The localized coupling of bacterial photophosphorylation: Effect of antimycin a and N,N-dicyclohexylcarbodiimide) in chromatophores from Rhodopseudomonas sphaeroides,Ga, studied by single turnover event analysis

1. The inhibition by antimycin A of the cyclic electron transfer has been studied in chromatophores from Rhodopseudomonas sphaeroides Ga following an approach based on the analysis of the relaxation kinetics of the reaction center optical changes in pulsed light. The recovery kinetics of the bacteriochlorophyll redox state have been found to be clearly biphasic. The half-times of the fast phase (13 ms) and slow phase (about 400 ms) were not modified by antimycin in a range of concentrations from 0.1 to 9 μM. On the other hand the percentage extent of the fast phase, which reflects the rate of the cyclic electron transfer, was monotonically decreased by increasing concentrations of the inhibitor. This indicates that antimycin decreases progressively the fraction of the photosynthetic units, active in cyclic electron transfer. 2. The ATP yield per flash observed under conditions of controlled inhibition of electron flow was strongly dependent upon the amount of active redox cycles. On the other hand, the amplitude of the carotenoid band shift, which has been demonstrated unequivocally to be correlated to the ATP yield per flash in uninhibited chromatophores, was not affected by antimycin up to a 40% inhibition of electron flow. 3. The effect of a progressive limitation by DCCD in the number of active ATP synthetase complexes on flash-induced phosphorylation has been examined. The decrease in ATP yield observed over a wide range of flash frequencies is related simply to the ATPase activity and to phosphorylation in continuous light, irrespective of the value of the membrane potential, which appears to be stabilized by this inhibitor. 4. As a whole, the results obtained at low concentrations of antimycin and under conditions of partial inhibition by DCCD evidence a localized coupling between the redox reactions and phosphorylation.     

Direct electrochemistry of spinach plastocyanin at a lipid bilayer-modified electrode: cyclic voltammetry as a probe of membrane-protein interactions.

The electron transfer reactions between a lipid bilayer-modified gold electrode and oxidized spinach plastocyanin have been studied by cyclic voltammetry, using either an electrically neutral phosphatidylcholine (PC) bilayer or a positively charged PC bilayer containing 40 mol% dimethyldioctadecylammonium chloride, at two ionic strengths of electrolyte (0.02 and 0.2 M NaClO4). Plastocyanin was found to interact strongly enough with the lipid membrane to support an efficient electron transfer reaction with the electrode. The interaction forces, and therefore the mode of diffusion of plastocyanin molecules to the electrode, which limits the electron transfer rate, could be controlled by the PC concentration. At low lipid concentrations (0-5 mg/ml), electrostatically attractive interactions between specific microelectroactive sites on the surface of the lipid membrane and plastocyanin molecules predominate, producing a radial mode of diffusion of the protein molecules to the electrode surface. On the other hand, at high lipid concentrations (greater than 5 mg/ml), interaction between plastocyanin and the lipid membrane occurs via hydrophobic forces, and a linear diffusion of protein molecules limits the electron transfer process. These observations support and extend other experimental and theoretical results which indicate two possible sites on the surface of the plastocyanin molecule, one hydrophobic and one negatively charged, which are able to participate in electron transfer reactions. We conclude that electrochemical measurements with the present system provide a new approach to the study of redox protein-membrane interactions.     

The pH-dependent redox inactivation of amicyanin from Paracoccus versutus as studied by rapid protein-film voltammetry

The redox properties of the blue copper protein amicyanin have been studied with slow and fast scan protein-film cyclic voltammetry. At slow scan rates, which reveal the thermodynamics of the redox reactions, the reduction potential of amicyanin depends on pH in a sigmoidal manner, and the data can be analysed in terms of electron transfer being coupled to a single protonatable group with pKa(red)=6.3 and pKa(ox) < or = 3.2 at 22 degrees C. Voltammetry at higher scan rates reveals the kinetics and shows that the low-pH reduced form of amicyanin is not oxidised directly; instead, oxidation occurs only after conversion to the high-pH form. Simulations show that this conversion, which gates the electron transfer, occurs with a rate constant >750 s-1 at 25 degrees C. In order to decrease the rate of the coupled reaction, the experiments were performed at 0 degrees C, at which the rate constant for this conversion was determined to be 35 +/- 20 s-1. Together with evidence from NMR, the results lead to a mechanism involving protonation and dissociation of the copper coordinating histidine-96 in the reduced form.     

