Evidence for change in proton delivery mechanism in D251N cytochrome P450_cam mutant

We have employed kinetic solvent isotope effect (KSIE) methodology to improve understanding of the mechanism of proton transfer in cytochrome P450fan]. It has been shown that the only step displaying a significant KSIK (1.8) in the P450cam wild type (WT) reaction cycle is the reduction of the fer rous dioxygen complex which is coupled with oxygen activation and product formation (Aikens, J., fe Sugar, S. G. (1994) .J. Am. Cfie.m. Sac. 116, 11431144). As expected, product formation rates for WT in a reconstituted system are similar to those of Aikens and Sligar. The rates of exo-5-hydroxycamphor production for D251N reconstituted system in protium oxide are much slower relative to those for the WT. Proton inventory analysis of the corresponding rates in various protium-deuterium mixtures indicates a larger number of pro tons involved in the protonation step of the D251N mutant with an overall KSIE of 10 at pl, = 6.3. This drastic change in the magnitude of solvent isotope effect reflects a major change in the activation barrier which suggests that: (i) different proton transfer mechanisms operate in these two proteins, and (ii) the rate limiting stop shifts from oxy-ferrous P450cam reduction to protona tion of a putative ferric peroxo intermediates in D251N mutant. This work was supported by grant from the National Institute of Health GM 31756.