Influence of pH on the steady state kinetics of electron transfer through the cytochrome chain of submitochondrial particles. Kinetic model for regulating the activity of carriers by the local concentration of hydrogen ions in the membrane]

The kinetic parameters of the submitochondrial particles cytochrome chain obtained from steady-state kinetics were studied for pH dependence. The life-times of the activated states (tau) for cytochrome pairs b leads to c1 and a leads to a3 are shown to bear dissimilar dependence on pH of the medium, while for cytochrome pairs c1 leads to c and c leads to a they display practically no pH dependence at all. The rate constants of the non-activated state (alphai-kiCo) decreased for the pair b leads to c1 and increased for a leads to a3 with the increase of pH from 6.5 to 8.5. The apparent pK values obtained therefrom were 7.2 and 8.9, respectively. A kinetic model is proposed suggesting that local pH in the mitochondrial membrane, dependent on the rate of electron transfer, may be a controlling factor for the ratio of activated and non-activated carrier states. The model is in good consistence with the experimental dependences of k'i on V and the pH dependences of alpha2 for b leads to c1 and a leads to a3. It also gives a qualitative prediction for the pH dependences of the ordinate intercepts of the straight lines in l/(k'i--alphai) vs. l/V plots. The rate constants for the diffusion of hydrogen and hydroxyl ions in the membrane are estimated on the basis of our kinetic data to be 10(4)--10(5) s-1 and 10(2)--10(3) s-i, respectively.     

Steady-state kinetics of electron transfer through cytochrome chain of uncoupled submitochondrial particles. II. Influence of pH on kinetics of electron transfer

pH Dependences of steady-state kinetic parameters of cytochrome chains of submitochondrial particles have been studied. It has been shown that the lifetimes of activated states (τ) of the pairs of cytochromes b → c₁ and a → a₃ have different pH dependences; those for the c₁ → c and c → a cytochrome pairs being similar. The rate constants for the non-activated state of the respiratory chains decreased for the b → c₁ pair and increased for the a → a₃ pair when the pH value was increased.The values of pK calculated from these dependences for the pairs b → c₁ and a → a₃ were 7.2 and 8.9, respectively. It has been supposed that the ratio of activated to non-activated electron carriers may be controlled by the local pH value in the mitochondrial membrane, the latter being dependent upon the rate of electron transfer. The kinetic model based on this assumption allows one to explain the experimental dependences on pH of the rate constants for cytochromes b → c, and a → a₃.The values of the diffusion rate constants for H⁺ and OH^? ions in the mitochondrial membrane estimated from these kinetic data obtained in this study weree 10⁴–10⁵ s^?1 and 10²–10³ s^?1, respectively.     

Steady-state kinetics of electron transfer through the cytochrome chain of uncoupled submitochondrial particles. General kinetic analysis]

Steady-state kinetics of electron transfer through the cytochrome chain of uncoupled ultrasonic submitochondrial particles at different pH values were studied. The rate constants calculated according to Pring's equation (k1=V/Prpoxt i+1) were found to increase linearly with the increase in the rate of electron transfer. Linearity was observed, however, only at relatively low rates of electron transfer. Several kinetic models were developed and analysed to fit the experimental data on the basis of the suggested activation of respiratory chains induced by their functioning. The best agreement with the experimental data was obtained with the model implying that the rate of activation of the electron carriers is directly proportional to the overall rate of electron transfer and the portion of non-activated respiratory chains in the system. It followed therefrom that electron transfer through already activated chains induced activation of adjacent non-activated chains. This model made it possiple to determine the rate constants for non-activated (ki) and activated (k) carrier states and the life-times of activated carriers (tau).     

The kinetics of electron transfer between pseudomonas aeruginosa cytochrome c-551 and its oxidase.

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.     

Ionic strength dependence of the kinetics of electron transfer from bovine mitochondrial cytochrome c to bovine cytochrome c oxidase.