Steady-state cyclic electron transfer through solubilized Rhodobacter sphaeroides reaction centres

The mechanism, thermodynamics and kinetics of light-induced cyclic electron transfer have been studied in a model energy-transducing system consisting of solubilized Rhodobacter sphaeroides reaction center/light harvesting-1 complexes (so-called core complexes), horse heart cytochrome c and a ubiquinone-0/ubiquinol-0 pool. An analysis of the steady-state kinetics of cytochrome c reduction by ubiquinol-0, after a light-induced steady-state electron flow had been attained, showed that the rate of this reaction is primarily controlled by the one-electron oxidation of the ubiquinol-anion. Re-reduction of the light-oxidized reaction center primary donor by cytochrome c was measured at different reduction levels of the ubiquinone-0/ubiquinol-0 pool. These experiments involved single turnover flash excitation on top of background illumination that elicited steady-state cyclic electron transfer. At low reduction levels of the ubiquinone-0/ubiquinol-0 pool, the total cytochrome c concentration had a major control over the rate of reduction of the primary donor. This control was lost at higher reduction levels of the ubiquinone/ubiquinol-pool, and possible reasons for this behaviour are discussed.     

Two molecular forms of pea ferredoxin in the electron transport chain of chloroplasts

The effects of two molecular forms of water-soluble ferredoxin (Fd I and Fd II) on the kinetics of electron transport in bean chloroplasts (class B) were studied. The light-induced redox transitions of the photosystem I reaction center P700 were measured by the intensity of the EPR signal I produced by P700+. Both forms of ferredoxin, Fd I and Fd II, when added to the chloroplasts in catalytic amounts, stimulate the light-induced electron transfer from P700 to NADP+. Nevertheless, Fd I is a better mediator of the back reactions from NADPH to P700+. This electron transfer pathway is sensitive to the cyclic electron transport inhibitor, antimycin A, and to DCMU inhibitor of electron transport between photosystem II and plastoquinone. It may be concluded that the two molecular forms of ferredoxin, Fd I and Fd II, differ in their ability to catalyze cyclic electron transport in photosystem I. The role of Fd I and Fd II in regulation of electron transport at the acceptor site of photosystem I is discussed.     

Myoglobin immobilization on electrodeposited nanometer-scale nickel oxide particles and direct voltammetry

Prosperity of information on the reactions of redox-active sites in proteins can be attained by voltammetric studies in which the protein sample is located on a suitable surface. This work reports the presentation of myoglobin/nickel oxide nanoparticles/glassy carbon (Mb/NiO NPs/GC) electrode, ready by electrochemical deposition of the NiO NPs on glassy carbon electrode and myoglobin immobilization on their surfaces by the potential cycling method. Images of electrodeposited NiO NPs on the surface of glassy carbon electrode were obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). A pair of well-defined redox peaks for Mb(Fe(III)-Fe(II)) was obtained at the prepared electrode by direct electron transfer between the protein and nanoparticles. Electrochemical parameters of immobilized myoglobin such as formal potential (E(0')), charge transfer coefficient (alpha) and apparent heterogeneous electron transfer rate constant (k(s)) were estimated by cyclic voltammetry and nonlinear regression analysis. Biocatalytic activity was exemplified at the prepared electrode for reduction of hydrogen peroxide.     

