Reduced oxy intermediate observed in D251N cytochrome P450(cam)

Cytochrome P450s are ubiquitous heme proteins responsible for various oxidative metabolic processes. The overall rate-determining step in the catalytic cycle of native cytochrome P450(cam) is the reduction of the dioxygen complex, which has made detection of catalytic intermediates after this reduction impossible. However, for the site-specific mutant D251N cytochrome P450(cam) (which affects proton transfer near the catalytic center), the overall rate-determining step occurs after the reduction of oxy-P450. As a consequence, we have observed in the UV-visible spectrum during catalytic turnover a new intermediate that is one electron reduced from oxy-P450 with an intact dioxygen bond.