Investigation of B-branch electron transfer by femtosecond time resolved spectroscopy in a Rhodobacter sphaeroides reaction centre that lacks the Q(A) ubiquinone

The dynamics of electron transfer in a membrane-bound Rhodobacter sphaeroides reaction centre containing a combination of four mutations were investigated by transient absorption spectroscopy. The reaction centre, named WAAH, has a mutation that causes the reaction centre to assemble without a Q(A) ubiquinone (Ala M260 to Trp), a mutation that causes the replacement of the H(A) bacteriopheophytin with a bacteriochlorophyll (Leu M214 to His) and two mutations that remove acidic groups close to the Q(B) ubiquinone (Glu L212 to Ala and Asp L213 to Ala). Previous work has shown that the Q(B) ubiquinone is reduced by electron transfer along the so-called inactive cofactor branch (B-branch) in the WAAH reaction centre (M.C. Wakeham, M.G. Goodwin, C. McKibbin, M.R. Jones, Photo-accumulation of the P(+)Q(B)(-) radical pair state in purple bacterial reaction centres that lack the Q(A) ubiquinone, FEBS Letters 540 (2003) 234-240). In the present study the dynamics of electron transfer in the membrane-bound WAAH reaction centre were studied by femtosecond transient absorption spectroscopy, and the data analysed using a compartmental model. The analysis indicates that the yield of Q(B) reduction via the B-branch is approximately 8% in the WAAH reaction centre, consistent with results from millisecond time-scale kinetic spectroscopy. Possible contributions to this yield of the constituent mutations in the WAAH reaction centre and the membrane environment of the complex are discussed.     

Generation of membrane potential during photosynthetic electron flow in chromatophores from Rhodopseudomonas capsulata

1. When cytochrome c₂ is available for oxidation by the photosynthetic reaction centre, the decay of the carotenoid absorption band shift generated by a short flash excitation of Rhodopseudomonas capsulata chromatophores is very slow (half-time approximately 10 s). Otherwise the decay is fast (half-time approximately 1 s in the absence and 0.05 s in the presence of 1,10-ortho-phenanthroline) and coincides with the photosynthetic back reaction.2. In each of these situations the carotenoid shift decay, but not electron transport, may be accelerated by ioniophores. The ionophore concentration dependence suggests that in each case the carotenoid response is due to a delocalised membrane potential which may be dissipated either by the electronic back reaction or by electrophoretic ion flux.3. At high redox potentials, where cytochrome c₂ is unavailable for photo-oxidation, electron transport is believed to proceed only across part of the membrane dielectric. Under such conditions it is shown that the driving force for carbonyl cyanide trifluoromethoxyphenyl hydrazone-mediated H⁺ efflux is nevertheless decreased by valinomycin/K⁺; demonstrating that the [BChl]₂ → Q electron transfer generates a delocalised membrane potential.     

Electron transfer to the active site of the bacterial nitric oxide reductase is controlled by ligand binding to heme b₃

The active site of the bacterial nitric oxide reductase from Paracoccus denitrificans contains a dinuclear centre comprising heme b? and non heme iron (Fe(B)). These metal centres are shown to be at isopotential with midpoint reduction potentials of E(m) ≈ +80 mV. The midpoint reduction potentials of the other two metal centres in the enzyme, heme c and heme b, are greater than the dinuclear centre suggesting that they act as an electron receiving/storage module. Reduction of the low-spin heme b causes structural changes at the dinuclear centre which allow access to substrate molecules. In the presence of the substrate analogue, CO, the midpoint reduction potential of heme b? is raised to a region similar to that of heme c and heme b. This leads us to suggest that reduction of the electron transfer hemes leads to an opening of the active site which allows substrate to bind and in turn raises the reduction potential of the active site such that electrons are only delivered to the active site following substrate binding.     

