l-Lactate cytochrome c reductase: Rapid kinetic studies of electron transfers within the flavocytochrome b2-cytochrome c assembly

Janot, Jean‐Marc; Capeillère‐Blandin, Chantal; Labeyrie, Françoise

doi:10.1016/0005-2728(90)90055-9

Cited by 15 publications

(14 citation statements)

References 15 publications

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“…According to the reaction scheme given above (see introduction), this would correspond to blockage of reaction 4. Consistent with this argument, Janot et al 1990) reported a slow reduction (~5 s-1) of a transiently oxidized cytochrome bi by the FMN in the flavocytochrome from Hansenula, which had been prepared by reduction of the enzyme in the presence of saturating concentrations of pyruvate. By this procedure, Janot et al produced an enzyme in which the FMN would be mainly in the semiquinone state.…”

Section: Discussionmentioning

confidence: 68%

“…In the case of ascorbate oxidase, it has also been shown that intramolecular electron transfer between the copper centers can increase from a value of ~150 s-1 for the one-electron reduced enzyme to 9500 s-1 in the twoelectron reduced species (Hazzard et al, 1994). For the flavocytochrome b2 from H. anómala, it has been proposed that there is a 4-fold decrease in the rate constant for the fully reduced FMN to heme electron transfer, relative to the reaction involving the FMN semiquinone (Janot et al, 1990), based on rapid mixing of oxidized cytochrome c with fully or partially reduced flavocytochrome b2.…”

Section: Discussionmentioning

confidence: 99%

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Intramolecular Electron Transfer in Yeast Flavocytochrome b2 upon One-Electron Photooxidation of the Fully Reduced Enzyme: Evidence for Redox State Control of Heme-Flavin Communication

Hazzard¹,

McDonough²,

Tollin³

1994

Biochemistry

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Flavocytochrome b2, which has been fully reduced using L-lactate, can be rapidly oxidized by 1 equiv using the laser-generated triplet state of 5-deazariboflavin. Parallel photoinduced oxidation occurs at the reduced heme and at the fully reduced FMN (FMNH2) prosthetic groups of different enzyme monomers, producing the anion semiquinone of FMN and a ferric heme. Following the initial oxidation reaction, rapid intramolecular reduction of the ferric heme occurs with concomitant oxidation of FMNH2, generating the neutral FMN semiquinone. The observed rate constant for this intramolecular electron transfer is 2200 s-1, which is 1 order of magnitude larger than the turnover number under these conditions. A slower reduction of the heme prosthetic group also occurs with an observed rate constant of approximately 10 s-1, perhaps due to intersubunit electron transfer from reduced FMN to heme. The rapid intramolecular electron transfer between the FMNH2 and ferric heme is eliminated upon addition of excess pyruvate (Ki = 3.8 mM). This latter result indicates that pyruvate inhibition of catalytic turnover apparently can occur at the FMNH2-->heme electron transfer step. These results markedly differ from those previously obtained (Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555) and confirmed here for electron transfer within the one-electron reduced enzyme and for the effect of pyruvate binding, suggesting that intramolecular communication between the heme and flavin prosthetic groups can be controlled by the redox state of the enzyme and by ligand binding to the active site.

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Intramolecular Electron Transfer in Yeast Flavocytochrome b2 upon One-Electron Photooxidation of the Fully Reduced Enzyme: Evidence for Redox State Control of Heme-Flavin Communication

Hazzard¹,

McDonough²,

Tollin³

1994

Biochemistry

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“…The conclusions presented above are also consistent with other previous experimental results. Reoxidation of fully reduced flavocytochrome b2 by cytochrome c has been examined using stopped-flow techniques in an effort to measure the rate constant for intramolecular electron transfer between the FMN and heme cofactors (Janot et al, 1990). During this study, biphasic reduction of cytochrome c by flavocytochrome b2 was obtained (a third, much slower phase was also observed which was ascribed to the turnover of enzyme when in the presence of L-lactate).…”

