Inhibition of L‐lactate: cytochrome‐c reductase (flavocytochrome b2) by product binding to the semiquinone transient

Tegoni, Mariella; Janot, Jean‐Marc; Labeyrie, Françoise

doi:10.1111/j.1432-1033.1990.tb15580.x

Cited by 25 publications

(55 citation statements)

References 37 publications

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“…It has been previously shown that cytochrome c can accept electrons only from the haem group of flavocytochrome b2 [14,21,22], whereas ferricyanide can accept electrons from both the haem and FMN groups [13,23]. This knowledge allows us to explain why the Y143F mutation has affected the electrontransfer rate to cytochrome c to a much greater extent than it has to ferricyanide.…”

Section: Discussionmentioning

confidence: 91%

Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2

Miles

Rouvière‐Fourmy

Lederer

et al. 1992

Biochemical Journal

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The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

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Section: Discussionmentioning

confidence: 91%

Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2

Miles

Rouvière‐Fourmy

Lederer

et al. 1992

Biochemical Journal

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“…On the basis of a detailed analysis of the pyruvate product binding to the three redox states of the flavin in the H.a. flavocytochrome b2 [26], the dissociation of the product after L-lactate oxidation from Fr species can be considered to be very fast and thus does not appear on the Scheme. Table 2 shows simulated steady-state values compared with the corresponding experimental data obtained under different conditions for substrate concentration, for different donors (L-lactate and L-[2H]lactate) and different acceptors (cytochrome c and ferricyanide).…”

Section: Fiavocytochrome B2 On L-12hilactate Concentrationmentioning

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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“…2. This mixed inhibition is reminiscent of that of pyruvate and oxalate with respect to Flb2, a behaviour which has been ascribed to the fact that pyruvate binds at the active site when the flavin is in the oxidized state as well as when it is in the semiquinone state (39)(40)(41)(42)(43). The respective K i values are presented in Table 2.…”

Section: Steady-state Kinetics With Hao and The Fdhmentioning

confidence: 94%