An electron transfer dependent membrane potential in chromaffin-vesicle ghosts

Adrenal medullary chromaffin-vesicle membranes contain a transmembrane electron carrier that may provide reducing equivalents for intravesicular dopamine beta-hydroxylase in vivo. This electron transfer system can generate a membrane potential (inside positive) across resealed chromaffin-vesicle membranes (ghosts) by passing electrons from an internal electron donor to an external electron acceptor. Both ascorbic acid and isoascorbic acid are suitable electron donors. As an electron acceptor, ferricyanide elicits a transient increase in membrane potential at physiological temperatures. A stable membrane potential can be produced by coupling the chromaffin-vesicle electron-transfer system to cytochrome oxidase by using cytochrome c. The membrane potential is generated by transferring electrons from the internal electron donor to cytochrome c. Cytochrome c is then reoxidized by cytochrome oxidase. In this coupled system, the rate of electron transfer can be measured as the rate of oxygen consumption. The chromaffin-vesicle electron-transfer system reduces cytochrome c relatively slowly, but the rate is greatly accelerated by low concentrations of ferrocyanide. Accordingly, stable electron transfer dependent membrane potentials require cytochrome c, oxygen, and ferrocyanide. They are abolished by the cytochrome oxidase inhibitor cyanide. This membrane potential drives reserpine-sensitive norepinephrine transport, confirming the location of the electron-transfer system in the chromaffin-vesicle membrane. This also demonstrates the potential usefulness of the electron transfer driven membrane potential for studying energy-linked processes in this membrane.     

Photoelectric currents across planar bilayer membranes containing bacterial reaction centers. Response under conditions of single electron turnover.

Light-induced electric current and potential responses have been measured across planar phospholipid membranes containing reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides. Under conditions in which the reaction centers are restricted to a single electron turnover, the responses can be correlated with the light-induced electron transfer reactions associated with the reaction center. The results indicate that electron transfer from the bacteriochlorophyll dimer to the primary ubiquinone molecule, and from ferrocytochrome c to the oxidized dimer occur in series across the planar membrane. Electron transfer from the primary to secondary ubiquinone molecule is not electrogenic.     

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: the phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster F(X)

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A(1), the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F(X) and the phylloquinone bound to either the PsaA (A(1A)) or the PsaB (A(1B)) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F(A) and F(B). The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A(1)) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F(X). A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A(1B) quinone and slightly endergonic, in the case of the A(1A) quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A(0) on both electron transfer branches and the reduction of F(A) by F(X).     

Regulation of the proton/electron stoichiometry of mitochondrial ubiquinol:cytochrome c reductase by the membrane potential.

The electron transfer reaction catalysed by mitochondrial ubiquinol:cytochrome c reductase is linked to the outwards translocation of protons with an H+ e- stoichiometry of 1 under non-membrane potential condition. The effect of the electrical membrane potential on the H+/e- stoichiometry was investigated. The enzyme was isolated from Neurospora crassa, reconstituted into phospholipid vesicles and electrical membrane potentials of various values were generated across the membranes by means of the valinomycin-induced potassium-diffusion method. Using lithium ions as counterions for the intravesicular potassium, the induced membrane potential was stable for minutes and was not significantly changed by the protons ejected by the working enzyme. This allowed the assay of steady-state reaction rates at pre-given values of electrical membrane potential. The rate ratio between electron transfer and proton translocation declined from 1 to 0.6 with increase of the membrane potential from 0 to 100 mV. The activity of the quinol/cytochrome c redox reaction followed a parabolic dependence, being activated by low (less than 50 mV) potential and inhibited by high (greater than 100 mV) potential. This apparent non-linear dependence was interpreted in terms of a linear flow/force relationship plus a membrane-potential-dependent slip. Evaluation of the parabolic course by means of a modified linear flow/force relation also indicated a decline of the H+/e- stoichiometry from 1 to 0.5 with increase of the membrane potential from 0 to 120 mV. These observations suggest that the membrane potential controls a change of ubiquinol:cytochrome c reductase between two states that have different reaction routes.     

