Cytochrome P450 reductase ratio

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... 

Grogan J, Shou M, Zhou D, et al. Use of aromatase (CYP19) metabolite ratios to characterize electron transfer from NADPH-cytochrome P450 reductase. Biochemistry 1993 32(45) 12007-12012. 

The cytochrome P450 system, found in microsomal and inner mitochondrial membranes (Figure IOC), consists of two enzymes NADPH-cytochrome P450 reductase and cytochrome 450 In each reaction two electrons are transferred one at a time from NADPH to a cytochrome P450 protein by NADPH cytochrome P450 reductase. The latter enzyme is a flavoprotein that contains both FAD and FMN in a ratio of 1 1 per mole of enzyme. 

Enzyme preparation Ratio of P450 reductase/CYP2C9 Ratio of cytochrome 1>5/CYP2C9... 

Even the optimum ratio of reductase to P450 depends on the substrate and the enzyme. Whereas most reactions are saturated by a reductase/P450 ratio of 10 1, testosterone metabolism by CYP2A1 saturates at much higher reductase ratios. In contrast, essentially all reactions that have a cytochrome b5 dependence are saturated at a h5/P450 ratio of 1 1. 

Thus, many P450 oxidations show a substantial and variable dependence on reaction conditions, which makes it impractical to optimize each reaction. In fact, the optimum reaction conditions may not represent the in vivo reaction environment. It would be difficult to justify a reaction temperature of 25 °C in an experiment that will be used for in vitro-in vivo correlations. A more practical approach would be to use a consistent set of reaction conditions that provide adequate velocities. Common reaction conditions include 100 mM KPi, pH 7.4, 37°C, a reductase/P450 ratio of 2 1, and a cytochrome b5/P450 ratio of 1 1. 

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