Kinetics of reduction by free flavin semiquinones of algal cytochromes and plastocyanin

It had been shown that plastocyanin and cytochrome c-553 are functionally interchangeable in algae and that the physiological electron transfer reactions are sensitive to ionic strength. The isoelectric points of these proteins range from very acidic to basic depending upon species, and naturally occurring amino acid substitutions of charged residues have been shown to affect the kinetics of electron transfer, presumably through alteration of protein net charge. We have now shown that these naturally occurring amino acid substitutions also affect the kinetics of nonphysiological electron transfer reactions, and that we can quantitate the extent of nonconservation of charge. The reduction of plant and algal proteins by FMN semiquinone is sensitive to ionic strength and the effects can be correlated with net protein charge with regard to sign, but not to magnitude, with the charge at the site of electron transfer varying from +3 through 0 to -3. We had previously observed in a large variety of electron transfer proteins from bacteria (G. Tollin, T. E. Meyer, and M. A. Cusanovich (1986) Biochim. Biophys. Acta 853, 29-41) that charge localized at the site of electron transfer, rather than net protein charge, was more likely to affect kinetics. This also appears to be the case with the algal proteins. By comparison of protein structures, we have been able to predict which substitutions are likely to be responsible for the kinetic effects in the algal proteins and to discuss the implications of such changes for function.     

Electron transfer through the acceptor side of photosystem I: Interaction with exogenous acceptors and molecular oxygen

This review considers the state-of-the-art on mechanisms and alternative pathways of electron transfer in photosynthetic electron transport chains of chloroplasts and cyanobacteria. The mechanisms of electron transport control between photosystems (PS) I and II and the Calvin–Benson cycle are considered. The redistribution of electron fluxes between the noncyclic, cyclic, and pseudocyclic pathways plays an important role in the regulation of photosynthesis. Mathematical modeling of light-induced electron transport processes is considered. Particular attention is given to the electron transfer reactions on the acceptor side of PS I and to interactions of PS I with exogenous acceptors, including molecular oxygen. A kinetic model of PS I and its interaction with exogenous electron acceptors has been developed. This model is based on experimental kinetics of charge recombination in isolated PS I. Kinetic and thermodynamic parameters of the electron transfer reactions in PS I are scrutinized. The free energies of electron transfer between quinone acceptors A_1A/A_1B in the symmetric redox cofactor branches of PS I and iron–sulfur clusters F_X, F_A, and F_B have been estimated. The second-order rate constants of electron transfer from PS I to external acceptors have been determined. The data suggest that byproduct formation of superoxide radical in PS I due to the reduction of molecular oxygen in the A1 site (Mehler reaction) can exceed 0.3% of the total electron flux in PS I.     

Electrocatalytic oxidation of NADH with Meldola's blue functionalized carbon nanotubes electrodes

Meldola's blue (MB) functionalized carbon nanotubes (CNT) nanocomposite film (MB/CNT) electrode was prepared by non-covalent adsorbing MB on the surface of a carbon nanotubes modified glassy carbon electrode (CNT/GCE). Electrochemical behaviors of the resulting electrode were investigated thoroughly with cyclic voltammetry in the potential range of -0.6 to 0.2V, and two well-defined redox couples were clearly visualized. We also studied the electron transfer kinetics of MB loaded on CNT (MB/CNT) in comparison with that of MB on conventional graphite powder (MB/GP). The heterogeneous electron transfer rate constant (k(s)) of MB/CNT was calculated to be about three times larger than that of MB/GP. The accelerated electron transfer kinetics was attributed to the unique electrical and nanostructural properties of CNT supports as well as the interaction between MB and CNT. In connection with the oxidation of nicotinamide adenine dinucleotide (NADH), excellent electrocatalytic activities were observed at MB/CNT/GCE compared with MB/GP modified glassy carbon electrode (MB/GP/GCE). Based on the results, a new NADH sensor was successfully established using the MB/CNT/GCE. Under a lower operation potential of -0.1V, NADH could be detected linearly up to a concentration of 500 microM with an extremely lower detection limit of 0.048+/-0.02 microM estimated at a signal-to-noise ratio of 3. Sensitivity, selectivity, reproducibility and stability of the NADH sensor were also investigated and the main analytical data were also compared with those obtained with the MB/GP/GCE.     

Biological electron transfer: progress and future directions.