The effect of ionic strength on the one-electron reduction of oxidized bovine cytochrome c oxidase by reduced bovine cytochrome c has been studied by using flavin semiquinone reductants generated in situ by laser flash photolysis. In the absence of cytochrome c, direct reduction of the heme a prosthetic group of the oxidase by the one-electron reductant 5-deazariboflavin semiquinone occurred slowly, despite a driving force of approximately +1 V. This is consistent with a sterically inaccessible heme a center. This reduction process was independent of ionic strength from 10 to 100 mM. Addition of cytochrome c resulted in a marked increase in the amount of reduced oxidase generated per laser flash. Reduction of the oxidase at the heme a site was monophasic, whereas oxidation of cytochrome c was multiphasic, the fastest phase corresponding in rate constant to the reduction of the heme a. During the fast kinetic phase, 2 equiv of cytochrome c was oxidized per heme a reduced. We presume that the second equivalent was used to reduce the Cua center, although this was not directly measured. The first-order rate-limiting process which controls electron transfer to the heme a showed a marked ionic strength effect, with a maximum rate constant occurring at mu = 110 mM (1470 s-1), whereas the rate constant obtained at mu = 10 mM was 630 s-1 and at mu = 510 mM was 45 s-1. There was no effect of "pulsing" the enzyme on this rate-limiting one-electron transfer process. These results suggest that there are structural differences in the complex(es) formed between mitochondrial cytochrome c and cytochrome c oxidase at very low and more physiologically relevant ionic strengths, which lead to differences in electron-transfer rate constants.     

Electron transfer between horse heart and Candida krusei cytochromes c in the free and bound states

Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540-560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20 degrees C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2-3).10(4) M-1.s-1 under the same conditions. The transfer rate for the bound form increased with increase in the binding ratio at ratios below half the maximum, and was almost constant at higher ratios up to the maximum. The maximum electron exchange rate was about 2.10(6) M-1.s-1, which is 60-70 times that for the free form at a given concentration of cytochrome c. The activation energy of the reaction for the bound cytochrome c was equal to that for the free form, being about 10 kcal/mol. The dependence of the exchange rate on temperature, cytochrome c concentration and solvent viscosity suggests that enhancement of the electron transfer rate between cytochromes c on binding to phosvitin is due to increase in the collision frequency between cytochromes c concentrated on the phosvitin molecule.     

The modulation of cytochrome c electron self-exchange by site-specific chemical modification and anion binding

The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5-dinitrobenzoic acid (CDNB) increases the electron self-exchange rate of the protein. In the presence of 0.24 M cacodylate (pH* 7.0) the electron self-exchange rate constants, kex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 X 10(3) and 6 X 10(4) M-1 X s-1 for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron self-exchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)3-6 catalyse electron self-exchange. At saturation a limiting value of approximately 1.4 X 10(5) M-1 X s-1 is observed for both anions.     

Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase.

1. A detailed study of cytochrome c oxidase activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome c concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholiptaining aid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD1 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2. The maximal velocities as low ionic strength increased with pH and were highest above ph 7.5. 3. The presence and properties of the low apparent Km phase of the kinetics were strongly dependent on the nature and concentration of the anions in the medium. The multivalent anions, phosphate, ADP, and ATP, greatly decreased the proportion of this phase and similarly decreased the amount of high affinity cytochrome c-cytochrome oxidase complex formed. The order of effectiveness was ATP greater than ADP greater than P1 and since phosphate binds to cytochrome c more strongly than the nucleotides, it is concluded that the inhibition resulted from anion interaction with the oxidase. 4mat low concentrations bakers' yeast iso-1, bakers' yeast iso-1, horse, and Euglena cytochromes c at high concentrations all attained the same maximal velocity. The different proportions of low apparent Km phase in the kinetic patterns of these cytochromes c correlated with the amounts of high affinity complex formed with purified cytochrome c oxidase. 5. The apparent Km for cytochrome c activity in the succinate-cytochrome c reductase system of Keilin-Hartree particles was identical with that obtained with the oxidase (5 x 10(-8) M), suggesting the same site serves both reactions. 6. It is concluded that the observed kinetics result from two catalytically active sites on the cytochrome c oxidase protein of different affinities for cytochrome c. The high affinity binding of cytochrome c to the mitochondrial membrane is provided by the oxidase and at this site cytochrome c can be reduced by cytochrome c1. Physiological concentrations of ATP decrease the affinity of this binding to the point that interaction of cytochrome c with numerous mitochondrial pholpholipid sites can competitively remove cytochrome c from the oxidase. It is suggested that this effect of ATP represents a possible mechanism for the control of electron flow to the oxidase.     