Kinetics of phyllosemiquinone oxidation in the Photosystem I reaction centre of Acaryochloris marina

Light-induced electron transfer reactions in the chlorophyll a/d-binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~5ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88±6ns and 345±10ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a-binding Photosystem I, which typically range in the 5-30ns and 200-300ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d-binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed.     

The flux-force relations across complex membranes with active transport

Equations are derived for the total material flux, and the total electric current flux, across a complex membrane system with active transport. The equations describe the fluxes as linear functions of forces across the system, and specifically of electrical potential, hydrostatic pressure, chemical potentials, and active transport rates. The equations can be simplified for experimental studies by making one or more of the forces equal to zero. The osmotic pressure difference across a membrane system is shown to be a function of the electrical potential and chemical potential differences and of the active transport rates. The transmembrane potential is shown to be the sum of a diffusion potential and an active transport potential. A simple equation is derived describing the current across a membrane as a linear function of the electrical potential and the active transport rate. Specific examples of the application of the equations to nerve membrane potentials are considered.     

Electroporation of curved lipid membranes in ionic strength gradients

A thermodynamic theory for the membrane electroporation of curved membranes such as those of lipid vesicles and cylindrical membrane tubes has been developed. The theory covers in particular the observation that electric pore formation and shape deformation of vesicles and cells are dependent on the salt concentration of the suspending solvent. It is shown that transmembrane salt gradients can appreciably modify the electrostatic part of Helfrich's spontaneous curvature, elastic bending rigidity and Gaussian curvature modulus of charged membranes. The Gibbs reaction energy of membrane electroporation can be explicitely expressed in terms of salt gradient-dependent contributions of bending, the ionic double layers and electric surface potentials and dielectric polarisation of aqueous pores. In order to cover the various physical contribution to the chemical process of electroporation-resealing, we have introduced a generalised chemophysical potential covering all generalised forces and generalised displacements in terms of a transformed Gibbs energy formalism. Comparison with, and analysis of, the data of electrooptical relaxation kinetic studies show that the Gibbs reaction energy terms can be directly determined from turbidity dichroism (Planck's conservative dichroism). The approach also quantifies the electroporative cross-membrane material exchange such as electrolyte release, electrohaemolysis of red blood cells or uptake of drugs and dyes and finally gene DNA by membrane electroporation.     

Lipids in photosynthetic reaction centres: structural roles and functional holes

Photosynthetic proteins power the biosphere. Reaction centres, light harvesting antenna proteins and cytochrome b(6)f (or bc(1)) complexes are expressed at high levels, have been subjected to an intensive spectroscopic, biochemical and mutagenic analysis, and several have been characterised to an informatively high resolution by X-ray crystallography. In addition to revealing the structural basis for the transduction of light energy, X-ray crystallography has brought molecular insights into the relationships between these multicomponent membrane proteins and their lipid environment. Lipids resolved in the X-ray crystal structures of photosynthetic proteins bind light harvesting cofactors, fill intra-protein cavities through which quinones can diffuse, form an important part of the monomer-monomer interface in multimeric structures and may facilitate structural flexibility in complexes that undergo partial disassembly and repair. It has been proposed that individual lipids influence the biophysical properties of reaction centre cofactors, and so affect the rate of electron transfer through the complex. Lipids have also been shown to be important for successful crystallisation of photosynthetic proteins. Comparison of the three types of reaction centre that have been structurally characterised reveals interesting similarities in the position of bound lipids that may point towards a generic requirement to reinforce the structure of the core electron transfer domain. The crystallographic data are also providing new opportunities to find molecular explanations for observed effects of different types of lipid on the structure, mechanism and organisation of reaction centres and other photosynthetic proteins.     

