Andrew W. Munro

Heme: the most versatile redox centre in biology?

Iron-porphyrin complexes, known as hemes, form the prosthetic groups of a number of proteins. These hemoproteins exhibit an impressive range of biological functions. These include: Simple electron transfer reactions, oxygen transport and storage, oxygen reduction to the level of hydrogen peroxide or water, oxygenations of organic substrates, and the reduction of peroxides. This diversity of function is often extended further by combining heme groups with other cofactors, e. g. flavins and/or metal ions. Such combinations frequently allow heme cofactors to couple electron transfers with other processes, such as the translocation of protons or the reduction/oxidation of other molecules. This versatility in function is made possible by a combination of differences in both the polypeptide and heme constituents of the various hemoproteins. The aim of this article is to illustrate how nature has used different protein frameworks to exploit the redox properties of heme. This is done by focusing on a carefully chosen selection of hemoproteins which exemplify the numerous redox functions performed by heme in biology.

Analysis of the structural stability of the multidomain enzyme flavocytochrome P-450 BM3.

The unfolding and refolding of flavocytochrome P-450 BM3 and its constituent haem and flavin domains have been analysed, using guanidinium chloride (GdnHCl) as a denaturant. Enzyme activities are lost at GdnHCl concentrations too low to cause major changes in secondary structure (0.1-0.5 M). The losses are primarily due to time-dependent FMN removal. Fluorescence and visible CD spectroscopies show that FMN dissociation is complete by 0.7 M GdnHCl, whereas FAD removal is complete by 1.5 M GdnHCl. Limited regain of activity is achieved by dilution of enzyme from solutions of < or = 0.75 M GdnHCl into fresh buffer. Supplementation of GdnHCl-free assay media with flavins (FAD and FMN) causes small additional regains in flavin domain (cytochrome-c reductase) activity lost at low [GdnHCl]. However, flavin addition during the denaturation step affords greater protection against inactivation, suggesting that conformational changes may occur subsequent to flavin loss and that these changes are not readily reversed on dilution of GdnHCl. Loss of catalytically competent haem ligation occurs over the same [GdnHCl] range for P-450 BM3 and its haem domain. In both cases, the denatured P-420 form accumulates in the reduced/carbon monoxide-bound visible spectrum from 0.5 to 2 M GdnHCl. Secondary structure loss also occurs over similar [GdnHCl] ranges for P-450 BM3 and its two domains (80-90% lost from 0.5-3 M GdnHCl), indicating that there is little mutual stabilisation of domains in the holoenzyme. Differential scanning calorimetry measurements support this conclusion, but show that the haem domain is more thermostable than the flavin domain.

Structural and enzymological analysis of the interaction of isolated domains of cytochrome P-450 BM3.

The interactions of the individually expressed haem‐ and flavin‐containing domains of cytochrome P‐450 BM3 have been analysed by enzymological and spectroscopic techniques. Electron transfer between the isolated domains occurs at a much lower rate than that occurring in the intact flavocytochrome. CD spectroscopic studies indicate that the linkage of the domains in intact P‐450 BM3 creates haem and amino acid environments suitable for efficient electron transfer from its flavin domain.

Inhibitor/fatty acid interactions with cytochrome P-450 BM3

The interaction of fatty acid substrate (palmitate) and inhibitor (metyrapone: 2‐methyl‐1,2‐di‐3‐pyridyl‐1‐propanone) with cytochrome P‐450 BM3 was analysed by UV‐visible and circular dichroism spectroscopy, and by surface‐enhanced resonance Raman scattering (SERRS). While visible spectroscopy provides information on the relative affinities of these compounds, SERRS provides additional novel data indicating palmitate‐induced structural changes in the haem environment. SERRS also demonstrates that binding of both palmitate and the large nitrogenous ligand metyrapone occurs simultaneously to P‐450 BM3 — highlighting the usefulness of this technique in probing haemoprotein active sites.

NADPH oxidase activity of cytochrome P-450 BM3 and its constituent reductase domain

Cytochrome P-450 BM3 from Bacillus megaterium catalyses NADPH oxidation in the absence of added substrate. This activity is also associated with the independently expressed flavin-containing reductase domain of the protein. The rates of these activities are more than two orders of magnitude lower than those in the presence of fatty acid P-450 substrates or artificial electron acceptors. Electrons derived from NADPH in this fashion are transferred onto oxygen, generating superoxide (O2-) anions. The formation of these active oxygen species is detectable by luminometry and the chemiluminescence can be inhibited through the addition of superoxide dismutase (but not catalase). This activity is reminiscent of the microbicidal NADPH oxidase activity associated with neutrophils and other leukocyte blood cell types. Diphenyliodonium, a potent inhibitor of the neutrophil NADPH oxidase, effectively inhibits fatty acid hydroxylase and electron transferase activities catalysed by P-450 BM3 and its reductase domain. CD studies on the native and NADPH-reduced P-450 BM3 and BM3 reductase indicate that no secondary structural alteration is caused by pre-incubation with the reductant. Therefore, the previously recognised reversible time-dependent inactivation of P-450 BM3 by NADPH may be attributed to the NADPH oxidase activity associated with the reductase domain of the enzyme.

