl ß-D-maltopyranoside) micelles. The 526 nm redox signal unexpectedly derives mainly from FMN (flavin mononucleotide), whose fluorescence in oxidized protein is fully quenched, but arises transiently upon reduction of Complex I by NADH. The paradoxical flare-up of FMN fluorescence is discussed in terms of conformational changes in the catalytic site upon NADH binding. The difficulties in revealing semiquinone fluorescent signal are considered.

Ca2+ stabilization of respiratory complex I from Escherichia coli.

Stability of the membrane-bound and purified H+-translocating NADH:ubiquinone oxidoreductase, Complex I, was studied. The loss of the enzyme activity is strongly increased by alkaline pH and dilution of the sample. Complex I inactivation is prevented specifically by a low concentration of Ca2+ and/or an intracellular stabilization factor (ISF). The action of both, Ca2+ and ISF, on Complex I stability is interdependent. The data are discussed in terms of a release of structural Ca2+ as a reason for Complex I decay and an effect of ISF on the affinity and/or accessibility of Ca2+-binding site.

Activation of respiratory Complex I from Escherichia coli studied by fluorescent probes.

Respiratory Complex I from E. coli may exist in two interconverting forms: resting (R) and active (A). The R/A transition of purified, solubilized Complex I occurring upon turnover was studied employing two different fluorescent probes, Annine 6+, and NDB-acetogenin. NADH-induced fluorescent changes of both dyes bound to solubilized Complex I from E. coli were characterized as a function of the protein:dye ratio, temperature, ubiquinone redox state and the enzyme activity. Analysis of this data combined with time-resolved optical measurements of Complex I activity and spectral changes indicated two ubiquinone-binding sites; a possibility of reduction of the tightly-bound quinone in the resting state and reduction of the loosely-bound quinone in the active state is discussed. The results also indicate that upon the activation Complex I undergoes conformational changes which can be mapped to the junction of the hydrophilic and membrane domains in the region of the assumed acetogenin-binding site.

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.

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.

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 ( approximately 1.5 x 10(7) m(-1) s(-1)), hydride ion transfer from NADH to FAD ( approximately 3.5 x 10(3) s(-1)), and partial electron separation and formation of equivalent fractions of reduced 2Fe-2S cluster and neutral semiquinone of FAD ( approximately 0.97 x 10(3) s(-1)). In the last step, a quasi-equilibrium is approached between the two states of FAD: two-electron reduced (50%) and one-electron reduced (the other 50%) species. The latter, neutral semiquinone of FAD, shares the second electron with the 2Fe-2S center. The transient midpoint redox potentials for the cofactors obtained during the fast kinetics measurements are very different from ones achieved during equilibrium redox titration and show that the functional states of the enzyme realized during its turning over cannot be modeled by the equilibrium approach.

Time-resolved ATR-FTIR spectroscopy of the oxygen reaction in the D124N mutant of cytochrome c oxidase from Paracoccus denitrificans.

Real-time measurements of the cytochrome c oxidase reaction with oxygen were performed by ATR-FTIR spectroscopy, using a mutant with a blocked D-pathway of proton transfer (D124N, Paracoccus denitrificans numbering). The complex spectrum of the ferryl-->oxidized transition together with other bands showed protonation of Glu 278 with a peak position at 1743 cm-1. Since our time resolution was not sufficient to follow the earlier reaction steps, the FTIR spectrum of the CO-inhibited fully reduced-->ferryl transition was obtained as a difference between the spectrum before the laser flash and the first spectrum after it. A trough at 1735 cm-1 due to deprotonation of Glu 278 was detected in this spectrum. These observations confirm the proposal [Smirnova I.A., et al. (1999) Biochemistry 38, 6826-6833] that the proton required for chemistry at the binuclear site is taken from Glu 278 in the perroxy-->ferryl step, and that the rate of the next step (ferryl-->oxidized) is limited by reprotonation of Glu 278 from the N-side of the membrane in the D124N mutant enzyme. The blockage of the D-pathway in this mutant for the first time allowed direct detection of deprotonation of Glu 278 and its reprotonation during oxidation of cytochrome oxidase by O2.

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 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.

Protolytic reactions on reduction of cytochrome c oxidase studied by ATR-FTIR spectroscopy.

Reduction of cytochrome c oxidase is coupled to proton uptake, and the reduced-minus-oxidized FTIR spectrum should include signatures of protonation of protolytic centers. The major part of the spectrum shows only small differences between acidic and alkaline conditions, which is consistent with the rather weak pH dependence of the proton uptake stoichiometry. Here we aim at revealing redox state-dependent protonatable sites and present a comprehensive investigation over a wide pH range. The reduced-minus-oxidized transition of cytochrome c oxidase from Paracoccus denitrificans was studied by means of Fourier transform infrared spectroscopy in the pH range 5.2-9.5. Effects of pH were analyzed as the difference between reduced-minus-oxidized FTIR spectra at different pH values. Two pH-dependent processes with apparent pKa values of 6.6 and 8.4 and Hill coefficients 0.9 and 0.1, respectively, were found by this methodology. A sharp OH band appears in the IR "water region" on reduction of the enzyme, independent of pH in the range 6.5-9.0, and downshifted by approximately 940 cm-1 on changing the solvent to D2O and by 10 cm-1 on H216O/H218O isotope exchange. This feature of an asymmetric water molecule may belong to water that is produced in the binuclear center upon reduction or to a structured water molecule that loses a hydrogen bond.