The rich diversity among bacterial cytochromes has played a key role in the development of our understanding of biological electron transfer. Although studies to date have allowed the elucidation of the contributions of driving force, electrostatics interactions and surface topology to electron transfer kinetics in collision-dependent reactions, much remains to be learned. Little is known about intramolecular and intracomplex electron transfer. Several factors controlling intramolecular and intracomplex electron transfer can be defined. These include driving force, the distance between redox centers, the relative orientation of prosthetic groups, the nature of the intervening media and the molecular dynamics within the electron transfer complex. However, at the present time, we have only a limited understanding of the contribution of these factors to electron transfer kinetics in biologically relevant systems. Nevertheless, a wide range of techniques are now available which should soon provide the information necessary to describe in molecular terms the mechanism of intramolecular and intracomplex electron transfer. Principal among these new approaches are site-directed mutagenesis and NMR spectroscopy.     

Design of photoactive ruthenium complexes to study electron transfer and proton pumping in cytochrome oxidase

This review describes the development and application of photoactive ruthenium complexes to study electron transfer and proton pumping reactions in cytochrome c oxidase (CcO). CcO uses four electrons from Cc to reduce O(2) to two waters, and pumps four protons across the membrane. The electron transfer reactions in cytochrome oxidase are very rapid, and cannot be resolved by stopped-flow mixing techniques. Methods have been developed to covalently attach a photoactive tris(bipyridine)ruthenium group [Ru(II)] to Cc to form Ru-39-Cc. Photoexcitation of Ru(II) to the excited state Ru(II*), a strong reductant, leads to rapid electron transfer to the ferric heme group in Cc, followed by electron transfer to Cu(A) in CcO with a rate constant of 60,000s(-1). Ruthenium kinetics and mutagenesis studies have been used to define the domain for the interaction between Cc and CcO. New ruthenium dimers have also been developed to rapidly inject electrons into Cu(A) of CcO with yields as high as 60%, allowing measurement of the kinetics of electron transfer and proton release at each step in the oxygen reduction mechanism.     

Functional homogeneity of P-700 in cyclic and non-cyclic electron transport reactions in thylakoids

The photoinduced turnover of P-700 (the reaction center chlorophyll a of photosystem I) in higher plant thylakoids was examined at room temperature by observation of the kinetics and amplitude of the transmission signal at 700 nm. The concentration of P-700 functional in cyclic and non-cyclic electron transfer reactions was compared. For the cyclic reactions mediated by N-methylphenazonium-p-methosulfate, 2,3,5,6-tetramethylphenylenediamine, 2,6-dichlorophenolindophenol and N,N,N′,N′-tetramethylphenylenediamine and non-cyclic reactions utilizing either methylviologen or NADP⁺ as acceptor, the illuminated steady-state concentration of P-700⁺ was shown to be similar. The data support the concept of a homogeneous pool of P-700 that is capable of interaction in both cyclic and non-cyclic electron transfer reactions and are consistent with previous data obtained in vivo.The amplitude and kinetics of the P-700 signal were found to be very dependent upon the composition of the reaction medium and differences were noted for turnover in the cyclic and non-cyclic reactions. Specifically, at white light saturation, the addition of low concentrations of divalent cations, such as Mg²⁺ or Ca²⁺, had no effect on the signal amplitude during the cyclic reactions, but, in confirmation of previous work, caused an attenuation of the signal amplitude during non-cyclic flow. At low light intensities, the divalent cations caused a similar reduction in redox level of P-700 in the steady-state during non-cyclic flow and also reduced the rate of P-700 photooxidation in the cyclic reactions. The concentration of divalent cation that reduced the signal amplitude of P-700⁺ during non-cyclic flow was compared with that required for the stimulation of the variable component of fluorescence, and it was shown to be similar with half maximal effects at 1 mM Mg²⁺. The observations confirm that divalent cations control non-cyclic electron transport by an activation of Photosystem II in addition to regulating the distribution of excitation energy between the two photosystems.     

The reduction kinetics of chlorophyll aI as an indicator for proton uptake between the light reactions in chloroplasts.

The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll aI (P-700) after far-red preillumination have been studied with high time resolution in spinach chloroplasts. 1. The kinetics of chlorophyll aI exhibits a pronounced lag phase of 2--3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll aI. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylkaloid membrane. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll aI reduction, is estimated as greater than 10(11) M-1s-1. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution. Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.     