Fatty Acid-Induced Alteration of the Porphyrin Macrocycle of Cytochrome P450 BM3

Surface-enhanced resonance Raman scattering (SERRS) of substrate-free and substrate-bound forms of the P450 domain of cytochrome P450 BM3 are reported and assigned. Substrate-free P450 yields mixed spin heme species in which the pentacoordinate high-spin arrangement is dominant. The addition of laurate or palmitate leads to an increase in high spin content and to an allosteric activation of heme mode v29, which is sensitive to peripheral heme/protein interactions. Differences between laurate and palmitate binding are observed in the relative intensities of a number of bands and the splitting of the heme vinyl modes. Laurate binding to P450 results in different protein environments being experienced by each vinyl mode, whereas palmitate binding produces a smaller difference. The results demonstrate the ability of SERRS to probe substrate/prosthetic group interactions within an active site, at low protein concentrations.

The K(+)-efflux system, KefC, in Escherichia coli: genetic evidence for oligomeric structure.

KefC is a glutathione-gated K(+)-efflux system that is widespread in Gram-negative bacteria and which plays a role in the protection of cells from the toxic effects of electrophilic reagents, such as N-ethylmaleimide (NEM). The KefC gene from Escherichia coli has been cloned and the DNA sequenced. A number of kefC mutants that affect K+ retention by the KefC system have been isolated and all retain activation by NEM. Cloned kefC was found to suppress the phenotype of two such mutants kefC121 and kefC103. Analysis of this phenomenon has shown that suppression is specific to the KefC system, but that cloned kefC from Klebsiella and Erwinia can also mediate suppression of the mutant phenotype. Plasmid constructs of the E. coli gene in which expression of the cloned gene was diminished showed induced ability to suppress the mutant phenotype. KefC-LacZ hybrid proteins were inserted in the membrane but did not suppress the mutant phenotype. Cloned kefC did not suppress a mutant kefB allele that exhibited a similar phenotype to the kefC121 allele. These data suggest that suppression is unlikely to arise from exclusion of the mutant form of the protein from the membrane. Furthermore, NEM-activated K+ efflux from a strain carrying both the mutant and cloned wild-type alleles was faster than when either allele was present in cells alone, suggesting that both forms of the protein are inserted into the membrane. These data are discussed in terms of a model for the KefC protein in which the protein is composed of one or more identical subunits that interact in the membrane.

Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from Myceliophthora thermophila

The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia coli and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridecanoic acid (KD = 2.7 μM). Reduced nicotinamide adenine dinucleotide phosphate is the preferred reductant for CYP505A30 (KM = 3.1 μM compared to 330 μM for reduced nicotinamide adenine dinucleotide in cytochrome c reduction). Electron paramagnetic resonance confirmed cysteine thiolate coordination of heme iron in CYP505A30 and its heme domain. Redox potentiometry revealed an unusually positive midpoint potential for reduction of the flavin adenine dinucleotide and flavin mononucleotide cofactors (E0′ ∼ −118 mV), and a large increase in the CYP505A30 heme domain FeIII/FeII redox couple (ca. 230 mV) on binding arachidonic acid substrate. This switch brings the ferric heme iron potential into the same range as that of the reductase flavins. Multiangle laser light scattering analysis revealed CYP505A30’s ability to dimerize, whereas the heme domain is monomeric. These data suggest CYP505A30 may function catalytically as a dimer (as described for Bacillus megaterium P450 BM3), and that binding interactions between CYP505A30 heme domains are not required for dimer formation. CYP505A30 catalyzed hydroxylation of straight chain fatty acids at the ω-1 to ω-3 positions, with a strong preference for ω-1 over ω-3 hydroxylation in the oxidation of dodecanoic and tetradecanoic acids (88 vs 2% products and 63 vs 9% products, respectively). CYP505A30 has important structural and catalytic similarities to P450 BM3 but distinct regioselectivity of lipid substrate oxidation with potential biotechnological applications.

Chapter 8:Structure, Mechanism and Function of Cytochrome P450 Enzymes

The cytochromes P450 constitute a superfamily of heme b-containing monoxygenase enzymes found in organisms from all of the domains of life. They catalyse the two-electron reduction of molecular oxygen bound to their ferrous heme iron, resulting in its scission and the introduction of a single atom of oxygen into a substrate molecule, with the remaining oxygen atom used in production of a molecule of water. The cytochromes P450 have numerous functions critical to human physiology — including key roles in steroid synthesis and in the metabolism of drugs and xenobiotics. In microbes they are increasingly recognized to be important enzymes for biotechnologically relevant transformations — for instance in the synthesis of polyketides and the oxidation of cholesterol. This chapter will focus on the structural and mechanistic properties of the cytochromes P450, including recent studies that have led to major breakthroughs in the spectroscopic characterization of the reactive iron-oxo species responsible for the substrate oxidation events. It will also consider wider aspects of cytochrome P450 catalysis, including the diverse redox partner systems used by these hemoprotein enzymes to facilitate catalysis. In addition, the biomedical relevance of various cytochromes P450 will be highlighted, and selected biotechnological applications of these enzymes reviewed. The cytochromes P450 are an important class of enzymes capable of highly regio- and stereoselective substrate oxidations that are often impossible to achieve using synthetic chemistry approaches. Their applicability for industrially relevant transformations is now well recognized and they are increasingly valued as tools in industrial biotechnology.

Surface Enhanced Resonance Raman Scattering from a Soluble Cytochrome P-450

The soluble bacterial cytochrome P-450BM3 catalyses the hydroxylation of long chain fatty acids such as palmitic acid and lauric acid. The haem domain exhibits 25–30% sequence identity to microsomal fatty acid co-hydroxylases and n-alkane cytochromes P-450(1) from eukaryotic systems. Recent publication of the haem crystal structure(2) makes this protein a particularly suitable one for the study of P-450 oxidation processes.