Rates and Equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase

Intramolecular electron transfer between CuA and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the CuA center and the concomitant reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration and its degree of reduction, demonstrating that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse heme a --> CuA process were found to be 20,400 s(-1) and 10,030 s(-1), respectively, at 25 degrees C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme's known three-dimensional structure, potential ET pathways, and the calculated reorganization energies.     

A new ruthenium complex to study single-electron reduction of the pulsed O(H) state of detergent-solubilized cytochrome oxidase

The first step in the catalytic cycle of cytochrome oxidase, the one-electron reduction of the fully oxidized enzyme, was investigated using a new photoactive binuclear ruthenium complex, [Ru(bipyrazine)2]2(quaterpyridine), (Ru2Z). The aim of the work was to examine differences in the redox kinetics resulting from pulsing the oxidase (i.e., fully reducing the enzyme followed by reoxidation) just prior to photoreduction. Recent reports indicate transient changes in the redox behavior of the metal centers upon pulsing. The new photoreductant has a large quantum yield, allowing the kinetics data to be acquired in a single flash. The net charge of +4 on Ru2Z allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. The photoexcited state Ru(II*) of Ru2Z is reduced to Ru(I) by the sacrificial electron donor aniline, and Ru(I) then reduces CuA with yields up to 60%. A stopped-flow-flash technique was used to form the pulsed state of cytochrome oxidase (the "OH" state) from several sources (bovine heart mitochondria, Rhodobacter sphaeroides, and Paracoccus denitrificans). Upon mixing the fully reduced anaerobic enzyme with oxygenated buffer containing Ru2Z, the oxidized OH state was formed within 5 ms. Ru2Z was then excited with a laser flash to inject one electron into CuA. Electron transfer from CuA --> heme a --> heme a3/CuB was monitored by optical spectroscopy, and the results were compared with the enzyme that had not been pulsed to the OH state. Pulsing had a significant effect in the case of the bovine oxidase, but this was not observed with the bacterial oxidases. Electron transfer from CuA to heme a occurred with a rate constant of 20,000 s-1 with the bovine cytochrome oxidase, regardless of whether the enzyme had been pulsed. However, electron transfer from heme a to the heme a3/CuB center in the pulsed form was 63% complete and occurred with biphasic kinetics with rate constants of 750 s-1 and 110 s-1 and relative amplitudes of 25% and 75%. In contrast, one-electron injection into the nonpulsed O form of the bovine oxidase was only 30% complete and occurred with monophasic kinetics with a rate constant of 90 s-1. This is the first indication of a difference between the fast form of the bovine oxidase and the pulsed OH form. No reduction of heme a3 is observed, indicating that CuB is the initial electron acceptor in the one-electron reduced pulsed bovine oxidase.     

Electron transfer process in cytochrome oxidase after pulse radiolysis

The reduction of bovine heart cytochrome oxidase by the 1-methylnicotinamide (MNA) radical was investigated by the use of pulse radiolysis. With the decay of the MNA radical, the absorption at 445 and 605 nm, a characteristic to ferrous heme a of the oxidase, increased. The kinetic difference spectrum obtained was similar to that of the fully reduced minus the fully oxidized form of the oxidase, and was not different from that obtained in the reaction of the MNA radical with the mixed valence CO complex of the oxidase, where heme a3 is the CO-bound reduced form with heme a oxidized. This suggests that the absorption changes at 445 and 605 nm arise from the reduction of heme a, not heme a3. In order to elucidate the contribution of "visible" copper in this reaction, the absorption of the oxidase in the near-infrared region was measured. A decrease of the 830 nm band due to the reduction of visible copper was detected with a half-life of 5 microseconds. This absorption change obeyed pseudo-first order kinetics and its rate constant increased with the concentration of the oxidase. This suggests that the absorption change at 830 nm is followed by a bimolecular reaction of the MNA radical with visible copper of the oxidase. After the first phase of the reduction, the return of the 830 nm band corresponding to oxidation of the copper was observed with a half-life of 100 microseconds. Concomitantly, the absorption at 605 and 445 nm due to the reduction of heme a increased. The rates of oxidation of the copper were identical to those of the reduction of heme a and independent of the oxidase concentration. This suggests that the MNA radical reacts with visible copper of the oxidase with a second order rate constant of 1.5 X 10(9) m-1 s-1 and subsequently the electron flows to heme a by intramolecular electron migration with a first order rate constant of 1.8 X 10(4) s-1. An activation energy of the intramolecular electron transfer was calculated to be 2.8 kcal/mol in the range 4-33 degrees C.     

