Two decades have passed since the discovery in liver microsomes of a haemprotein that forms a reduced-CO complex with the absorptive maximum of the Soret at 450 nm (Klingenberg, 1958; Garfinkel, 1958) and the identification of this protein as a new cytochrome: pigment cytochrome, P-450 (Omura and Sato, 1962, 1964a). In the intervening years, the study of cytochrome P-450 dependent monoxygenases has expanded exponentially. From the first crude attempts to solubilise a P-450 (Omura and Sato, 1963, 1964b) to the determination of the primary, secondary, and tertiary structure of cytochrome P-450cam by amino acid sequencing (Haniu et al., 1982a,b) and x-ray crystallography (Poulos et al., 1984) our understanding of this unique family of proteins has been advancing on all fronts. Since, perhaps, the greatest understanding of the structure and mechanism of P-450s has come from concentrated study of P-450cam of the Pseudomonas putida camphor-5-exo-hydroxylase, this review will concentrate on findings with P-450cam; attention will be drawn to parallel and contrasting examples from other P-450s as appropriate.

Structure and Chemistry of Cytochrome P450

Hydrogen peroxide emerged as major redox metabolite operative in redox sensing, signaling and redox regulation. Generation, transport and capture of H2O2 in biological settings as well as their biological consequences can now be addressed. The present overview focuses on recent progress on metabolic sources and sinks of H2O2 and on the role of H2O2 in redox signaling under physiological conditions (1–10 nM), denoted as oxidative eustress. Higher concentrations lead to adaptive stress responses via master switches such as Nrf2/Keap1 or NF-κB. Supraphysiological concentrations of H2O2 (>100 nM) lead to damage of biomolecules, denoted as oxidative distress. Three questions are addressed: How can H2O2 be assayed in the biological setting? What are the metabolic sources and sinks of H2O2? What is the role of H2O2 in redox signaling and oxidative stress?

Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress.

The biophysical properties that enable proteins to so readily evolve to perform diverse biochemical tasks are largely unknown. Here, we show that a protein’s capacity to evolve is enhanced by the mutational robustness conferred by extra stability. We use simulations with model lattice proteins to demonstrate how extra stability increases evolvability by allowing a protein to accept a wider range of beneficial mutations while still folding to its native structure. We confirm this view experimentally by mutating marginally stable and thermostable variants of cytochrome P450 BM3. Mutants of the stabilized parent were more likely to exhibit new or improved functions. Only the stabilized P450 parent could tolerate the highly destabilizing mutations needed to confer novel activities such as hydroxylating the antiinflammatory drug naproxen. Our work establishes a crucial link between protein stability and evolution. We show that we can exploit this link to discover protein functions, and we suggest how natural evolution might do the same.

Protein stability promotes evolvability.

In chemical terms, the regio- and stereoselective hydroxylation of hydrocarbon C-H bonds is a very difficult transformation. Nevertheless, these reactions are deftly catalyzed by a variety of metalloenzymes, among which the most diverse are the many members of the cytochrome P450 family. Cytochrome P450 enzymes are found in most classes of organisms, including bacteria, fungi, plants, insects, and mammals. Thousands of such proteins are now known (http://drnelson.utmem.edu/cytochromeP450.html), including 57 in the human genome (1), 20 in Mycobacterium tuberculosis (2), 272 in Arabidopsis (3), and the amazing number of 457 in rice (4). The nomenclature for these enzymes is based on their sequence similarity when appropriately aligned, a somewhat arbitrary similarity cutoff (approximately >40% identity) being used to define members of a family and a higher cutoff (approximately >55% identity) members of a subfamily (5). Thus CYP3A4 corresponds to the fourth enzyme in family 3, subfamily A. This nomenclature allows the naming of enzymes without regard to their origin or specific properties.

The mammalian, plant, and fungal proteins are commonly membrane bound and are relatively difficult to manipulate, but the bacterial proteins are usually soluble, monomeric proteins. For that reason, much of the early research on mechanisms of cytochrome P450 enzymes was carried out with bacterial enzymes, particularly with the prototypical enzyme CYP101 (P450cam) from Pseudomonas putida (6, 7). From a chemist's point of view, there is a particular interest in the thermophilic enzymes, which currently include CYP119 (8-10), P450st (11), CYP174A1 (12), and CYP231A2 (13). The thermal stability of these enzymes makes them attractive starting points for the development of industrially useful catalysts. In this context, particular attention has also focused on CYP102 (P450BM3), a self-sufficient enzyme from Bacillus megaterium in which the flavoprotein protein required for transfer of electrons from NADPH is fused to the hemoprotein (14). The resulting simplicity and high catalytic rate have led to extensive efforts to engineer this protein for practical catalytic purposes (15-19). Although these proteins have properties that make them particularly attractive for engineering purposes, the large reservoir of P450 enzymes that collectively catalyze an astounding diversity of reactions suggests that P450 catalysis will develop into a highly useful technology.

The cytochrome P450 enzymes are defined by the presence in the proteins of a heme (iron protoporphyrin IX) prosthetic group coordinated on the proximal side by a thiolate ion (20, 21). This feature gives rise to the spectroscopic signature that defines these enzymes, as the thiolate-ligated ferrous-CO complex is characterized by a Soret absorption maximum at ∼450 nm (21). A thiolate-coordinated heme group is present in all P450 enzymes, although not all proteins with such coordination are members of this superfamily. One obvious exception, for example, is chloroperoxidase, which has a thiolate-coordinated heme group but normally catalyzes a very different reaction than the P450 enzymes (21-23).

