NEW INSIGHTS INTO THE CATALYTIC CYCLE OF FLAVOCYTOCHROME B(2)

Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate d ehydrogenation to cytochrome c reduction in the mitochondrial intermem brane space. The catalytic cycle for this process can be described in terms of five consecutive electron-transfer events. L-Lactate dehydrog enation results in the two-electron reduction of FMN. The two electron s are individually passed to b(2)-heme (intramolecular electron transf er) 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 r ate constant 104 (+/- 5) s(-1) [per L-lactate oxidized; Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapm an, S. K. (1992) Biochem. J. 285, 187-192]. Stopped-flow spectrophotom etry has been used to show that the major rate-limiting step in the ca talytic cycle is electron transfer from flavin semiquinone to b(2)-hem e. This conclusion is based on the observation that pre-steady-state f lavin oxidation by ferricytochrome c takes place at 120 s(-1). Althoug h flavin oxidation involves several other electron transfer steps, the se are considered too fast to contribute significantly to the rate con stant. It was also shown that the reaction product, pyruvate, is able to inhibit pre-steady-state flavin oxidation (K-i = 40 +/- 17 mM) cons istent with reports that it acts as a noncompetitive inhibitor in the steady state at high concentrations [K-i = 30 mM; Lederer, F. (1978) E ur. 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 als o on data collected for enzyme reduction [Miles, C. S., Rouviere-Fourm y, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (199 2) Biochem. J. 285, 187-192] and its interaction with cytochrome c [Da ff, 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 b y demonstrating that during steady-state turnover of the enzyme approx imately 75% of the flavin is in the semiquinone oxidation state.