Electron Transfer in Flavocytochrome P450 BM3:  Kinetics of Flavin Reduction and Oxidation, the Role of Cysteine 999, and Relationships with Mammalian Cytochrome P450 Reductase†

Biochemistry ◽  
2003 ◽  
Vol 42 (36) ◽  
pp. 10809-10821 ◽  
Author(s):  
Olivier Roitel ◽  
Nigel S. Scrutton ◽  
Andrew W. Munro
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Cytochrome P450 Reductase ◽  
P450 Bm3 ◽  
P450 Reductase ◽  
Reduction And Oxidation ◽  
Kinetics Of

Relaxation Kinetics of Cytochrome P450 Reductase:  Internal Electron Transfer Is Limited by Conformational Change and Regulated by Coenzyme Binding†

Biochemistry ◽  
2002 ◽  
Vol 41 (14) ◽  
pp. 4626-4637 ◽  
Author(s):  
Aldo Gutierrez ◽  
Mark Paine ◽  
C. Roland Wolf ◽  
Nigel S. Scrutton ◽  
Gordon C. K. Roberts
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Conformational Change ◽  
Cytochrome P450 Reductase ◽  
Relaxation Kinetics ◽  
Coenzyme Binding ◽  
P450 Reductase ◽  
Internal Electron ◽  
Kinetics Of

scholarly journals Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH–cytochrome P450 reductase

2011 ◽  
Vol 435 (1) ◽  
pp. 197-206 ◽  
Author(s):  
Louise Aigrain ◽  
Denis Pompon ◽  
Gilles Truan
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Cytochrome P450 Reductase ◽  
Nadph Cytochrome ◽  
Catalytic Domains ◽  
P450 Reductase ◽  
Internal Electron ◽  
Nadph Cytochrome P450 Reductase ◽  
The Impact

CPR (NADPH–cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the impact of interdomain interactions on the redox potentials of the flavins, the internal electron-transfer efficiency and the global conformation and dynamic equilibrium of the CPRs.

Determination of the Role of Target Tissue Metabolism in Lung Carcinogenesis Using Conditional Cytochrome P450 Reductase-Null Mice

2007 ◽  
Vol 67 (16) ◽  
pp. 7825-7832 ◽  
Author(s):  
Yan Weng ◽  
Cheng Fang ◽  
Robert J. Turesky ◽  
Melissa Behr ◽  
Laurence S. Kaminsky ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Target Tissue ◽  
Cytochrome P450 Reductase ◽  
Lung Carcinogenesis ◽  
Tissue Metabolism ◽  
P450 Reductase

scholarly journals Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

2002 ◽  
Vol 367 (1) ◽  
pp. 19-30 ◽  
Author(s):  
Kirsty KNIGHT ◽  
Nigel S. SCRUTTON
Keyword(s):  
Nitric Oxide ◽  
Electron Transfer ◽  
Cytochrome P450 ◽  
Nitric Oxide Synthase ◽  
Neuronal Nitric Oxide Synthase ◽  
Stopped Flow ◽  
Cytochrome P450 Reductase ◽  
P450 Reductase ◽  
Oxide Synthase ◽  
Reductase Domain

The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+. The second and third steps represent further reduction of the enzyme flavins and NADP+ release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of cytochrome P450 reductase, we attribute these slow fluorescence and absorption changes to enzyme disproportionation and/or conformational change. Unlike for the functionally related cytochrome P450 reductase, transfer of the first hydride equivalent from NADPH to nNOS reductase does not generate the flavin di-semiquinoid state. This indicates that internal electron transfer is relatively slow and is probably gated by NADP+ release. Release of calmodulin from the nNOS reductase does not affect the kinetics of inter-flavin electron transfer under stopped-flow conditions, although the observed rate of formation of the equilibrium between the NADPH-oxidized enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+ is modestly slower in calmodulin-depleted enzyme. Our studies indicate the need for significant re-interpretation of published kinetic data for electron transfer in the reductase domain of neuronal nitric oxide synthase.

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