tailieunhanh - Báo cáo khoa học: Inter-flavin electron transfer in cytochrome P450 reductase – effects of solvent and pH identify hidden complexity in mechanism

This study on human cytochrome P450 reductase (CPR) presents a com-prehensive analysis of the thermodynamic and kinetic effects of pH and solvent on two- and four-electron reduction in this diflavin enzyme. pH-dependent redox potentiometry revealed that the thermodynamic equilibrium between various two-electron reduced enzyme species (FMNH • ,FADH • ; FMN,FADH2; FMNH2 ,FAD) is independent of pH. | ễFEBS Journal Inter-flavin electron transfer in cytochrome P450 reductase - effects of solvent and pH identify hidden complexity in mechanism Sibylle Brenner Sam Hay Andrew W. Munro and Nigel S. Scrutton Manchester Interdisciplinary Biocentre and Faculty of Life Sciences University of Manchester UK Keywords electron transfer pH dependence redox potentiometry solvent kinetic isotope effect stopped-flow Correspondence N. S. Scrutton Manchester Interdisciplinary Biocentre and Faculty of Life Sciences University of Manchester 131 Princess Street Manchester M1 7DN UK Fax 44 161 306 8918 Tel 44 161 306 5152 E-mail Received 4 June 2008 revised 8 July 2008 accepted 15 July 2008 doi This study on human cytochrome P450 reductase CPR presents a comprehensive analysis of the thermodynamic and kinetic effects of pH and solvent on two- and four-electron reduction in this diflavin enzyme. pH-dependent redox potentiometry revealed that the thermodynamic equilibrium between various two-electron reduced enzyme species FmNH FADH FMN FADH2 FMNH2 FAD is independent of pH. No shift from the blue neutral di-semiquinone FMNH FADH towards the red anionic species is observed upon increasing the pH from to . Spectrophotometric analysis of events following the mixing of oxidized CPR and NADPH 1 to 1 in a stopped-flow instrument demonstrates that the establishment of this thermodynamic equilibrium becomes a very slow process at elevated pH indicative of a pH-gating mechanism. The final level of blue di-semiquinone formation is found to be pH independent. Stopped-flow experiments using excess NADPH over CPR provide evidence that both pH and solvent significantly influence the kinetic exposure of the blue di-semiquinone intermediate yet the observed rate constants are essentially pH independent. Thus the kinetic pH-gating mechanism under stoichiometric conditions is of no significant kinetic relevance for four-electron .