Deactivation of the Photosystem II oxidation (S) states by 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT2p) and the putative role of carotenoid

Addition of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT2p) to detergent-solubilised Photosystem II (PS II) particles results in the photo-oxidation of carotenoid and inhibition of the steady-state oxygen-evolution rate. It has been proposed that ANT2p may modify the water-splitting reactions by mediating the transfer of reducing equivalents from endogenous electron donors, such as carotenoid, to the S₂ and S₃ oxidation states of PS II. In this paper we present evidence indicating that ANT2p can interact with PS II at two separate loci. The water-splitting complex is shown to be the primary site of attack by ANT2p, since artificial electron donors, such as 1,5-diphenylcarbazide (DPC), can restore PS II photochemical activity by feeding reducing equivalents directly to the reaction centre. The ANT2p interaction at this site is light-intensity dependent. A second inhibitory site close to the reaction centre P-680 chlorophyll is detected at slightly higher ANT2p concentrations. The inhibition at this site is unaffected either by changes in the actinic light intensity or by the addition of electron donors. The flash-induced oxidation of carotenoid has an ANT2p concentration dependence and an insensitivity to DPC which suggests that it results from the inhibition of the reaction centre and not with that of the water-splitting complex.     

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: The phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster FX

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A₁, the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F_X and the phylloquinone bound to either the PsaA (A_1A) or the PsaB (A_1B) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F_A and F_B. The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A₁) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F_X. A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A_1B quinone and slightly endergonic, in the case of the A_1A quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A₀ on both electron transfer branches and the reduction of F_A by F_X.     

Thermodynamics of electron transfer and its coupling to vectorial processes in biological membranes.

A method is developed to express the flux of an electron transfer reaction as a function of the conjugate force, the redox potential difference, throughout the nonlinear region. The flux can be expressed by a product of the hyperbolic sine of the force, a factor ("redox-poising parameter") determined by the redox potentials of subsystem (in certain cases by local pH's and pK's of subsystems), and some constants. This is analogous to the expression of the flux of a diffusion process by the product of its force and the concentration of the diffusing species. The redox-poising parameter corresponds to the concentration term. The expression is applied to redox chains in which electron transfers are coupled to vectorial processes such as proton translocation or electric current.     

The membrane potential and its representation by a constant electric field in computer simulations

A theoretical framework is elaborated to account for the effect of a transmembrane potential in computer simulations. It is shown that a simulation with a constant external electric field applied in the direction normal to the membrane is equivalent to the influence of surrounding infinite baths maintained to a voltage difference via ion-exchanging electrodes connected to an electromotive force. It is also shown that the linearly-weighted displacement charge within the simulation system tracks the net flow of charge through the external circuit comprising the electromotive force and the electrodes. Using a statistical mechanical reduction of the degrees of freedom of the external system, three distinct theoretical routes are formulated and examined for the purpose of characterizing the free energy of a protein embedded in a membrane that is submitted to a voltage difference. The W-route is constructed from the variations in the voltage-dependent potential of mean force along a reaction path connecting two conformations of the protein. The Q-route is based on the average displacement charge as a function of the conformation of the protein. Finally, the G-route considers the relative charging free energy of specific residues, with and without applied membrane potentials. The theoretical formulation is illustrated with a simple model of an ion crossing a vacuum slab surrounded by two aqueous bulk phases and with a fragment of the voltage-sensor of the KvAP potassium channel.     

Determination of depth of immersion of chlorophyll P680, pheophytin, and a secondary electron donor in the subchloroplast preparations of photosystem II from pea

The immersion depths (r) of the main functional components of the reaction centres of the Photosystem 2 in the thylakoid membranes were determined by ESR at 77K. It was shown that P680(+), Pheo(-) (pheophytin), and Z(+) (secondary electron donor) the r value was 2-4, 4-7 and 14-20 A, respectively. On the basis of these and reference data a model of location of the Photosystem 2 reaction centre components in the photosynthetic membrane was suggested.     

On Bioelectric Potentials in an Inhomogeneous Volume Conductor

Green's theorem is used to derive two sets of expressions for the quasi-static potential distribution in an inhomogeneous volume conductor. The current density in passive regions is assumed to be linearly related instantaneously to the electric field. Two equations are derived relating potentials to an arbitrary distribution of impressed currents. In one, surfaces of discontinuity in electrical conductivity are replaced by double layers and in the other, by surface charges. A multipole equivalent generator is defined and related both to the potential distribution on the outer surface of the volume conductor and to the current sources. An alternative result involves the electric field at the outer surface rather than the potential. Finally, the impressed currents are related to electrical activity at the membranes of active cells. The normal component of membrane current density is assumed to be equal at both membrane surfaces. One expression is obtained involving the potentials at the inner and outer surfaces of the membrane. A second expression involves the transmembrane potential and the normal component of membrane current.     