On the involvement of electron transfer reactions in the fluorescence decay kinetics heterogeneity of proteins

Time-resolved fluorescence study of single tryptophan-containing proteins, nuclease, ribonuclease T1, protein G, glucagon, and mastoparan, has been carried out. Three different methods were used for the analysis of fluorescence decays: the iterative reconvolution method, as reviewed and developed in our laboratory, the maximum entropy method, and the recent method that we called "energy transfer" method. All the proteins show heterogeneous fluorescence kinetics (multiexponential decay). The origin of this heterogeneity is interpreted in terms of current theories of electron transfer process, which treat the electron transfer process as a radiationless transition. The theoretical electron transfer rate was calculated assuming the peptide bond carbonyl as the acceptor site. The good agreement between experimental and theoretical electron-transfer rates leads us to suggest that the electron-transfer process is the principal quenching mechanism of Trp fluorescence in proteins, resulting in heterogeneous fluorescence kinetics. Furthermore, the origin of apparent homogeneous fluorescence kinetics (monoexponential decay) in some proteins also can be explained on the basis of electron-transfer mechanism.     

Reaction kinetics of electron phototransfer in an eosin-casein complex

The role of protein matrix in the process of charge photorespiration on the model of eosin-casein complex was studied. We have also studied the kinetics of the electron transfer reactions being photosensitized by free eosin and eosin sorbed on casein in the donor (cystein) - acceptor (methylviologen) system. When eosin is sorbed on protein a 10-fold increase of the probability relation of non-retrospective direct electron transfer to the restrospective one was observed.     

Laser-flash photolysis indicates that internal electron transfer is triggered by proton uptake by Alcaligenes xylosoxidans copper-dependent nitrite reductase

Enzyme-catalysed electron transfer reactions are often controlled by protein motions and coupled to chemical change such as proton transfer. We have investigated the nature of this control in the blue copper-dependent nitrite reductase from Alcaligenes xylosoxidans (AxNiR). Inter-Cu electron transfer from the T1Cu site to the T2Cu catalytic site in AxNiR occurs via a proton-coupled electron transfer mechanism. Here we have studied the kinetics of both electron and proton transfer independently using laser-flash photolysis for native AxNiR and its proton-channel mutant N90S. In native AxNiR, both inter-Cu electron transfer and proton transfer exhibit similar rates, and show an unusual dependence on the nitrite concentration. An initial decrease in the observed rates at low nitrite concentrations is followed by an increase in the observed rates at high nitrite concentrations (> 5 mm). In N90S, in which the T1Cu reduction potential is elevated by 60 mV, no inter-Cu electron transfer or proton transfer was observed in the absence of nitrite. Only in the presence of nitrite were both processes detected, with similar [nitrite] dependence, but the nitrite dependence was different compared with native enzyme. The substrate dependence in N90S was similar to that observed in steady-state assays, suggesting that this substitution resulted in proton-coupled electron transfer becoming rate-limiting. A pH perturbation experiment with native AxNiR revealed that protonation triggers inter-Cu electron transfer and generation of NO. Our results show a strong coupling of inter-Cu electron transfer and proton transfer for both native AxNiR and N90S, and provide novel insights into the controlled delivery of electrons and protons to the substrate-utilization T2Cu active site of AxNiR.     

Immobilization of hemoglobin on electrodeposited cobalt-oxide nanoparticles: direct voltammetry and electrocatalytic activity

