The Ferredoxin-NADP(H) Reductase from Rhodobacter capsulatus: Molecular Structure and Catalytic Mechanism
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- 作者：Isabel Nogué ; s ; Inmaculada Pé ; rez-Dorado ; Susana Frago ; Cristian Bittel ; Stephen G. Mayhew ; Carlos Gó ; mez-Moreno ; Juan A. Hermoso ; Milagros Medina ; Né ; stor Cortez ; and Né
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：September 6, 2005
- 年：2005
- 卷：44
- 期：35
- 页码：11730 - 11740
- 全文大小：460K
- 年卷期：v.44,no.35(September 6, 2005)
- ISSN：1520-4995
文摘
The photosynthetic bacterium Rhodobacter capsulatus contains a ferredoxin (flavodoxin)-NADP(H) oxidoreductase (FPR) that catalyzes electron transfer between NADP(H) and ferredoxin orflavodoxin. The structure of the enzyme, determined by X-ray crystallography, contains two domainsharboring the FAD and NADP(H) binding sites, as is typical of the FPR structural family. The FADmolecule is in a hairpin conformation in which stacking interactions can be established between thedimethylisoalloxazine and adenine moieties. The midpoint redox potentials of the various transitionsundergone by R. capsulatus FPR were similar to those reported for their counterparts involved in oxygenicphotosynthesis, but its catalytic activity is orders of magnitude lower (1-2 s-1 versus 200-500 s-1) asis true for most of its prokaryotic homologues. To identify the mechanistic basis for the slow turnover inthe bacterial enzymes, we dissected the R. capsulatus FPR reaction into hydride transfer and electrontransfer steps, and determined their rates using stopped-flow methods. Hydride exchange between theenzyme and NADP(H) occurred at 30-150 s-1, indicating that this half-reaction does not limit FPRactivity. In contrast, electron transfer to flavodoxin proceeds at 2.7 s-1, in the range of steady-state catalysis.Flavodoxin semiquinone was a better electron acceptor for FPR than oxidized flavodoxin under bothsingle turnover and steady-state conditions. The results indicate that one-electron reduction of oxidizedflavodoxin limits the enzyme activity in vitro, and support the notion that flavodoxin oscillates betweenthe semiquinone and fully reduced states when FPR operates in vivo.