Rate of electron transfer between cytochrome b561 and extravesicular ascorbic acid

Cytochrome b561 transfers electrons across secretory vesicle membranes in order to regenerate intravesicular ascorbic acid. To show that cytosolic ascorbic acid is kinetically competent to function as the external electron donor for this process, electron transfer rates between cytochrome b561 in adrenal medullary chromaffin vesicle membranes and external ascorbate/semidehydroascorbate were measured. The reduction of cytochrome b561 by external ascorbate may be measured by a stopped-flow method. The rate constant is 450 (+/- 190) M-1 s-1 at pH 7.0 and increases slightly with pH. The rate of oxidation of cytochrome b561 by external semidehydroascorbate may be deduced from rates of steady-state electron flow. The rate constant is 1.2 (+/- 0.5) x 10(6) M-1 s-1 at pH 7.0 and decreases strongly with pH. The ratio of the rate constants is consistent with the relative midpoint reduction potentials of cytochrome b561 and ascorbate/semidehydroascorbate. These results suggest that cytosolic ascorbate will reduce cytochrome b561 rapidly enough to keep the cytochrome in a mostly reduced state and maintain the necessary electron flux into vesicles. This supports the concept that cytochrome b561 shuttles electrons from cytosolic ascorbate to intravesicular semidehydroascorbate, thereby ensuring a constant source of reducing equivalents for intravesicular monooxygenases.     

Kinetics of electron transfer between cytochrome c and laccase.

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting Rhus vernicifera laccase as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 125 M-1 s-1 at 25 degrees C in 0.2 M phosphate buffer at pH 6.0 with the activation parameters delta H++ = 16.2 kJ mol-1 and delta S++ = 28.9 J mol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that electron transfer from cytochrome c to laccase is favored by the local electrostatic interaction (ZAZB = -0.9 at pH 6 and -1.3 at pH 4.8) between the basic proteins with positive net charges. From the increase of the rate of electron transfer with decreasing pH, one of the driving forces of the reaction was suggested to be the difference in the redox potentials between the type 1 copper in laccase and the central iron in cytochrome c. Further, on addition of one hexametaphosphate anion per cytochrome c molecule, the rate of the electron transfer was increased, probably because the association of both proteins became more favorable.     

Characterization of electron donation to cytochrome c-555 in Chromatium vinosum from ferrocyanide, tetramethylphenylenediamine and reduced dimethylquinone. Effects of redox potential, pH and salt concentration

1. The dependences of the reduction of ferricytochrome c-555 in the reaction center-cytochrome c complex on the redox potential and pH were investigated using N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), ferrocyanide, and reduced 2,5-dimethyl-p-quinone as electron donors. 2. In the reduction of cytochrome c-555 by TMPD, the unprotonated form was the exclusive electron donor to the cytochrome with a second-order rate constant of 1.0 X 10(5) M-1.s-1. 3. Ferrocyanide reduced cytochrome c-555 slowly with a rate constant of 7.8 X 10(3) M-1.s-1 at infinite salt concentration. The value of -5.2 X 10(-4) elementary charge/A2 was estimated as the surface charge density in the vicinity of cytochrome c-555 by analyzing the salt effect on the cytochrome reduction using the Gouy-Chapman theory. 4. The characteristics of the dependences of the reduction of cytochrome c-555 by reduced 2,5-dimethyl-p-quinone on the redox potential and pH were well explained by the redox potential and pH dependences of the formation of the semiquinone. In the neutral-to-alkaline pH range the anionic semiquinone was the main electron-donating species with a second-order rate constant of 6.0 X 10(7) m-1.s-1.     

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.     

Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5.