Reconstitution of biological molecular generators of electric current. Cytochrome oxidase.

1. Direct measurement of the electric current generation by cytochrome oxidase has been carried out. To this end, two procedures were used. The simpler one consists in formation of planar artificial membrane from the mixture of decane solution of soya bean phospholipids and beef heart cytochrome oxidase. Addition of cytochrome c and ascorbate to one of the two compartments separated by the cytochrome oxidase-containing planar membrane was found to result in a transmembrane electric potential difference being formed (plus on cytochrome c side of the membrane). Maximal values of potential differences obtained by this method were about 40 mV. Much higher potentials were observed when another ("photeoliposome-planar membrane") method was applied. In this case cytochrome oxidase was reconstituted with phospholipid to form proteoliposomes which adhered to planar phospholipid membrane in the presence of Ca2+ ions. Addition of cytochrome c and ascorbate to the proteoliposome-containing compartment gives rise to generation of an electric potential difference across the planar membrane, which reached 100 mV at a current of about 1 X 10(-11) A (minus in the proteoliposome-free compartment). The electromotive force of this generator was estimated as being about 0.2 V. If ascorbate and proteoliposomes were added into different compartments, a penetrating hydrogen atom carrier (phenazine methosulfate, (PMS) or tetramethyl-p-phenylenediamine (TMPD)) was required for a membrane potential to be formed. Generation of an electric potential difference of the opposite direction (plus in the proteoliposome-free compartment) was revealed in experiments with cytochrome oxidase proteoliposome containing cytochrome c in their interior. In this case, addition of PMS or TMPD was necessary. 2. In the suspension of cytochrome oxidase proteoliposome the uptake of a cationic penetrant (tetraphenyl phosphonium cation) was found to be coupled with electron transfer via external cytochrome c. Electron transfer via intraproteoliposomal cytochrome c induced the uptake of anionic penetrants (tetraphenyl borate and phenyldicarbaundecaborane anions). 3. All the above effects were sensitive to cyanide and protonophorous uncouplers. 4. In proteoliposomes containing both cytochrome oxidase and bacteriorhodopsin, the light- and oxidation-dependent generations of membrane potential have been revealed. 5. The data obtained are in agreement with Mitchell's idea of transmembrane electron flow in the cytochrome oxidase segment of the respiratory chain.     

Mutations that modify or exclude binding of the QA ubiquinone and carotenoid in the reaction center from Rhodobacter sphaeroides

Three single-site mutations have been introduced at positions close to the Q_A ubiquinone in the reaction centre from Rhodobacter sphaeroides. Two of these mutations, Ala M260 to Trp and Ala M248 to Trp, result in a reaction centre that does not support photosynthetic growth of the bacterium, and in which electron transfer to the Q_A ubiquinone is abolished. In the reaction centre with an Ala to Trp mutation at the M260 residue, electron transfer from the primary donor to the acceptor bacteriopheophytin is not affected by the mutation, but electron transfer from the acceptor bacteriopheophytin to Q_A is not observed. The most likely basis for these effects is that the mutation produces a structural change that excludes binding of the Q_A ubiquinone. A third mutation, Leu M215 to Trp, produces a reaction centre that has an impaired capacity for supporting photosynthetic growth. The mutation changes the nature of ubiquinone binding at the Q_A site, and renders the site sensitive to quinone site inhibitors such as o- phenanthroline. Adopting a similar approach, in which a small residue located close to a cofactor is changed to a more bulky residue, we show that the reaction centre can be rendered carotenoid-less by the mutation Gly M71 to Leu.     

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

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.