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.     

Reaction of dioxygen with cytochrome c oxidase reduced to different degrees: indications of a transient dioxygen complex with copper-B.

The reactions of the fully reduced, three-electron-reduced, and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, starting from the CO complexes, at 445 and 830 nm at pH 7.4 and 25 degrees C. With the fully reduced and the three-electron-reduced enzyme, four kinetic phases with rate constants in the range from 1 x 10(5) to 10(3) s-1 can be observed. The initial fast phase is associated with an absorbance increase at 830 nm. This is followed by an absorbance decrease (2.8 x 10(4) s-1), the amplitude of which increases with the degree of reduction of the oxidase. The third phase (6 x 10(3) s-1) displays the largest absorbance change at both wavelengths in the fully reduced enzyme and is not seen in the mixed-valence oxidase at 830 nm; a change with opposite sign but with a similar rate constant is found at 445 nm in this enzyme form. The slowest phase (10(3) s-1) is also largest in the fully reduced oxidase and not seen in the mixed-valence enzyme. It is suggested that O2 initially binds to reduced CuB and is then transferred to cytochrome a3 before electron transfer from cytochrome a/CuA takes place. The fast oxidation of cytochrome a seen with the fully reduced enzyme is suggested not to occur during natural turnover. A reaction cycle for the complete turnover of the enzyme is presented. In this cycle, the oxidase oscillates between electron input and output states of the proton pump, characterized by cytochrome a having a high and a low reduction potential, respectively.     

Electron transfer among the CuA-, heme b- and a3-centers of Thermus thermophilus cytochrome ba3

The 1-methyl-nicotinamide radical (MNA(*)), produced by pulse radiolysis has previously been shown to reduce the Cu(A)-site of cytochromes aa(3), a process followed by intramolecular electron transfer (ET) to the heme a but not to the heme a(3) [Farver, O., Grell, E., Ludwig, B., Michel, H. and Pecht, I. (2006) Rates and equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase. Biophys. J. 90, 2131-2137]. Investigating this process in the cytochrome ba(3) of Thermus thermophilus (Tt), we now show that MNA(*) also reduces Cu(A) with a subsequent ET to the heme b and then to heme a(3), with first-order rate constants 11200 s(-1), and 770 s(-1), respectively. The results provide clear evidence for ET among the three spectroscopically distinguishable centers and indicate that the binuclear a(3)-Cu(B) center can be reduced in molecules containing a single reduction equivalent.     

Chicken liver sulfite oxidase. Kinetics of reduction by laser-photoreduced flavins and intramolecular electron transfer

Laser flash photolysis was used to study the reaction of photoproduced 5-deazariboflavin (dRFH.), lumiflavin (LFH.), and riboflavin (RFH.) semiquinone radicals with the redox centers of purified chicken liver sulfite oxidase. Kinetic studies of the native enzyme with dRFH. yielded a second-order rate constant of 4.0 X 10(8) M-1 s-1 for direct reduction of the heme and a first-order rate constant of 310 s-1 for intramolecular electron transfer from the Mo center to the heme. The reaction with LFH. gave a second-order rate constant of 2.9 X 10(7) M-1 s-1 for heme reduction. Reoxidation of the reduced heme due to intramolecular electron transfer to the Mo center gave a first-order rate constant of 155 s-1. The direction of intramolecular electron transfer using dRFH. and LFH. was independent of the buffer used for the experiment. The different first-order rate constants observed for intramolecular electron transfer using dRFH. and LFH. are proposed to result from chemical differences at the Mo site. Flash photolysis studies with cyanide-inactivated sulfite oxidase using dRFH. and LFH. resulted in second-order reduction of the heme center with rate constants identical with those obtained with the native enzyme, whereas the first-order intramolecular electron-transfer processes seen with the native enzyme were absent. The isolated heme peptide of sulfite oxidase gave only second-order kinetics upon laser photolysis and confirmed that the first-order processes observed with the native enzyme involve the Mo site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser flash photolysis studies of the kinetics of electron-transfer reactions of Saccharomyces flavocytochrome b2: evidence for conformational gating of intramolecular electron transfer induced by pyruvate binding

