Caroline S. Miles

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.

Octaheme tetrathionate reductase is a respiratory enzyme with novel heme ligation

We have isolated a soluble cytochrome from Shewanella oneidensis that contains eight covalently attached heme groups and determined its crystal structure. One of these hemes exhibits novel ligation of the iron atom by the ε-amino group of a lysine residue, despite its attachment via a typical CXXCH motif. This heme is most likely the active site for tetrathionate reduction, a reaction catalyzed efficiently by this enzyme.

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.

Importance of the domain-domain interface to the catalytic action of the NO synthase reductase domain

Calmodulin (CaM) activates NO synthase (NOS) by binding to a 20 amino acid interdomain hinge in the presence of Ca (2+), inducing electrons to be transferred from the FAD to the heme of the enzyme via a mobile FMN domain. The activation process is influenced by a number of structural features, including an autoinhibitory loop, the C-terminal tail of the enzyme, and a number of phosphorylation sites. Crystallographic and other recent experimental data imply that the regulatory elements lie within the interface between the FAD- and FMN-binding domains, restricting the movement of the two cofactors with respect to each other. Arg1229 of rat neuronal NOS is a conserved residue in the FAD domain that forms one of only two electrostatic contacts between the domains. Mutation of this residue to Glu reverses its charge and is expected to induce an interdomain repulsion, allowing the importance of the interface and domain-domain motion to be probed. The charge-reversal mutation R1229E has three dramatic effects on catalysis: (i) hydride transfer from NADPH to FAD is activated in the CaM-free enzyme, (ii) FAD to FMN electron transfer is inhibited in both forms, and (iii) electron transfer from FMN to the surrogate acceptor cytochrome c is activated in the CaM-free enzyme. As a result, during steady-state turnover with cytochrome c, calmodulin now deactivates the enzyme and causes cytochrome c-dependent inhibition. Evidently, domain-domain separation is large enough in the mutant to accommodate another protein between the cofactors. The effects of this single charge reversal on three distinct catalytic events illustrate how each is differentially dependent on the enzyme conformation and support a model for catalytic motion in which steps i, ii, and iii occur in the hinged open, closed, and open states, respectively. This model is also likely to apply to related enzymes such as cytochrome P450 reductase.

Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.

A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina.

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their kcat values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1Å resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.

Catalysis in fumarate reductase

In the absence of oxygen many bacteria are able to utilise fumarate as a terminal oxidant for respiration. In most known organisms the fumarate reductases are membrane-bound iron-sulfur flavoproteins but Shewanella species produce a soluble, periplasmic flavocytochrome c(3) that catalyses this reaction. The active sites of all fumarate reductases are clearly conserved at the structural level, indicating a common mechanism. The structures of fumarate reductases from two Shewanella species have been determined. Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis. Conversion of fumarate to succinate requires hydride transfer from FAD and protonation by an active site acid. The identity of the proton donor has been open to question but we have used structural considerations to suggest that this function is provided by an arginine side chain. We have confirmed this experimentally by analysing the effects of site-directed mutations on enzyme activity. Substitutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis.

Substitution of a haem-iron axial ligand in flavocytochrome b2

The importance of haem-iron axial coordination in flavocytochrome b2 (L-lactate: cytochrome-c oxidoreductase) has been examined by replacing one of the ligating histidines, His-43, with methionine. The His-43-->Met mutation (H43M) results in a distinct colour change from red in the wild-type enzyme to green in the mutant enzyme. The electronic absorption spectrum indicates that only approx. 5% of the haem binding sites are occupied. There is no evidence of any absorption band at 695 nm (characteristic of methionine ligation) suggesting that methionine does not act as an axial ligand in the mutant enzyme. The H43M-mutant enzyme shows a band around 640-650 nm which is usually associated with high-spin ferric-haem proteins, either five coordinate or with a weak-field ligand in the sixth position. The EPR spectrum of the H43M-enzyme at 7 K shows a g-value near 6.0, indicating that the haem-iron is high-spin in contrast to its low-spin state in the wild-type enzyme. The His-43-->Met mutation has only a small effect on the lactate dehydrogenase activity of the enzyme as measured with ferricyanide as external electron acceptor, but greatly reduces its cytochrome-c reductase activity.

Oxygen activation in neuronal NO synthase: resolving the consecutive mono-oxygenation steps.

The vital signalling molecule NO is produced by mammalian NOS (nitric oxide synthase) enzymes in two steps. L-arginine is converted into NOHA (Nω-hydroxy-L-arginine), which is converted into NO and citrulline. Both steps are thought to proceed via similar mechanisms in which the cofactor BH4 (tetrahydrobiopterin) activates dioxygen at the haem site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochrome P450, a haem-iron oxo species may be formed, or direct reaction between a haem-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. In the present paper we analyse the two reaction steps using the G586S mutant of nNOS (neuronal NOS), which introduces an additional hydrogen bond in the active site and provides an additional proton source. In the mutant enzyme, BH4 activates dioxygen as in the wild-type enzyme, but an interesting intermediate haem species is then observed. This may be a stabilized form of the active oxygenating species. The mutant is able to perform step 2 (reaction with NOHA), but not step 1 (with L-arginine) indicating that the extra hydrogen bond enables it to discriminate between the two mono-oxygenation steps. This implies that the two steps follow different chemical mechanisms.