Review
Fatty acid metabolism, conformational change, and electron transfer in cytochrome P-450BM-3

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Abstract

Crystal structure-based mutagenesis studies on cytochrome P-450BM-3 have confirmed the importance of R47, Y51, and F87 in substrate binding. Replacing F87 has profound effects on regioselectivity. In contrast, changing either R47 or Y51 alone to other residues results in limited impact on substrate binding affinity. Mutating both, however, leads to large changes. Substrate-induced protein conformational changes not only lead to specific substrate binding in the heme domain, but also affect interactions with the FMN domain. Unlike the microsomal P-450 reductase, the FMN semiquinone is the active electron donor to the heme iron in P-450BM-3. The crystal structure of P-450BM-3 heme/FMN bidomain provides important insights into why the FMN semiquinone is the preferred electron donor to the heme as well as how substrate-induced structural changes possibly affect the FMN and heme domain-domain interaction.

Introduction

Cytochromes P-450 are a large family of enzymes that catalyze the monooxygenation of a wide variety of hydrophobic substrates [1]. P-450s not only metabolize exogenous compounds as the key enzymes in drug metabolism and xenobiotic detoxification in mammals, but also are involved in endogenous compound metabolism such as steroids and fatty acids. The second role of microsomal P-450s in the production of important metabolic intermediates was more fully appreciated when it was established in the early 1980s that P-450s participate in arachidonic acid (AA) metabolism as part of the arachidonic cascade [2]. The metabolites of AA by P-450s appeared to be the endothelium-derived hyperpolarization factor (EDHF) that mediates the endothelium-dependent vasodilation by hyperpolarizing smooth muscle through activation of the K+ channel [3]. Therefore, this would-be factor that can, in addition to nitric oxide (NO), regulate vascular tone becomes an important target for physiological investigations.

Because bacterial P-450s are soluble and often can be prepared in large quantities using recombinant expression systems, much of what we know regarding P-450 structure-function relationships derives from studies with bacterial P-450s. To date, the best available bacterial P-450 that closely resembles eukaryotic microsomal P-450s is P-450BM-3 (CYP102) from Bacillus megaterium. P-450BM-3 is the first bacterial P-450 found to use a FAD/FMN diflavin reductase as the redox partner like the other class II microsomal P-450s. It functions as a highly efficient ω-hydroxylase for long chain fatty acids and epoxygenase for unsaturated fatty acids. In addition to being a soluble protein, the main difference between P-450BM-3 and microsomal P-450s is that the catalytic P-450 domain and diflavin reductase domain are fused into a single polypeptide of 119 kDa [4] while the reductase and heme proteins are separate polypeptides in the microsomal systems. The heme domain of P-450BM-3 is one of the five P-450s that has had its crystal structure determined ([5] and references therein). Based on both substrate-free and -bound structures, significant information has accumulated for this enzyme via site-directed mutagenesis coupled with NMR and other biophysical techniques. This review will not attempt to cover all the research progress regarding P-450BM-3, but will focus on three aspects: (1) the relationship between structure and regioselectivity of fatty acid metabolism; (2) the function of substrate binding induced protein conformational change; and (3) the intramolecular electron transfer complex.

Section snippets

Regiospecificity of fatty acid hydroxylation and epoxidation

P-450BM-3 catalyzes the hydroxylation of long chain fatty acids (12–18 carbons) at ω-1, ω-2, and ω-3 positions [6], but never at the ω-methyl group which is in contrast to regioselectivity of some mammalian microsomal P-450s in the 4A subfamily [7]. Epoxygenase activity for long chain unsaturated fatty acids (up to 20 carbons) was also characterized for P-450BM-3 [8]. The crystal structure of the substrate-free P-450BM-3 heme domain [9] revealed an open channel from the protein surface deep

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