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Volume 272, Number 12, Issue of March 21, 1997 pp. 7915-7921
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Domain Architecture of Cytochrome P450BM-3

(Received for publication, October 2, 1996, and in revised form, December 5, 1996)

From the Departments of Molecular Biology & Biochemistry and Physiology & Biophysics, University of California, Irvine, California 92697-3900

Cytochromes P450 utilize redox partners to deliver electrons from NADPH/NADH to the P450 heme center. Microsomal P450s utilize an FAD/FMN reductase. The bacterial fatty acid hydroxylase, P450BM-3, is similar except the P450 heme and FAD/FMN proteins are linked together in a single polypeptide chain arranged as heme-FMN-FAD. Sequence comparisons indicate that the P450BM-3 FMN and FAD domains are similar to flavodoxin and ferredoxin reductase, respectively. Previous work has shown that the heme and FMN/FAD domains can be separately expressed and purified. In this study we have expressed, purified, and characterized the following additional domains: heme-FMN, FMN, and FAD. Each domain retains their prosthetic groups although the FMN domain is more labile. The FAD domain retains a high level of ferricyanide reductase activity but no cytochrome c reductase activity. In addition, we have deleted a 110-residue stretch in the FAD domain that is not present in ferredoxin reductase. This protein retains both FAD and heme but not FMN. We also have investigated the dimerization pattern of the individual domains that lead to the following conclusions. Holo-P450BM-3 appears to dimerize via interactions that do not involve disulfide bond formation, whereas the reductase and FAD domains form intermolecular disulfides. This indicates that the Cys residues not available for dimerization in holo-P450BM-3 are unmasked in the individual domains.

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