New insights into the catalytic cycle of flavocytochrome b(2)

Simon Daff, W John Ingledew, Graeme A Reid, Stephen K Chapman

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    Abstract

    Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction in the mitochondrial intermembrane space. The catalytic cycle for this process can be described in terms of five consecutive electron-transfer events. L-Lactate dehydrogenation results in the two-electron reduction of FMN. The two electrons are individually passed to b(2)-heme (intramolecular electron transfer) and then onto cytochrome c (intermolecular electron transfer). At 25 degrees C, I 0.10, in the presence of saturating concentrations of ferricytochrome c and L-lactate, the catalytic cycle progresses with rate constant 104 (+/- 5) s(-1) [per L-lactate oxidized; Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. Stopped-flow spectrophotometry has been used to show that the major rate-limiting step in the catalytic cycle is electron transfer from flavin semiquinone to b(2)-heme. This conclusion is based on the observation that pre-steady-state flavin oxidation by ferricytochrome c takes place at 120 s(-1). Although flavin oxidation involves several other electron transfer steps, these are considered too fast to contribute significantly to the rate constant. It was also shown that the reaction product, pyruvate, is able to inhibit pre-steady-state flavin oxidation (K-i = 40 +/- 17 mM) consistent with reports that it acts as a noncompetitive inhibitor in the steady state at high concentrations [K-i = 30 mM; Lederer, F. (1978) Eur. J. Biochem. 88, 425-431]. This novel way of measuring the electron transfer rate constant is directly applicable to the catalytic cycle and has enabled us to derive a self-consistent model for it, based also on data collected for enzyme reduction [Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192] and its interaction with cytochrome c [Daff, S., Sharp, R. E., Short, D. M., Bell, C., White, P., Manson, F. D. C., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6351-6357]. Rapid-freezing quenched-flow EPR has been used to confirm the model by demonstrating that during steady-state turnover of the enzyme approximately 75% of the flavin is in the semiquinone oxidation state.

    Original languageEnglish
    Pages (from-to)6345-6350
    Number of pages6
    JournalBiochemistry
    Volume35
    Issue number20
    DOIs
    Publication statusPublished - 21 May 1996

    Keywords

    • PROSTHETIC GROUPS
    • FLAVODEHYDROGENASE
    • B2
    • CYTOCHROME-C REDUCTASE
    • REACTIVITY
    • L-LACTATE
    • BAKERS-YEAST
    • SIMULATION
    • PRODUCT BINDING
    • INTRAMOLECULAR ELECTRON-TRANSFER

    Cite this

    Daff, S., Ingledew, W. J., Reid, G. A., & Chapman, S. K. (1996). New insights into the catalytic cycle of flavocytochrome b(2). Biochemistry, 35(20), 6345-6350. https://doi.org/10.1021/bi9522559