Although there are unusual P450 enzymes, such as the thromboxane and prostacyclin synthases (24), or CYP152 from Sphingomonas paucimobilis or Bacillus subtilis (25, 26), that normally utilize peroxides as substrates, the defining reaction for P450 enzymes is the reductive activation of molecular oxygen. In this reaction, one of the oxygen atoms of molecular oxygen is inserted into the substrate and the other oxygen atom is reduced to a molecule of water. With one exception to date (27, 28), the electrons required for this reduction of molecular oxygen derive from reduced pyridine nucleotides (NADH or NADPH). The overall equation for the reaction thus adheres to the formula: RH + NAD(P)H + O2 + H+ -> ROH + NAD(P)+ + H2O, where RH stands for a substrate with a hydroxylatable site. P450 enzymes therefore belong to the monooxygenase class of enzymes that only insert one of the oxygen atoms of molecular oxygen into their substrates. However, under appropriate circumstances or with specific substrates, other P450-catalyzed reactions can be observed, including desaturation, carbon-carbon bond scission, and carbon-carbon bond formation (29, 30). This review specifically focuses on P450-catalyzed hydrocarbon hydroxylation, the reaction that is most characteristic of P450 enzymes and that has been most extensively investigated. However, the principles that apply in these reactions also apply to other hydroxylation reactions, including those that occur on carbons adjacent to nitrogen, sulfur, or oxygen.

Hydrocarbon hydroxylation by cytochrome P450 enzymes.

The utilization of CO2 via electrochemical reduction constitutes a promising approach toward production of value-added chemicals or fuels using intermittent renewable energy sources. For this purpose, molecular electrocatalysts are frequently studied and the recent progress both in tuning of the catalytic properties and in mechanistic understanding is truly remarkable. While in earlier years research efforts were focused on complexes with rare metal centers such as Re, Ru, and Pd, the focus has recently shifted toward earth-abundant transition metals such as Mn, Fe, Co, and Ni. By application of appropriate ligands, these metals have been rendered more than competitive for CO2 reduction compared to the heavier homologues. In addition, the important roles of the second and outer coordination spheres in the catalytic processes have become apparent, and metal–ligand cooperativity has recently become a well-established tool for further tuning of the catalytic behavior. Surprising advances have also been made ...

Homogeneously Catalyzed Electroreduction of Carbon Dioxide-Methods, Mechanisms, and Catalysts

https://www.cell.com/trends/biochemical-sciences/pdf/S0968-0004(02)02086-8.pdf

P450 BM3: the very model of a modern flavocytochrome.

NO synthase: structures and mechanisms.

Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polypeptide whose heme and diflavin domains are fused to produce a catalytically self-sufficient fatty acid monooxygenase. Redox potentiometry studies have been performed with intact flavocytochrome P-450 BM3 and with its component heme, diflavin, FAD, and FMN domains. Results indicate that elctron flow occurs from the NADPH donor through FAD, then FMN and on to the heme center where fatty acid substrate is bound and monooxygenation occurs. Prevention of futile cycling of electrons is avoided through an increase in redox potential of more than 100 mV caused by binding of fatty acids to the active site of P-450. Redox potentials are little altered for the component domains with respect to their values in the larger constructs, providing further evidence for the discrete domain organization of this flavocytochrome. The reduction potentials of the 4-electron reduced diflavin domain and 2-electron reduced FAD domain are considerably lower than th...

Redox Control of the Catalytic Cycle of Flavocytochrome P-450 BM3†

Rapid events in the processes of electron transfer and substrate binding to cytochrome P-450 BM3 from Bacillus megaterium and its constituent haem-containing and flavin-containing domains have been investigated using stopped-flow spectrophotometry. The formation of a blue semiquinone flavin form occurs during the NADPH-dependent reduction of the flavin domain and a species with a similar absorption maximum is also seen during reduction of the holoenzyme by NADPH. EPR spectroscopy confirms the formation of the flavin semiquinone. The formation of this semiquinone is transient during fatty acid monooxygenation by the holoenzyme, but in the presence of excess NADPH the species reforms once fatty acid is exhausted. Electron transfers through the reductase domain are too rapid to limit the fatty acid monooxygenation reaction. The substrate-binding-induced haem iron spin-state shift also occurs much faster than the k,,, at 25°C. The rate of first electron transfer to the haem domain is also rapid; but it is of the order of 5-10-times larger than the k,,, for the enzyme (dependent on the fatty acid used). Given that two successive electron transfers to haem iron are required for the oxygenation reaction, these rates are likely to exert some control over the rate of fatty acid oxygenation reactions. The presence of large amounts of NADPH also results in decreased rates of electron transfer from flavin to haem iron. In the difference spectrum of the active fatty acid hydroxylase, features indicative of a high-spin iron haem accumulate. These are in accordance with the presence of large amounts of an Fe’+-product bound enzyme during turnover and indicate that product release may also contribute to rate limitation. Taken together, these data suggest that the catalytic rate is not determined by the accumulation of a single intermediate in the reaction scheme, but rather that it is controlled in a series of steps.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1996.0403u.x

Probing electron transfer in flavocytochrome P-450 BM3 and its component domains.

In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be s...

Simon Daff

Papers

P450 BM3: the very model of a modern flavocytochrome.

NO synthase: structures and mechanisms.

Redox Control of the Catalytic Cycle of Flavocytochrome P-450 BM3†

Probing electron transfer in flavocytochrome P-450 BM3 and its component domains.

Oxygen Activation and Electron Transfer in Flavocytochrome P450 BM3