title: Real-time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochrome c.
abstract: Protein domain dynamics and electron transfer chemistry are often associated, but real-time analysis of domain motion in enzyme-catalysed reactions and the elucidation of mechanistic schemes that relate these motions to the reaction chemistry are major challenges for biological catalysis research. Previously we suggested that reduction of human cytochrome P450 reductase with the reducing coenzyme NADPH is accompanied by major structural re-orientation of the FMN- and FAD-binding domains through an inferred dynamic cycle of 'open' and 'closed' conformations of the enzyme (PLoS Biol, 2011, e1001222). However, these studies were restricted to stopped-flow/FRET analysis of the reductive half-reaction, and were compromised by fluorescence quenching of the acceptor by the flavin cofactors. Here we have improved the design of the FRET system, by using dye pairs with near-IR fluorescence, and extended studies on human cytochrome P450 reductase to the oxidative half-reaction using a double-mixing stopped-flow assay, thereby analysing in real-time conformational dynamics throughout the complete catalytic cycle. We correlate redox changes accompanying the reaction chemistry with protein dynamic changes observed by FRET, and show that redox chemistry drives a major re-orientation of the protein domains in both the reductive and oxidative half-reactions. Our studies using the tractable (soluble) surrogate electron acceptor cytochrome c provide a framework for analysing mechanisms of electron transfer in the endoplasmic reticulum between cytochrome P450 reductase and cognate P450 enzymes. More generally, our work emphasizes the importance of protein dynamics in intra- and inter-protein electron transfer, and establishes methodology for real-time analysis of structural changes throughout the catalytic cycle of complex redox proteins.
authors: Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, UK.
authors: Tobias M
authors: TM
authors: Hedison
authors: Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, UK.
authors: Sam
authors: S
authors: Hay
authors: Manchester Institute of Biotechnology and Faculty of Life Sciences, University of Manchester, UK.
authors: Nigel S
authors: NS
authors: Scrutton
mesh_terms: D045304:Cytochromes c / Q000737:chemistry*; D004579:Electron Transport; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D045304:Cytochromes c; D009251:NADPH-Ferrihemoprotein Reductase
keywords: Förster resonance energy transfer; cytochrome P450 reductase; diflavin oxidoreductase; electron transfer; protein domain dynamics