The kinetics of reduction of the flavocytochrome from Saccharomyces cerevisiae by exogenous deazaflavin semiquinones have been investigated by using laser flash photolysis. Direct reduction by deazaflavin semiquinone of both the b2 heme and the FMN cofactor occurred via second-order kinetics with similar rate constants (9 x 10(8) M-1 s-1). A slower, monoexponential, phase of FMN reoxidation was also observed, concurrent with a slow phase of heme reduction. The latter accounted for approximately 20-25% of the total heme absorbance change. Both of these slow phases were protein concentration dependent, yielding identical second-order rate constants (1.1 x 10(7) M-1 s-1), and were interpreted as resulting from intermolecular electron transfer from the FMN semiquinone on one protein molecule to an oxidized heme on a second molecule. Consistent with this conclusion, no slow phase of heme reduction was observed with deflavo-flavocytochrome b2. Upon the addition of pyruvate (but not D-lactate or oxalate), the second-order rate constant for heme reduction was unaffected, but direct reduction of the FMN cofactor was no longer observed. Reduction of the heme cofactor was followed by a slower partial reoxidation, which occurred concomitantly with a monoexponential phase of FMN reduction. Both processes were protein concentration independent and were interpreted as the result of intramolecular electron transfer from reduced b2 heme to oxidized FMN. Potentiometric titrations of the flavocytochrome in the absence and presence of pyruvate demonstrated that the thermodynamic driving force for electron transfer from FMN to heme is much greater in the absence of pyruvate. Despite this, intramolecular electron transfer was only observed in the presence of pyruvate. This result is interpreted in terms of a conformational change induced by pyruvate binding which permits electron transfer between the cofactors. The rate constant for intramolecular electron transfer in the presence of pyruvate was dependent on ionic strength, suggesting the occurrence of electrostatic effects which influence this process.     

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen.

The reaction of the electrostatic cytochrome c-cytochrome oxidase complex with oxygen is measured by transient absorption spectroscopy. The oxygen reaction is initiated by photolytic removal of CO from cytochrome oxidase, using a flash-pumped dye laser. The subsequent reaction of the cytochrome c-cytochrome oxidase complex with oxygen is reported at 550, 605, 744, and 830 nm at different cytochrome c:cytochrome oxidase ratios and different oxygen concentrations. In the absence of cytochrome c the time course of the reaction of the oxidase is well described by a triple exponential process at any of the measured wavelengths. The three processes are well resolved at high O2 levels (i.e. greater than 200 microM), where they reach first-order rate limits of 2.4 x 10(4), 7.5 x 10(3), and 650 s-1. When cytochrome c is added the oxidation of cytochrome a and one of the redox active cooper centers (CuA) are interrupted. The maximal effect of cytochrome c on the oxidation of the oxidase occurs at a c:aa3 ratio of 1. Cytochrome c reacts in a biphasic process with rates of up to 7 x 10(3) and 550 s-1 at high oxygen. The fast phase takes up 60% of the process, and this is independent of the cytochrome c:cytochrome oxidase ratio. The results are discussed in the context of a model in which electron entry into cytochrome oxidase from cytochrome c is via CuA, and cytochrome a functions to mediate electron transfer from CuA to the oxygen binding site. The role of CuA as initial electron acceptor in cytochrome c oxidase is related to its physical proximity to cytochrome c is the cytochrome c-cytochrome oxidase complex.     

Reduction of cytochrome oxidase by 5,10-dihydro-5-methylphenazine: kinetic parameters from rapid-scanning stopped-flow experiments

