Review
P450 BM3: the very model of a modern flavocytochrome

https://doi.org/10.1016/S0968-0004(02)02086-8Get rights and content

Abstract

Flavocytochrome P450 BM3 is a bacterial P450 system in which a fatty acid hydroxylase P450 is fused to a mammalian-like diflavin NADPH—P450 reductase in a single polypeptide. The enzyme is soluble (unlike mammalian P450 redox systems) and its fusion arrangement affords it the highest catalytic activity of any P450 mono-oxygenase. This article discusses the fundamental properties of P450 BM3 and how progress with this model P450 has affected our comprehension of P450 systems in general.

Section snippets

Model P450 system

In searching for what really makes a P450 ‘tick’, we therefore have a quandary. Mammalian P450s are the ones for which we desire detailed enzymatic and structural detail, owing to their physiological importance. However, soluble bacterial P450 systems are most suited to the kinds of spectroscopic examination that allow a fundamental understanding of P450 properties. The Pseudomonas putida camphor hydroxylase P450cam (CYP101) has provided crucial information about the properties of the P450 heme

P450 BM3 structure, function and mechanism

P450 BM3 (called CYP102A1 according to Nelson’s classification system [2]) has proved to be an excellent system with which to analyze structural factors that govern substrate selectivity and electron transfer in P450s. Early studies of P450 BM3 confirmed that it hydroxylated a range of fatty acids near the ω terminus, most frequently at the ω-2 position [8]. The precise physiological function of P450 BM3 remains elusive, although a potential role in detoxification of polyunsaturated fatty acids

Can we redesign P450 substrate selectivity?

The structure of the palmitoleate-bound form of P450 BM3 provided clear pictures of the binding mode for long-chain fatty acids and of the key amino acids that define the substrate-binding pocket [14] (Fig. 3). The site-directed mutant F87G catalyzed the accelerated oxidation of polycyclic aromatics (including pyrene and benzo-a-pyrene), as well as affecting the regioselectivity of fatty acid oxidation [16]. In addition, mutant F87V specifically catalyzed the production of 14S,15R

How are electron transfer and catalysis regulated?

At the enzyme level, there are several regulatory features to consider for P450s. The most important is their ability to control electron transfer to the heme iron and to couple this to the reduction of molecular oxygen and substrate oxidation so that a productive catalytic cycle ensues. The consequences of a lack of control are wastage of energy from NAD(P)H and the production of reactive oxygen species (ROS), primarily superoxide and peroxide. Uncoupled turnovers will thus lead to cellular

Electron transfer engineering

The structures of redox proteins generally reveal that their redox cofactors come close to one another, providing rapid, highly directional electron transfer and removing the need for extensive ‘through-bond’ pathways using specific secondary and tertiary protein motifs [29]. For instance, in the four-heme flavocytochrome fumarate reductase (fcc3) from Shewanella frigidimarina, the longest edge-to-edge distance between adjacent cofactors is <9 Å, which guarantees overall electron tunneling

Future prospects

Although P450 BM3 was discovered ∼20 years ago, only the past decade has done justice to its ‘model P450’ tag. With a large amount of structural and biochemical information already collected, the question might be raised of what else can we learn about P450 BM3. However, when we remember just what an experimentally tractable enzyme P450 BM3 is, this question really becomes ‘what do we still need to learn about the mechanism and properties of P450s and related oxidoreductases that engage in

Acknowledgements

We thank the BBSRC and EPSRC for financial support in this area. S.D. is the recipient of a Royal Society research fellowship. P.L.D. acknowledges NIH grant GM27309.

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