Interaction of Cytochrome c with Flavocytochrome b2

详细信息    查看全文

• 作者：Simon Daff ; R. Eryl Sharp ; Duncan M. Short ; Cameron Bell ; Patricia White ; Forbes D. C. Manson ; Graeme A. Reid ; and Stephen K. Chapman
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：May 21, 1996
• 年：1996
• 卷：35
• 期：20
• 页码：6351 - 6357
• 全文大小：575K
• 年卷期：v.35,no.20(May 21, 1996)
• ISSN：1520-4995

文摘

Flavocytochrome b₂ from Saccharomycescerevisiae couples L-lactate dehydrogenationtocytochrome c reduction. At 25 C, 0.10 M ionicstrength, and saturating L-lactate concentration,theturnover rate is 207 s^-1 [per cytochromec 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-orderrate constant for cytochrome c reduction in thepre-steady-state has been determined by stopped-flowspectrophotometry to be 34.8 (± 0.9) M^-1s^-1 in the presence of 10 mML-lactate. This rate constanthas been found to be dependent entirely on the rate of complexformation, the electron-transfer rate in thepre-formed complex being in excess of 1000s^-1. Inhibition of the pre-steady-statereduction of cytochromec by either zinc-substituted cytochrome c orferrocytochrome c has led to the estimation of aK_d for thecatalytically competent complex of 8 M, and from this thedissociation rate constant of 280 s^-1, avaluemuch less than the actual electron-transfer rate. The inhibitionobserved is only partial which indicatesthat electron transfer from the 1:1 complex to another cytochromec can occur and that alternative electrontransfer sites exist. The cytochrome c binding siteproposed by Tegoni et al. [Tegoni, M., White, S. A.,Roussel, A., Mathews, F. S., & Cambillau, C. (1993) Proteins16, 408-422] has been tested using site-directed mutagenesis. Mutations designed to affect the complexstability and putative electron-transferpathway had little effect, suggesting that the primary cytochromec binding site on flavocytochromeb₂lies elsewhere. The combination of tight binding and multipleelectron-transfer sites gives flavocytochromeb₂ a low K_m and a highk_cat, maximizing its catalytic efficiency.In the steady-state, the turnover rate istherefore largely limited by other steps in the catalytic cycle, aconclusion which is discussed in thepreceding paper in this issue [Daff, S., Ingledew, W. J., Reid, G. A.,& Chapman, S. K. (1996) Biochemistry35, 6345-6350].