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(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.
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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