Cytochromes P450s utilize a heme cofactor and atmospheric dioxygen, O<sub>2</sub>, to activate C–H bonds These enzymes play essential roles in drug detoxification as well as the biosynthesis of steroids and natural products. Critical to their catalytic mechanism is the heterolytic cleavage of the dioxygen O–O bond which, to occur efficiently, requires the delivery of externally supplied protons and electrons. Much of what we know about this mechanism derives from studies of the model bacterial P450cam, CYP101A1. Despite years of investigation, the timing and regulation of proton and electron delivery has been challenging to elucidate in part because it is controlled by P450cam’s redox partner Putidaredoxin, Pdx. Here, we utilize X-ray crystallography, molecular dynamics (MD), and NMR spectroscopy to illuminate the complex interplay necessary for catalysis between CYP101A1 with its substrate, redox-partner, and dioxygen. Specifically, we identify the location of a secondary allosteric substrate binding site on the surface of P450cam. Through MD simulations, we find that camphor binding to this secondary site assists in the opening of two active site access channels. These molecular dynamics data were then utilized to reconcile pseudo-contact shift (PCS) NMR data that was previously unable to back predict the location of several “mobile” residues. Finally, as a mimic of a dioxygen bound intermediate of the catalytic cycle, the “oxy-complex”, we present the X-ray crystal structure of P450cam complexed with its redox partner, Pdx, substrate, and cyanide. Remarkably, the structure of P450cam undergoes ordered changes originally proposed by NMR including multiple proline <i>cis/trans</i> isomerizations. These changes provide a channel for water entry and product egress consistent with the channel formation observed in our simulations. Together, these data present a dynamic but tightly regulated picture of P450 catalysis.

Conformational plasticity and redox partner binding in cytochrome P450cam

ACS Spring 2022

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