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  • Title: An Intermediate Conformational State of Cytochrome P450cam-CN in Complex with Putidaredoxin.
    Author: Chuo SW, Wang LP, Britt RD, Goodin DB.
    Journal: Biochemistry; 2019 May 07; 58(18):2353-2361. PubMed ID: 30994334.
    Abstract:
    Cytochrome P450cam is an archetypal example of the vast family of heme monooxygenases and serves as a model for an enzyme that is highly specific for both its substrate and reductase. During catalysis, it undergoes significant conformational changes of the F and G helices upon binding its substrate and redox partner, putidaredoxin (Pdx). Recent studies have shown that Pdx binding to the closed camphor-bound form of ferric P450cam results in its conversion to a fully open state. However, during catalytic turnover, it remains unclear whether this same conformational change also occurs or whether it is coupled to the formation of the critical compound I intermediate. Here, we have examined P450cam bound simultaneously by camphor, CN-, and Pdx as a mimic of the catalytically competent ferrous oxy-P450cam-Pdx state. The combined use of double electron-electron resonance and molecular dynamics showed direct observation of intermediate conformational states of the enzyme upon CN- and subsequent Pdx binding. This state is coupled to the movement of the I helix and residues at the active site, including Arg-186, Asp-251, and Thr-252. These movements enable occupation of a water molecule that has been implicated in proton delivery and peroxy bond cleavage to give compound I. These findings provide a detailed understanding of how the Pdx-induced conformational change may sequentially promote compound I formation followed by product release, while retaining stereoselective hydroxylation of the substrate of this highly specific monooxygenase.
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