The ionic strength dependence of the electron self-exchange rate constants of cytochromes c, c551, and b5 has been analyzed in terms of a monopole-dipole formalism (van Leeuwen, J.W. 1983. Biochim. Biophys. Acta. 743:408-421). The dipole moments of the reduced and oxidized forms of Ps. aeruginosa cytochrome c551 are 190 and 210 D, respectively (calculated from the crystal structure). The projections of these on the vector from the center of mass through the exposed heme edge are 120 and 150 D. For cytochrome b5, the dipole moments calculated from the crystal structure are 500 and 460 D for the reduced and oxidized protein; the projections of these dipole moments through the exposed heme edge are -330 and -280 D. A fit of the ionic strength dependence of the electron self-exchange rate constants gives -280 (reduced) and -250 (oxidized) D for the center of mass to heme edge vector. The self-exchange rate constants extrapolated to infinite ionic strength of cytochrome c, c551, and b5 are 5.1 x 10(5), 2 x 10(7), and 3.7 x 10(5) M-1 s-1, respectively. The extension of the monopole-dipole approach to other cytochrome-cytochrome electron transfer reactions is discussed. The control of electron transfer by the size and shape of the protein is investigated using a model which accounts for the distance of the heme from each of the surface atoms of the protein. These calculations indicate that the difference between the electrostatically corrected self-exchange rate constants of cytochromes c and c551 is due only in part to the different sizes and heme exposures of the two proteins.     

Kinetics of electron transfer between mitochondrial cytochrome c and iron hexacyanides

The reduction of horse and Candida krusei cytochromes c by ferrocyanide has been studied by 1H NMR spectroscopy and the reaction found to involve a precursor complex of ferrocyanide bound to ferricytochrome c (pH* 7.4, 2H2O, I = 0.12, and 25 degrees C). The electron transfer rate constants for the reduction of the two ferricytochromes by associated ferrocyanide were found to be the same at 780 +/- 80 sec-1 but the association constants for binding of ferrocyanide to ferricytochrome c were significantly different: horse, 90 +/- 20 M-1 and Candida, 285 +/- 30 M-1. The different association constants partly accounts for the previously observed reactivity difference between horse and Candida cytochromes c. Comparison of the NMR data with data obtained by other kinetic methods has allowed the electron transfer rate constant for the oxidation of ferrocytochrome c by associated ferricyanide to be determined. This was found to be 4.6 +/- 1 X 10(4) sec-1.     

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles.

1. Beef heart mitochondria have a cytochrome c1:c:aa3 ratio of 0.65:1.0:1.0 as isolated; Keilin-Hartree submitochondrial particles ahve a ratio of 0.65:0.4:1.0. More than 50% of the submitochondrial particle membrane is in the 'inverted' configuration, shielding the catalytically active cytochrome c. The 'endogenous' cytochrome c of particles turns over at a maximal rate between 450 and 550 s-1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300-400 s-1, at 28 degrees-30 degrees C, pH 7.4. 2. Ascorbate plus N,N,N',N'-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c1 and cytochrome c steady states at 552.5-547 nm and 550-556.5 nm, respectively. 3. Cytochrome c1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate + N,N,N',N'-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c1 and c in the aerobic steady state, with a rate constant for the c1 leads to c reduction step greater than 10(3) s-1. 4. The greater apparent response of the c/aa3 electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the c1/c step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa3 units in situ. Cytochrome c1 can either reduce the cytochrome c-cytochrome aa3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.     

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. I. The interaction of cytochrome c2 with the reaction center

1. The kinetics of the interaction of cytochrome c2 and photosynthetic reaction centers purified from Rhodobacter capsulatus were studied in proteoliposomes reconstituted with a mixture of phospholipids simulating the native membrane (i.e. containing 25% L-alpha-phosphatidylglycerol). 2. At low ionic strength, the kinetics of cytochrome-c2 oxidation induced by a single turnover flash was very different, depending on the concentration of cytochrome c2: at concentrations lower than 1 microM, the process was strictly bimolecular (second-order rate constant, k = 1.7 x 10(9) M-1 s-1), while at higher concentrations a fast oxidation process (half-time lower than 20 microseconds) became increasingly dominant and encompassed the total process at a cytochrome c2 concentration around 10 microM. From the concentration dependence of the amplitude of this fast phase an association constant for a reaction-center--cytochrome-c2 complex of about 10(5) M-1 was evaluated. From the fraction of photo-oxidized reaction centers promptly re-reduced in the presence of saturating concentrations of externally added cytochrome c2, it was found that in approximately 60% of the centers the cytochrome-c2 site was exposed to the external compartment. 3. Both the second-order oxidation reaction and the formation of the reaction-center--cytochrome-c2 complex were very sensitive to ionic strength. In the presence of 180 mM KCl, the value of the second-order rate constant was decreased to 7.0 x 10(7) M-1 s-1 and no fast oxidation of cytochrome c2 could be observed at 10 microM cytochrome c2. 4. The kinetics of exchange of oxidized cytochrome c2 bound to the reaction center with the reduced form of the same carrier, following a single turnover flash, was studied in double-flash experiments, varying the dark time between photoactivations over the range 30 microseconds to 5ms. The experimental results were analyzed according to aminimal kinetic model relating the amounts of oxidized cytochrome c2 and reaction centers observable after the second flash to the dark time between flashes. This model included the rate constants for the electron transfer between the primary and secondary ubiquinone acceptors of the complex (k1) and for the exchange of cytochrome c2 (k2). Fitting to the experimental results indicated a value of k1 equal to 2.4 x 10(3) s-1 and a lower limit for k2 of approximately 2 x 10(4) s-1 (corresponding to a second-order rate constant of approximately 3 x 10(9) M-1 s-1).     