The kinetics of the reduction of resting cytochrome oxidase and of its cyanide complex by 5,10-dihydro-5- methylphenazine (MPH) have been characterized by rapid-scan and fixed-wavelength stopped-flow spectrophotometry in the Soret, visible, and near-IR spectral regions. In this study, we focused on a form of the resting enzyme that is characterized by a Soret absorption maximum at 424 nm. These experiments complement earlier work on the reduction of a 418 nm absorbing form of the resting enzyme [ Halaka , F.G., Babcock , G. T., & Dye, J. L. (1981) J. Biol. Chem. 256, 1084-1087]. The reduction of cytochrome a is accomplished in a second-order reaction with a rate constant of 3 X 10(5) M-1 s-1. The reduction of the 830-nm absorber, Cua, is closely coupled to but lags the reduction of cytochrome a; we have resolved a rate constant of about 20 s-1 for the copper reduction. The reduction of cytochrome a proceeds with a rate constant that is nearly independent of the spectral properties of the resting enzyme and of the ligation state of cytochrome a3. The reduction of cytochrome a3 occurs by slow, intramolecular electron transfer. We have resolved two phases for this process that have rate constants of approximately 0.2 s-1 and approximately 0.02 s-1 for both the 418- and 424-nm forms of the resting enzyme. It appears, therefore, that spectroscopic heterogeneity at the cytochrome a3 site in the resting enzyme exerts very little influence on the kinetics of the anaerobic reduction of the oxidase metal centers. From this we conclude that the rate of electron transfer to the a3 site is probably controlled by the protein conformation and not primarily by local factors within the a3 environment.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a2+ a23+-CO) complex and the mixed-valence carboxycytochrome c oxidase (a3+ a23+-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a23+-CO complex. This would confirm that the spectral properties of cytochrome a3 are not affected by the redox state of cytochrome a and CuA. When the absorbance changes after photolysis of cytochrome a23+-CO with a laser flash were followed on a time scale from 1 mus to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7 X 10(3) s-1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a3. Concomitantly, a decrease of the 830 nm band was observed due to reduction of CuA. This demonstrates that in the partially reduced enzyme a pathway is present between CuA and the cytochrome a3-CuB pair, via which electrons are transferred rapidly.     

Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing.

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

Electrochemical and kinetic analysis of electron-transfer reactions of Chlorella nitrate reductase.

Assimilatory nitrate reductase (NR) from Chlorella is homotetrameric, each subunit containing FAD, heme, and Mo-pterin in a 1:1:1 stoichiometry. Measurements of NR activity and steady-state reduction of the heme component under conditions of NADH limitation or competitive inhibition by nitrite suggested intramolecular electron transfer between heme and Mo-pterin was a rate-limiting step and provided evidence that heme is an obligate intermediate in the transfer of electrons between FAD and Mo-pterin. In addition to the physiological substrates NADH and nitrate, various redox mediators undergo reactions with one or more of the prosthetic groups. These reactions are coupled by NR to NADH oxidation or nitrate reduction. To test whether intramolecular redox reactions of NR were rate-determining, rate constants for redox reactions between NR and several chemically diverse mediators were measured by cyclic voltammetry in the presence of NADH or nitrate. Reduction of ferrocenecarboxylic acid, dichlorophenolindophenol, and cytochrome c by NADH-reduced NR was coupled to reoxidation at a glassy carbon electrode (ferrocene and dichlorophenolindophenol) or at a bis(4-pyridyl) disulfide modified gold electrode (cytochrome c), yielding rate constants of 10.5 x 10(6), 1.7 x 10(6), and 2.7 x 10(6) M-1 s-1, respectively, at pH 7. Kinetics were consistent with a second-order reaction, implying that intramolecular heme reduction by NADH and endogenous FAD was not limiting. In contrast, reduction of methyl viologen and diquat at a glassy carbon electrode, coupled to oxidation by NR and nitrate, yielded similar kinetics for the two dyes. In both cases, second-order kinetics were not obeyed, and reoxidation of dye-reduced Mo-pterin of NR by nitrate became limiting at low scan rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of electron gating in proton pumping cytochrome c oxidase: the effect of pH and temperature on internal electron transfer

Electron-transfer reactions following flash photolysis of the mixed-valence cytochrome oxidase-CO complex have been measured at 445, 598 and 830 nm between pH 5.2 and 9.0 in the temperature range of 0-25 degrees C. There is a rapid electron transfer from the cytochrome a3-CuB pair to CuA (time constant: 14200 s-1), which is followed by a slower electron transfer to cytochrome a. Both the rate and the amplitude of the rapid phase are independent of pH, and the rate in the direction from CuA to cytochrome a3-CuB is practically independent of temperature. The second phase depends strongly on pH due to the titration of an acid-base group with pKa = 7.6. The equilibrium at pH 7.4 corresponds to reduction potentials of 225 and 345 mV for cytochrome a and a3, respectively, from which it is concluded that the enzyme is in a different conformation compared to the fully oxidized form. The results have been used to suggest a series of reaction steps in a cycle of the oxidase as a proton pump. Application of the electron-transfer theory to the temperature-dependence data suggests a mechanism for electron gating in the pump. Reduction of both cytochrome a and CuA leads to a conformational change, which changes the structure of cytochrome a3-CuB in such a way that the reorganizational barrier for electron transfer is removed and the driving force is increased.     

