Single turnover kinetics of the reaction between oxycytochrome P-450cam and reduced putidaredoxin

Abstract

A study of the single turnover kinetics of the reaction between oxycytochrome P-450cam and reduced putidaredoxin was performed using the inhibitor metyrapone to trap the cytochrome immediately after release of the product, 5-exo-hydroxycamphor. EPR determinations of the concentrations of reduced putidaredoxin and ferric metyrapone-bound cytochrome at the same time points showed that there is no time lag between the oxidation of reduced putidaredoxin and the appearance of metyrapone-bound cytochrome. This implies that the rate constant for electron transfer is smaller than the rate constant for the later processes involved in product formation and release, lumped into a single step. Taking this restriction into account and doing computer simulation of absorbance versus time curves, previously obtained at various putidaredoxin concentrations using stopped-flow spectrophotometry, allowed bounds to be determined for rate constants of the processes within the reaction. At 4 degrees C in buffer at pH 7.4 with 0.50 M KCl, the rate constant for the bimolecular association of the two enzymes is between 3 and 20/microM.s; the rate constant for dissociation is between 12 and 600/s; the rate constant for electron transfer is between 60 and 100/s; and the rate constant for the later processes is at least 200/s.