Tobias W. B. Ost

P450 BM3: the very model of a modern flavocytochrome

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.

Rational re-design of the substrate binding site of flavocytochrome P450 BM3

Bacillus megaterium P450 BM3 is a fatty acid hydroxylase with selectivity for long chain substrates (C12–C20). Binding or activity with substrates of chain length 13‐fold with butyrate, while the L75T/L181K double mutant has k cat/K M increased >15‐fold with hexanoate and binding (K d) improved >28‐fold for butyrate. Removing the arginine 47/lysine 51 carboxylate binding motif at the mouth of the active site disfavours binding of all fatty acids, indicating its importance in the initial recognition of substrates.

Protein engineering of cytochromes P-450

The cytochromes P-450 are an immensely important superfamily of heme-containing enzymes. They catalyze the monooxygenation of an enormous range of substrates. In bacteria, cytochromes P-450 are known to catalyze the hydroxylation of environmentally significant substrates such as camphor, phenolic compounds and many herbicides. In eukaryotes, these enzymes perform key roles in the synthesis and interconversion of steroids, while in mammals hepatic cytochromes P-450 are vital for the detoxification of many drugs. As such, the cytochromes P-450 are of considerable interest in medicine and biotechnology and are obvious targets for protein engineering. The purpose of this article is to illustrate the ways in which protein engineering has been used to investigate and modify the properties of cytochromes P-450. Illustrative examples include: the manipulation of substrate selectivity and regiospecificity, the alteration of membrane binding properties, and probing the route of electron transfer.

Analysis of the domain properties of the novel cytochrome P450 RhF.

The properties of the heme, flavin mononucleotide (FMN) and FeS domains of P450 RhF, from Rhodococcus sp. NCIMB 9784, expressed separately and in combination are analysed. The nucleotide preference, imidazole binding and reduction potentials of the heme and FMN domains are unaltered by their separation. The intact enzyme is monomeric and the flavin is confirmed to be FMN. The two one‐electron reduction potentials of the FMN are −240 and −270 mV. The spectroscopic and thermodynamic properties of the FeS domain, masked in the intact enzyme, are revealed for the first time, confirming it as a 2Fe–2S ferredoxin with a reduction potential of −214 mV.

Thermodynamic and Kinetic Analysis of the Nitrosyl, Carbonyl, and Dioxy Heme Complexes of Neuronal Nitric-oxide Synthase THE ROLES OF SUBSTRATE AND TETRAHYDROBIOPTERIN IN OXYGEN ACTIVATION

Mammalian NO synthases catalyze the monooxygenation of l-arginine (l-Arg) to N-hydroxyarginine (NOHA) and the subsequent monooxygenation of this to NO and citrulline. Both steps proceed via formation of an oxyferrous heme complex and may ultimately lead to a ferrous NO complex, from which NO must be released. Electrochemical reduction of NO-bound neuronal nitricoxide synthase (nNOS) oxygenase domain was used to form the ferrous heme NO complex, which was found to be stable only in the presence of low NO concentrations, due to catalytic degradation of NO at the nNOS heme site. The reduction potential for the heme-NO complex was approximately –140 mV, which shifted to 0 mV in the presence of either l-Arg or NOHA. This indicates that the complex is stabilized by 14 kJmol–1 in the presence of substrate, consistent with a strong H-bonding interaction between NO and the guanidino group. Neither substrate influenced the reduction potential of the ferrous heme CO complex, however. Both l-Arg and NOHA appear to interact with bound NO in a similar way, indicating that both bind as guanidinium ions. The dissociation constant for NO bound to ferrous heme in the presence of l-Arg was determined electrochemically to be 0.17 nm, and the rate of dissociation was estimated to be 10–4 s–1, which is much slower than the rate of catalysis. Stopped-flow kinetic analysis of oxyferrous formation and decay showed that both l-Arg and NOHA also stabilize the ferrous heme dioxy complex, resulting in a 100-fold decrease in its rate of decay. Electron transfer from the active-site cofactor tetrahydrobiopterin (H4B) has been proposed to trigger the monoxygenation process. Consistent with this, substitution by the analogue/inhibitor 4-amino-H4B stabilized the oxyferrous complex by a further two orders of magnitude. H4B is required, therefore, to break down both the oxyferrousand ferrous nitrosyl complexes of nNOS during catalysis. The energetics of these processes necessitates an electron donor/acceptor operating within a specific reduction potential range, defining the role of H4B.

Flavocytochrome P450 BM3 Substrate Selectivity and Electron Transfer in a Model Cytochrome P450

The cytochromes P450 (P450s) are haem b-containing monooxygenase enzymes that play crucial roles in the processes of drug metabolism, steroid and fatty acid metabolism in mammals (Munro and Lindsay, 1996). They were first recognised as carbon monoxide-binding haem pigments in mammalian liver microsomes. The fact that treatment of reduced microsomes with carbon monoxide led to loss of drug-metabolising activity and that this

Cytochrome P450 BM3, NO binding and real-time NO detection.

Nitric oxide is known to coordinate to ferrous heme proteins very tightly, following which it is susceptible to reaction with molecular oxygen or free NO. Its coordination to ferric heme is generally weaker but the resultant complexes are more stable in the presence of oxygen. Here we report determination of the binding constants of Cytochrome P450 BM3 for nitric oxide in the ferric state in the presence and absence of substrate. Compared to other 5-coordinate heme proteins, the K(d) values are particularly low at 16 and 40 nM in the presence and absence of substrate respectively. This most likely reflects the high hydrophobicity of the active site of this enzyme. The binding of NO is tight enough to enable P450 BM3 oxygenase domain to be used to determine NO concentrations and in real-time NO detection assays, which would be particularly useful under conditions of low oxygen concentration, where current methods break down.