Steady-state redox behavior of cytochrome c, cytochrome a, and CuA of cytochrome c oxidase in intact rat liver mitochondria.

We have examined the steady-state redox behavior of cytochrome c (Fec), Fea, and CuA of cytochrome c oxidase during steady-state turnover in intact rat liver mitochondria under coupled and uncoupled conditions. Ascorbate was used as the reductant and TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine) as the redox mediator. After elimination of spectroscopic interference from the oxidized form of TMPD, we found that Fea remains significantly more oxidized than previously thought. During coupled turnover, CuA always appears to be close to redox equilibrium with Fec. By increasing the amount of TMPD, both centers can be driven to fairly high levels of reduction while Fea remains relatively oxidized. The reduction level at Fea is close to a linear function of the enzyme turnover rate, but the levels at Fec and CuA do not keep pace with enzyme turnover. This behavior can be explained in terms of a redox equilibrium among Fec, CuA, and Fea, where Fea is the electron donor to the oxygen reduction site, but only if Fea has an effective Em (redox midpoint potential) of 195 mV. This is too low to be accounted for on the basis of nonturnover measurements and the effects of the membrane potential. However, if there is no equilibrium, the internal CuA----Fea electron-transfer rate constant must be slow in the time average (about 200 s-1). Other factors which might contribute to such a low Em are discussed. In the presence of uncoupler, this situation changes dramatically. Both Fec and CuA are much less reduced; within the resolution of our measurements (about 10%), we were unable to measure any reduction of CuA. Fea and CuA remain too oxidized to be in redox equilibrium with Fec during steady-state turnover. Furthermore, our results indicate that, in the uncoupled system, the (time-averaged) internal electron-transfer rate constants in cytochrome oxidase must be of the order of 2500 s-1 or higher. When turnover is slowed by azide, the relative redox levels at Fea and Fec are much closer to those predicted from nonturnover measurements. In presence of uncouplers, Fea is always more reduced than Fec, but in the absence of uncouplers, the two centers track together. Unlike the uninhibited, coupled system, the redox behavior here is consistent with the known effect of the electrical membrane potential on electron distribution in the enzyme. Interestingly, in these circumstances (azide and uncoupler present), Fea behaves as if it were no longer the kinetically controlling electron donor to the bimetallic center.     

Photoinduced electron-transfer kinetics of singly labeled ruthenium bis(bipyridine) dicarboxybipyridine cytochrome c derivatives

Cytochrome c derivatives labeled at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine [RuII(bpy)2(dcbpy)] were prepared by using the procedure described previously [Pan, L. P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Four additional singly labeled derivatives were purified, bringing the total number to 10. These derivatives have a strong luminescence emission centered at 662 nm arising from the excited state, RuII*. Transient absorption spectroscopy was used to directly measure the rate constants for the photoinduced electron-transfer reaction from RuII* to the ferric heme group (k1) and for the thermal back-reaction from the ferrous heme group to RuIII (k2). The rate constants were found to be k1 = 14 X 10(6) s-1 and k2 = 24 X 10(6) s-1 for the derivative modified at lysine 72, which has a distance of 8-16 A between the ruthenium and heme groups. Similar rate constants were found for the derivatives modified at lysines 13 and 27, which have distances of 6-12 A separating the ruthenium and heme groups. The rate constants were significantly slower for the derivatives modified at lysine 25 (k1 = 1 X 10(6) s-1, k2 = 1.5 X 10(6) s-1) and lysine 7 (k1 = 0.3 X 10(6) s-1, k2 = 0.5 X 10(6) s-1), which have distances of 9-16 A. Transients due to photoinduced electron transfer could not be detected for the remaining derivatives, which have larger distances between the ruthenium and heme groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational change accompanies redox reactions of the tetraheme cytochrome c-554 of Nitrosomonas europaea

