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Cytochrome P450: Nature's Aircraft Carrier

  • Author(s): Follmer, Alec Hoffman
  • Advisor(s): Poulos, Thomas L
  • et al.
Abstract

Cytochromes P450 are heme-containing enzymes that utilize O2 for C–H bond activation and play essential roles in drug detoxification and biosynthesis of steroids and a variety of natural products. A number of P450s now have been shown to adopt both an open and closed conformational state. In the open state, the active site is solvent exposed. Upon substrate binding, the P450 shifts to the closed state and sequesters the active site from bulk solvent. Since its discovery, cytochrome P450cam has served as a paradigm for mechanistic and structure-function studies. Over many years of investigation, a wealth of data has suggested that P450cam may possess two camphor binding sites, the active site pocket and an additional site that shifts P450cam toward the open state. However, location of this secondary site was never determined. Here, molecular dynamics simulations were performed that revealed the location of a secondary site on the surface of P450cam. Binding to this allosteric site assists in the opening of both the primary and new secondary active site access channel. Related to these observations is the recent finding that the binding of P450cam’s redox partner, Pdx, favors the open conformation. This shift towards the open state has led to the hypothesis that in order to provide the proton relay network required for O2 activation, P450cam must undergo a structural rearrangement from the closed form. Here, we present the X-ray crystal structure of P450cam complexed with its redox partner, Pdx, substrate, and cyanide as a mimic of a critical intermediate of the catalytic cycle, the “oxy-complex”. The structure of P450cam undergoes ordered changes proposed originally by NMR but never observed crystallographically. These changes provide a channel for water entry and product egress in agreement with the channel formation hypothesized by our simulations. These redox partner interactions studies are extended to a homologue of P450cam, P450terp, which exhibits a less stringent selectivity for its native redox partner Tdx, whose binding may still induce a conformational change in P450terp. Finally, a new P450, CYP102L1, was classified and characterized from Mycobacterium phage Adler and its potential role in viruses is discussed.

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