Cyclic voltammetry at potential range − 1.1 to 0.5 V from aqueous buffer solution (pH 7) containing CoCl₂ produced a well defined cobalt oxide (CoOx) nanoparticles deposited on the surface of glassy carbon electrode. The morphology of the modified surface and cobalt oxide formation was examined with SEM and cyclic voltammetry techniques. Hemoglobin (Hb) was successfully immobilized in cobalt-oxide nanoparticles modified glassy carbon electrode. Immobilization of hemoglobin onto cobalt oxide nanoparticles have been investigated by cyclic voltammetry and UV–visible spectroscopy. The entrapped protein can take direct electron transfer in cobalt-oxide film. A pair of well defined, quasi-reversible cyclic voltammetric peaks at about − 0.08 V vs. SCE (pH 7), characteristic of heme redox couple (Fe(III)/Fe(II)) of hemoglobin, and the response showed surface controlled electrode process. The dependence of formal potential (E^0′) on the solution pH (56 mV pH^− 1) indicated that the direct electron transfer reaction of hemoglobin was a one-electron transfer coupled with a one proton transfer reaction process. The average surface coverage of Hb immobilized on the cobalt oxide nanoparticles was about 5.2536 × 10^− 11 mol cm^− 2_, indicating high loading ability of nanoparticles for hemoglobin entrapment. The heterogeneous electron transfer rate constant (k_s) was 1.43 s^− 1, indicating great of facilitation of the electron transfer between Hb and electrodeposited cobalt oxide nanoparticles. Modified electrode exhibits a remarkable electrocatalytic activity for the reduction of hydrogen peroxide and oxygen. The Michaels–Menten constant K_m of 0.38 mM, indicating that the Hb immobilized onto cobalt oxide film retained its peroxidases activity. The biosensor exhibited a fast amperometric response < 5 s, a linear response over a wide concentration range 5 μM to 700 μM and a low detection limit 0.5 μM. According to the direct electron transfer property and enhanced activity of Hb in cobalt oxide film, a third generation reagentless biosensor without using any electron transfer mediator or specific reagent can be constructed for determination of hydrogen peroxide in anaerobic solutions.     

Electron transfer and conformational change in complexes of trimethylamine dehydrogenase and electron transferring flavoprotein.

The trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH.ETF) electron transfer complex has been studied by fluorescence and absorption spectroscopies. These studies indicate that a series of conformational changes occur during the assembly of the TMADH.ETF electron transfer complex and that the kinetics of assembly observed with mutant TMADH (Y442F/L/G) or ETF (alpha R237A) complexes are much slower than are the corresponding rates of electron transfer in these complexes. This suggests that electron transfer does not occur in the thermodynamically most favorable state (which takes too long to form), but that one or more metastable states (which are formed more rapidly) are competent in transferring electrons from TMADH to ETF. Additionally, fluorescence spectroscopy studies of the TMADH.ETF complex indicate that ETF undergoes a stable conformational change (termed structural imprinting) when it interacts transiently with TMADH to form a second, distinct, structural form. The mutant complexes compromise imprinting of ETF, indicating a dependence on the native interactions present in the wild-type complex. The imprinted form of semiquinone ETF exhibits an enhanced rate of electron transfer to the artificial electron acceptor, ferricenium. Overall molecular conformations as probed by small-angle x-ray scattering studies are indistinguishable for imprinted and non-imprinted ETF, suggesting that changes in structure likely involve confined reorganizations within the vicinity of the FAD. Our results indicate a series of conformational events occur during the assembly of the TMADH.ETF electron transfer complex, and that the properties of electron transfer proteins can be affected lastingly by transient interaction with their physiological redox partners. This may have significant implications for our understanding of biological electron transfer reactions in vivo, because ETF encounters TMADH at all times in the cell. Our studies suggest that caution needs to be exercised in extrapolating the properties of in vitro interprotein electron transfer reactions to those occurring in vivo.     

Kinetic measurements of electron transfer in coupled chromatophores from photosynthetic bacteria. A method of correction for the electrochromic effects

A quantitative study of the kinetics of electron transfer under coupled conditions in photosynthetic bacteria has so far been prevented by overlap of the electrochromic signals of carotenoids and bacteriochlorophyll with the absorbance changes of cytochromes and reaction centers. In this paper a method is presented by which the electrochromic contribution at any wavelength can be calculated from the electrochromic signal recorded at 505 nm, using a set of empirically determined polynomial functions. The electrochromic contribution to kinetic changes at any wavelength can then be subtracted to leave the true kinetics of the redox changes. The corrected redox changes of the reaction center measured at 542 and 605 nm mutually agree, thus providing an excellent test of self-consistency of the method. The corrected traces for reaction center and of cytochrome b-566 demonstrate large effects of the membrane potential on the rate and poise of electron transfer. It will be possible to study the interrelation between proton gradient and individual electron reactions under flash or steady-state illumination.