INTERACTION OF CYTOCHROME-C WITH FLAVOCYTOCHROME B(2)

Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate d ehydrogenation to cytochrome c reduction. At 25 degrees C, 0.10 M ioni c strength, and saturating L-lactate concentration, the turnover rate is 207 s(-1) [per cytochrome c reduced; Miles, C. S., Rouviere, N., Le derer, F., Mathews, F. S., Reid, G. A., Black, M. T., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. The second-order rate constant for cytochrome c reduction in the pre-steady-state has been determined by stopped-flow spectrophotometry to be 34.8 (+/- 0.9) mu M(-1) s(-1) in the presence of 10 mM L-lactate. This rate constant has been found to be dependent entirely on the rate of complex formation, the electron-t ransfer rate in the pre-formed complex being in excess of 1000 s(-1). Inhibition of the pre-steady-state reduction of cytochrome c by either zinc-substituted cytochrome c or ferrocytochrome c has led to the est imation of a K-d for the catalytically competent complex of 8 mu M, an d from this the dissociation rate constant of 280 s(-1), a value much less than the actual electron-transfer rate. The inhibition observed i s only partial which indicates that electron transfer from the 1:1 com plex to another cytochrome c can occur and that alternative electron t ransfer sites exist. The cytochrome c binding site proposed by Tegoni et al. [Tegoni, M., White, S. A., Roussel, A., Mathews, F. S., & Cambi llau, C. (1993) Proteins 16, 408-422] has been tested using site-direc ted mutagenesis. Mutations designed to affect the complex stability an d putative electron-transfer pathway had little effect, suggesting tha t the primary cytochrome c binding site on flavocytochrome b(2) lies e lsewhere. The combination of tight binding and multiple electron-trans fer sites gives flavocytochrome b(2) a low K-m and a high k(cat), maxi mizing its catalytic efficiency. In the steady-state, the turnover rat e is therefore largely limited by other steps in the catalytic cycle, a conclusion which is discussed in the preceding paper in this issue [ Daff, S., Ingledew, W. J., Reid, G. A., & Chapman, S. K. (1996) Bioche mistry 35, 6345-6350].