Inter-flavin electron transfer in cytochrome P450 reductase – effects of solvent and pH identify hidden complexity in mechanism

Authors


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: nigel.scrutton@manchester.ac.uk

Abstract

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 6.5 to 8.5. 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 reduction, but rather modulates the observed semiquinone absorbance at 600 nm in a pH-dependent manner. The use of proton inventory experiments and primary kinetic isotope effects are described as kinetic tools to disentangle the intricate pH-dependent kinetic mechanism in CPR. Our analysis of the pH and isotope dependence in human CPR reveals previously hidden complexity in the mechanism of electron transfer in this complex flavoprotein.

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