P450, catalysis, electron transfer

Central to P450 catalysis is electron transfer. The catalytic cycle (Scheme 1) involves two one-electron reductions catalysis is initiated with the first, while... 

One of the most intriguing reactions in the chytochrome P450 catalysis is the transfer of second electron and dioxygen activation, which appears to be a key step of the entire process. The chemical nature of reactive oxidizing species appears in the coordination sphere of heme iron and the mechanism of hydroxylation of organic compounds, saturated hydrocarbons in particular, is a much debated question in the field of the cytochrome P450 catalysis. To solve this problem, an entire arsenal of modern experimental and theoretical methods are employed. The catalytic pathway of cytochrome P450cam from Pseudomonas putida obtained on the basis of X-ray analysis at atomic resolution is presented in Fig. 3.10. 

It is becoming increasingly obvious that the activity of P450 BM3 is controlled both thermodynamically and by structural changes triggered by substrate (fatty acid or NADP(H)) or redox state of the enzyme. The availability of the crystal structures of substrate-free and bound forms allows investigation of the roles of various amino acids in the processes of substrate binding, electron transfer and oxidative catalysis. This can be achieved... 

NADPH cytochrome P450 reductase, an enzyme containing which a complex flavoenzyme that contains two flavins, one electron is first intramolecularly transferred from FAD to FMN, before the reaction with cytochrome P450 takes place. With FNR, NADP+ first has to bind to the oxidized form, before the very fast one-electron transfer from the specifically interacting reduced ferredoxin (Fdred) occurs (8). Subsequent dissociation of the oxidized ferredoxin (Fdox) is rate-limiting in catalysis. The enzyme semiquinone-NADP" complex then reacts with another reduced ferredoxin molecule to yield the flavin hydroquinone state. In the final steps of the catalytic cycle, the NADP+ is reduced and the NADPH dissociates ... 

Natural proteides like hemoglobin, myoglobin, peroxidase, catalase, cytochrom a, b, P450, desoxigenase and chlorophyll contain the porphyrin system as a prostetic group. The porphyrin is bound coordinatively through the metal atom to the natural polymer or inserted in lipid-protein layers. Important properties are binding of small molecules, catalysis and electron transfer reactions. 

Hanna IH, Kim MS, Guengerich FP. Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation the role of aspartate 301 in structural integrity. Arch Biochem Biophys 2001 393 255-61. 

In order to sustain catalysis, the P450 enzymes require a reductase to supply two additional electrons from NAD(P)H needed for dioxygen activation. The nature of the reductase varies depending on the specific P450, ranging from self-contained redox cofactors such as FAD and FMN present in the same protein as the iron-heme cofactor to multi-protein systems in which separate proteins containing redox cofactors such as FAD and iron-sulfur clusters perform the electron-transfer function [12]. 

All of our studies with MMO thus far indicate that the regulatory scheme [5] differs from that described for P450 m [14]. In particular, there seems to be no effect of methane and most other substrates on the kinetics of formation of any of the intermediates in the reaction cycle prior to Q [60]. On the other hand, the decay rate of Q depends linearly on the concentration of substrate and leads to product formation, so it is likely that substrate enters the reaction cycle at this stage. This is supported by our recent observation that electron transfer from the reduced MMOR to MMOH occurs readily in the absence of substrate at rates that are not rate limiting for the overall reaction (X.-Y. Zhang and J. D. Lipscomb, unpublished observations). Despite the apparent lack of substrate gating in the MMO catalysis, the reconstituted enzyme system is nearly 100% efiScient in methane turnover. This appears to be due to the effects of component complex formation on the rates of specific steps in the cycle and on the redox potentials of the components [25-27, 60]. 

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