Cytochrome P450 reductase electron transfer rates

A single turnover study of the conversion of the heme-HO-1 complex to free biliverdin has elucidated the relative rates of the catalytic steps 129). This transient kinetic study indicates that the conversion of Fe heme to Fe verdoheme is biphasic. Electron transfer to the Fe -heme HO-1 complex occurred at a rate of 0.11 s at 4°C and 0.49 s at 25°C with a 0.1 1 ratio of NADPH-cytochrome P450 reductase to heme HO-l complex. Oxygen binding to the reduced iron was sufficiently rapid im-der the experimental conditions that the species actually monitored... 

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 ... 

This would indicate that the reduction of the enzyme-substrate complex is the rate-limiting step of the overall reaction sequence. Two other observations are in accord with this assumption. Firstly, in a reconstituted system the turnover number can be increased up to 600 when the reductase concentration has reached a saturation value (Coon, M. J., priv. commun.). Secondly, NADH can also reduce cytochrome P450 via cytochrome bs in liver microsomes (Chap. 5.3) and this electron transfer adds to the rate observed with NADPH which would not be the case if any other reaction except electron transfer would be rate limiting. Since NADH with some substrates often exerts a synergistic effect, it may be that in those cases the second electron transfer becomes rate limiting. This possibility will be discussed in the next chapter. 

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