Interaction of cytochrome c with flavocytochrome b2.
Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. At 25 degrees C, 0.10 M ionic strength, and saturating L-lactate concentration, the turnover rate is 207 s-1 [per cytochrome c reduced; Miles, C. S., Rouviere, N., Lederer, 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) muM-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-transfer 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 estimation of a Kd for the catalytically competent complex of 8 microM, and from this the dissociation rate constant of 280 s-1, a value much less than the actual electron-transfer rate. The inhibition observed is only partial which indicates that electron transfer from the 1:1 complex to another cytochrome c can occur and that alternative electron transfer sites exist. The cytochrome c binding site proposed by Tegoni et al. [Tegoni, M., White, S. A., Roussel, A., Mathews, F. S. & Cambillau, C. (1993) Proteins 16, 408-422] has been tested using site-directed mutagenesis. Mutations designed to affect the complex stability and putative electron-transfer pathway had little effect, suggesting that the primary cytochrome c binding site on flavocytochrome b2 lies elsewhere. The combination of tight binding and multiple electron-transfer sites gives flavocytochrome b2 a low K(m) and a high kcat, maximizing its catalytic efficiency. In the steady-state, the turnover rate 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) Biochemistry 35, 6345-6350].
Daff
S
S
Department of Chemistry, University of Edinburgh, Scotland, UK.
Sharp
R E
RE
Short
D M
DM
Bell
C
C
White
P
P
Manson
F D
FD
Reid
G A
GA
Chapman
S K
SK
D001483:Base Sequence; D001665:Binding Sites; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D015032:Zinc / Q000737:chemistry
D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006418:Heme; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D015032:Zinc