Interaction of cytochrome c with flavocytochrome b(2)

S Daff, R E Sharp, D M Short, C Bell, P White, F D C Manson, G A Reid, S K Chapman

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Abstract

Flavocytochrome b(2) 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) 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-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 K-d for the catalytically competent complex of 8 mu M, 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 b(2) lies elsewhere. The combination of tight binding and multiple electron-transfer sites gives flavocytochrome b(2) a low K-m and a high k(cat), 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].

Original languageEnglish
Pages (from-to)6351-6357
Number of pages7
JournalBiochemistry
Volume35
Issue number20
Publication statusPublished - 21 May 1996

Keywords

  • YEAST FLAVOCYTOCHROME-B2-CYTOCHROME-C COMPLEX
  • INTRAMOLECULAR ELECTRON-TRANSFER
  • IONIC-STRENGTH-DEPENDENCE
  • L-LACTATE DEHYDROGENASE
  • ESCHERICHIA-COLI
  • EMISSION-SPECTRA
  • BAKERS-YEAST
  • B2
  • ABSORPTION
  • KINETICS

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