Cytochrome c-554 of the ammonia-oxidizing chemolithoautotropic bacteria is thought to mediate electron transfer from hydroxylamine oxidoreductase to a terminal oxidase and/or to ammonia monooxygenase. The cytochrome has four c hemes which interact magnetically and have the same redox potential. We report that the kinetics of reduction of ferric cytochrome c-554 by dithionite or the oxidation of ferrous cytochrome c-554 by O2 or H2O2 are complex and multiphasic. Transient rapid-scan difference spectra indicate discrete maxima at approximately 418 nm, 425 nm and 432 nm. Absorbance changes at all three difference maxima appear to occur in all kinetic phases, although not in equal amounts for each wavelength. Reduction by 20 mM dithionite was biphasic. At pH 7.5 the first phase, which involved approximately 50% of the total absorbance change, had a rate constant (20 degrees C) of 140 s-1 and energy of activation of 20 kJ X mol-1. The slow phase had a rate constant 0.43 s-1 and a relatively high energy of activation, 87 kJ X mol-1, suggesting that a change in protein configuration accompanied the reaction. As the pH of the solution increased, the rate constant for both phases decreased and the fraction of absorbance change in the rapid phase increased. Oxidation of ferrous cytochrome c-554 by O2 involved a discrete rapid phase with a rate constant of 14 s-1, accounting for 6% of the absorbance. The remainder of the reaction was multiphasic with rate constants in the range 0.1-0.01 s-1. With H2O2 as the oxidant, the rapid phase involved 39% of the change in absorbance with a rate constant of 19 s-1. The remainder of the reoxidation was multiphasic with rate constants ranging over 0.4-0.01 s-1.     

Electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli revealed by pulse radiolysis

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b558, b595, and d as the redox metal centers. Taking advantage of spectroscopic properties of three hemes which exhibit distinct absorption peaks, we investigated electron transfer within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in mainly an oxygenated form and partially an oxoferryl and a ferric low-spin forms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b558 and b595 with a second-order rate constant of 3 x 10(8) M-1 s-1, suggesting that a rapid equilibrium occurs for electron transfer between two b-type hemes long before 10 micros. In the slower phase, an intramolecular electron transfer from heme b to the oxoferryl and the ferric heme d occurred with the first-order rate constant of 4.2-5.6 x 10(2) s-1. In contrast, the oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated that the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the ferric and oxoferryl forms of heme d to the ferrous and ferric forms, respectively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s-1, respectively.     

The reaction of fully reduced cytochrome c oxidase with oxygen studied by flow-flash spectrophotometry at room temperature. Evidence for new pathways of electron transfer.

Absorption changes during the O2 reaction of reduced bovine cytochrome c oxidase were investigated by the rapid-reaction technique of flow-flash spectrophotometry in the Soret, visible and near-i.r. spectral regions. New features in the time courses of absorption change were observed relative to the earlier findings reported by Greenwood & Gibson [(1967) J. Biol. Chem. 242, 1782-1787]. These new features arise in the Soret and near-i.r. regions and allow the reaction to be described at all wavelengths as a composite of three exponential processes. There is a rapid O2-sensitive phase detectable in the Soret and visible region. The second phase has a rate that is somewhat less dependent on O2 concentration than is the fastest phase rate and is detectable in all three spectral regions. The rate of the third phase is almost independent of the O2 concentration and is also detectable in all spectral regions. Analysis of the three phases gives their rates and absorption amplitudes. The fast phase reaches a rate of 2.5 X 10(4) s-1 at the highest O2 concentration available at 20 degrees C, whereas the phase of intermediate rate is limited at a value of 7 X 10(3) s-1 and the slow phase rate is limited at 700 s-1. The ratios of the kinetic difference spectra for the fast phase and the slow phase do not correspond to the spectra of the individual haem centres. A branched mechanism is advanced that is able to reconcile the kinetic and static difference spectra. This mechanism suggests that some of the cytochrome a is oxidized along with cytochrome a3 in the initial O2-sensitive phase. In addition, the model requires that CuA is oxidized heterogeneously. This fits with the complex time course of oxidation observed at 830 nm while retaining CuA as virtually the sole contributor to absorbance at this wavelength.     

Single electron reduction of 'slow' and 'fast' cytochrome-c oxidase.

Evidence is presented that single electron reduction is sufficient for rapid electron transfer (k greater than 20 s-1 at pH 8.0 in 0.43 M potassium EDTA) between haem a/CuA and the binuclear centre in 'fast' oxidase, whereas in 'slow' oxidase intramolecular electron transfer is slow even when both CuA and haem a are reduced (k congruent to 0.01 s-1). However, while a single electron can equilibrate rapidly between CuA, haem a and CuB in 'fast' oxidase, it seems that equilibration with haem a3 is relatively slow (k congruent to 2 s-1). Electron transfer between cytochrome c and CuA/haem a is similar for both types of enzyme (k congruent to 2.4 x 10(5) M-1.s-1).