Cytochrome P450BM-3, a bacterial fatty acid monoxygenase, resembles the eukaryotic microsomal P450's and their flavoprotein reductase in primary structure and function. The three-dimensional structure of the hemoprotein domain of P450BM-3 was determined by x-ray diffraction and refined to an R factor of 16.9 percent at 2.0 angstrom resolution. The structure consists of an alph and a beta domain. The active site heme is accessible through a long hydrophobic channel formed primarily by the beta domain and the B' and F helices of the alpha domain. The two molecules in the asymmetric unit differ in conformation around the substrate binding pocket. Substantial differences between P450BM-3 and P450cam, the only other P450 structure available, are observed around the substrate binding pocket and the regions important for redox partner binding. A general mechanism for proton transfer in P450's is also proposed.

Based on this comparison we believe that all P450s will be found to possess the same tertiary structure. The ability to precisely predict other P450 substrate-contact residues is limited by the extreme structural heterogeneity in the substrate-recognition regions. The central I-helix structures of P450terp and P450BM-3 suggest a role for helix-associated solvent molecules as a source of catalytic protons, distinct from the mechanism for P450cam. We suggest that the P450 molecular dipole might aid in both redox-partner docking and proton recruitment for catalysis.

The crystal structure of the complex between the heme-and FMN-binding domains of bacterial cytochrome P450BM-3, a prototype for the complex between eukaryotic microsomal P450s and P450 reductase, has been determined at 2.03 Å resolution. The f lavodoxin-like f lavin domain is positioned at the proximal face of the heme domain with the FMN 4.0 and 18.4 Å from the peptide that precedes the heme-binding loop and the heme iron, respectively. The hemebinding peptide represents the most efficient and coupled through-bond electron pathway to the heme iron. Substantial differences between the FMN-binding domains of P450BM-3 and microsomal P450 reductase, observed around the f lavinbinding sites, are responsible for different redox properties of the FMN, which, in turn, control electron f low to the P450.Cytochromes P450, a gene superfamily of heme proteins found in all eukaryotes, most prokaryotes, and Archaea (1), catalyze the monooxygenation of a wide variety of organic molecules. P450 reactions of biological significance include steroid biogenesis, drug metabolism, procarcinogen activation, xenobiotic detoxification, and fatty acid metabolism (2, 3). Electron transfer from a redox partner to the P450 is a key step in the P450 catalytic cycle. Bacterial and mitochondrial P450s receive electrons from a small soluble iron-sulfur protein, whereas the redox partner for mammalian microsomal enzymes is an FAD͞FMN-dependent NADPH-cytochrome P450 oxidoreductase (CPR). In CPR, FAD serves as an electron acceptor from NADPH, whereas the FMN moiety interacts with and reduces the P450. The problem of redox partner recognition and mechanism of electron transfer has been one of the most important and intriguing in the area of P450 research, in particular, and in biological electron-transfer reactions, in general. The involvement of both electrostatic and hydrophobic forces in protein-protein interactions between P450s and their redox partners has been demonstrated (4-9). Although the structures of four bacterial P450s, putidaredoxin, adrenodoxin, and a soluble form of rat CPR are known (10-16), the questions of where and how P450s interact with electron donors and the precise nature of the electron-transfer mechanism remain to be answered.Flavocytochrome P450BM-3 (119 kDa), a self-sufficient fatty acid monooxygenase from Bacillus megaterium (17, 18), consists of a heme-(BMP) and FMN͞FAD-containing reductase domains linked together on a single polypeptide. Being a soluble multidomain electron-transfer protein, this enzyme represents an excellent model system for studying structure͞ function relationships in P450s and the mechanism of electron transfer. Expression of the individual domains and subdomains of P450BM-3 significantly facilitated studies on the mechanism of domain-domain interaction and interdomain electron transfer (19 -25). The heme͞FMN-containing domain of P450BM-3 (BMP͞FMN, missing the FAD domain) was found to be the simplest model to follow the FMN to heme intramolecular electron transfer (24, 25). Here we rep...

Cytochrome P450s constitute a superfamily of enzymes that catalyze the oxidation of a vast number of structurally and chemically diverse hydrophobic substrates. Herein, we describe the crystal structure of a complex between the bacterial P450BM-3 and the novel substrate N-palmitoylglycine at a resolution of 1.65 A, which reveals previously unrecognizable features of active site reorganization upon substrate binding. N-palmitoylglycine binds with higher affinity than any other known substrate and reacts with a higher turnover number than palmitic acid but with unaltered regiospecificity along the fatty acid moiety. Substrate binding induces conformational changes in distinct regions of the enzyme including part of the I-helix adjacent to the active site. These changes cause the displacement by about 1 A of the pivotal water molecule that ligands the heme iron, resulting in the low-spin to high-spin conversion of the iron. The water molecule is trapped close to the heme group, which allows it to partition between the iron and the new binding site. This partitioning explains the existence of a high-spin-low-spin equilibrium after substrate binding. The close proximity of the water molecule to the heme iron indicates that it may also participate in the proton-transfer cascade that leads to heterolytic bond scission of oxygen in P450BM-3.

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