Intramolecular electron transfer and binding constants in iron hexacyanide-cytochrome c complexes as studied by pulse radiolysis.

Internal oxidation and reduction rates of horse cytochrome c in the complexes CII . Fe(III)(CN)6(3)- and CIII . Fe(II)(CN)6(4)-, are 4.6 . 10(4)s-1 and 3.3 . 10(2)s-1, respectively. The binding site of the iron hexacyanide ions on either CII or CIII are kinetically almost indistinguishable; binding constants range from 0.87 . 10(3) to 2 . 10(3)M-1. The present pulse radiolytic kinetic data is compared with that from NMR, T-jump and equilibrium dialysis studies.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Internal electron transfer in cytochrome c oxidase: evidence for a rapid equilibrium between cytochrome a and the bimetallic site.

Internal electron-transfer reactions in cytochrome oxidase following flash photolysis of the CO compounds of the enzyme reduced to different degrees (2-4 electron equiv) have been followed at 445, 605, and 830 nm. Apart from CO dissociation and recombination, two kinetic phases are seen both at 445 and at 605 nm with rate constants of 2 x 10(5) and 1.3 x 10(4) s-1, respectively; at 605 nm, an additional phase with a rate constant of 400 s-1 is resolved. At 830 nm, only the second reaction phase (rate constant of 1.3 x 10(4) s-1) is observed. The amplitude of the first phase is largest with the two-electron-reduced enzyme, whereas that of the second phase is maximal at the three-electron-reduction level. Neither phase shows any marked pH dependence. The reaction in the first phase has a free energy of activation of 41 kJ mol-1 and an entropy of activation of -14 JK-1 mol-1. Analysis suggests that the two rapid reaction phases represent internal electron redistributions between the bimetallic site and cytochrome a, and between cytochrome a and CuA, respectively. The slow phase (400 s-1) probably involves a structural rearrangement.     

Genetic engineering of redox donor sites: measurement of intracomplex electron transfer between ruthenium-65-cytochrome b5 and cytochrome c.

The de novo design and synthesis of ruthenium-labeled cytochrome b5 that is optimized for the measurement of intracomplex electron transfer to cytochrome c are described. A single cysteine was substituted for Thr-65 of rat liver cytochrome b5 by recombinant DNA techniques [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. The single sulfhydryl group on T65C cytochrome b5 was then labeled with [4-(bromomethyl)-4'-methylbipyridine] (bisbipyridine)ruthenium2+ to form Ru-65-cyt b5. The ruthenium group at Cys-65 is only 12 A from the heme group of cytochrome b5 but is not located at the binding site for cytochrome c. Laser excitation of the complex between Ru-65-cyt b5 and cytochrome c results in electron transfer from the excited state Ru(II*) to the heme group of Ru-65-cyt b5 with a rate constant greater than 10(6) s-1. Subsequent electron transfer from the heme group of Ru-65-cyt b5 to the heme group of cytochrome c is biphasic, with a fast-phase rate constant of (4 +/- 1) x 10(5) s-1 and a slow-phase rate constant of (3 +/- 1) x 10(4) s-1. This suggests that the complex can assume two different conformations with different electron-transfer properties. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased, indicating dissociation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)