Section: Fox-hredmentioning

confidence: 99%

Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding

Walker

Tollin²

1992

Biochemistry

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Intramolecular electron transfer between the heme and flavin cofactors of flavocytochrome b2 is an obligatory step during the enzymatic oxidation of L-lactate and subsequent reduction of cytochrome c. Previous kinetic studies using both steady-state and transient methods have suggested that such intramolecular electron transfer is inhibited when pyruvate, the two-electron oxidation product of L-lactate, is bound at the active site of Hansenula anomala flavocytochrome b2. In contrast to this, we have recently demonstrated using laser flash photolysis that intramolecular electron transfer could be observed in the flavocytochrome b2 from Saccharomyces cerevisiae only when pyruvate was present [Walker, M., & Tollin, G. (1991) Biochemistry 30, 5546-5555], despite a large thermodynamic driving force of 100 mV and apparently favorable cofactor geometry as indicated by crystallographic studies. In the present study, we have utilized laser flash photolysis to investigate intramolecular electron transfer in the flavocytochrome b2 from H. anomala in an effort to address these apparently conflicting interpretations with respect to the influence of pyruvate on enzyme properties. The results obtained are closely comparable to those we reported using the protein from Saccharomyces. Thus, in the absence of pyruvate, bimolecular reduction of both the heme and FMN cofactors by deazaflavin semiquinone occurs (k approximately 10(9) M-1 s-1), followed by a protein concentration dependent intermolecular electron transfer from the semiquinone form of the FMN cofactor to the heme (k approximately 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The resulting complex behaved as a dead-end complex in steadystate kinetics [26]. The reactivity of pyruvate-liganded Fsq species towards Ho in H-enzyme was determined to be reduced to 5s-1, instead of 200 s-1, for non-liganded F5q species by stopped-flow measurements at 5°C [36]. These results provide an explanation for the steady-state plateau reached by Fsq and continued over 200ms, when two electrons per protomer have been delivered to the enzyme [14].…”

Section: Slow Final Reduction Stages Of Flavocytochrome B2mentioning

confidence: 99%

Electron-transfer steps involved in the reactivity of Hansenula anomala flavocytochrome b2 as deduced from deuterium isotope effects and simulation studies

Capeillère‐Blandin

1991

Biochemical Journal

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The L-lactate-flavocytochrome b2-ferricyanide electron-transfer system from the yeast Hansenula anomala was investigated by rapid-reaction techniques. The kinetics of reduction of oxidized flavocytochrome b2 by L-lactate and L-[2H]lactate were biphasic both for flavin and haem prosthetic groups and at all concentrations tested. The first-order rate constants of the rapid and slow phases depended upon substrate concentrations, a saturation behaviour being exhibited. Substitution of the C alpha-H atom by 2H was found to cause appreciable changes in the rate constants for the initial reduction of flavin and haem (phase I), which were respectively about 3-fold and 2-fold less than with L-lactate. In contrast, no significant isotope effect was noted on the apparent reduction rate constants of the slow phase, phase II. Under steady-state conditions an isotope effect of 2.0 was found on the overall electron transfer from L-lactate to ferricyanide. These transient reduction results were discussed in terms of a kinetic model implying intra- and inter-protomer electron exchanges between flavin and haem b2, all of which have been experimentally described. Computer simulations indicate that the reaction scheme provides a reasonable explanation of the fast-reduction phase, phase I (in absence of acceptor). The pseudo-first-order rate constant for oxidation of reduced haem b2 in flavocytochrome b2 increased with increasing ferricyanide concentration in a hyperbolic fashion. The limiting value at infinite ferricyanide concentration, which was attributed to the intramolecular electron-transfer rate from ferroflavocytochrome b2 to the iron of ferricyanide within a complex, was 920 +/- 50 s-1 at pH 7.0 and 5 degrees C. Stopped-flow and rapid-freezing measurements showed haem b2 and flavin to be 90 and 44% oxidized respectively under steady-state conditions in presence of ferricyanide. Simulation studies were carried out to check the participation of the proposed reduction sequence in the overall catalytic reaction together with the role of reduced haem b2 (Hr) and flavin semiquinone (Fsq) as electron donors to ferricyanide. When the rate of the intramolecular electron-transfer exchange between Fsq and ferricyanide was adjusted to 200 s-1, simulated data accounted for molar activities defined under various conditions of L-lactate, [2H]lactate and ferricyanide concentrations. Simulation studies were extended to data obtained using cytochrome c as acceptor and reaction catalysed by Saccharomyces cerevisiae flavocytochrome b2. The differences in reactivity observed for Hr and Fsq with ferricyanide and cytochrome c were discussed in terms of redox potentials, electrostatic interactions, distances and accessibility of the participating groups.

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l-Lactate cytochrome c reductase: Rapid kinetic studies of electron transfers within the flavocytochrome b2-cytochrome c assembly

Cited by 15 publications

References 15 publications

Intramolecular Electron Transfer in Yeast Flavocytochrome b2 upon One-Electron Photooxidation of the Fully Reduced Enzyme: Evidence for Redox State Control of Heme-Flavin Communication

Intramolecular Electron Transfer in Yeast Flavocytochrome b2 upon One-Electron Photooxidation of the Fully Reduced Enzyme: Evidence for Redox State Control of Heme-Flavin Communication

Laser flash photolysis study of the kinetics of electron transfer reactions of flavocytochrome b2 from Hansenula anomala: further evidence for intramolecular electron transfer mediated by ligand binding

Electron-transfer steps involved in the reactivity of Hansenula anomala flavocytochrome b2 as deduced from deuterium isotope effects and simulation studies

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