Search

In this study, the effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL (bifunctional spin-label) onto the F or G helices and using DEER (double electron-electron resonance) to measure the distance between labels. Recent EPR and crystallographic studies have observed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state. However, this change was not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound P450cam (Fe2+CO). In addition, recent NMR studies have failed to observe a change in P450cam conformation upon binding Pdx. Hence, resolving these issues is important for a full understanding the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformation when labeled on either the F or G helices with MTSL. BSL at these sites can either narrow the distance distribution widths dramatically or alter the extent of the conformational change. In addition, we report DEER spectra on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed. This indicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted by Pdx binding. Finally, we report the open form P450cam crystal structure with substrate bound, which suggests that crystal packing effects may prevent conformational conversion. Using multiple labeling approaches, DEER provides a unique perspective to resolve how the conformation of P450cam depends on Pdx and ligand states. [ABSTRACT FROM AUTHOR]

CYP125 from Mycobacterium tuberculosis catalyzes sequential oxidation of the cholesterol side-chain terminal methyl group to the alcohol, aldehyde, and finally acid. Here, we demonstrate that CYP125 simultaneously catalyzes the formation of five other products, all of which result from deformylation of the sterol side chain. The aldehyde intermediate is shown to be the precursor of both the conventional acid metabolite and the five deformylation products. The acid arises by protonation of the ferric-peroxo anion species and formation of the ferryl-oxene species, also known as Compound I, followed by hydrogen abstraction and oxygen transfer. The deformylation products arise by addition of the same ferric-peroxo anion to the aldehyde intermediate to give a peroxyhemiacetal that leads to C-C bond cleavage. This bifurcation of the catalytic sequence has allowed us to examine the effect of electron donation by the proximal ligand on the properties of the ferric-peroxo anion. Replacement of the cysteine thiolate iron ligand by a selenocysteine results in UV-vis, EPR, and resonance Raman spectral changes indicative of an increased electron donation from the proximal selenolate ligand to the iron. Analysis of the product distribution in the reaction of the selenocysteine substituted enzyme reveals a gain in the formation of the acid (Compound I pathway) at the expense of deformylation products. These observations are consistent with an increase in the pKa of the ferric-peroxo anion, which favors its protonation and, therefore, Compound I formation. [ABSTRACT FROM AUTHOR]

Electrode potentials for every intermediate in the cytochrome P450 cycle were estimated and evaluated by means of an oxidation state diagram. By this approach, and within the uncertainties of the approximations, the superoxide complex of cytochrome P450 at pH 7 is oxidizing: E°′ (P450FeO22+, H+/P450FeOOH2+) = +0.93 V, and the Gibbs energy for the reaction of the hydroperoxo complex of cytochrome P450 to form compound I and water, P450FeOOH2+ + H+ = P450FeO2+ por•+ + H2O, is 0 kJ/mol. Although cytochrome P450FeOOH2+ and cytochrome P450FeO2+por•+ are approximately isoenergetic, they are likely to react at different rates with substrates and may yield different products. Homolysis of the hydroperoxo complex of cytochrome P450 to compound II and the hydroxyl radical, P450FeOOH2+ = P450FeO2+ + HO•, is unfavorable (ΔG°′ = +92 kJ/mol), as is the dissociation into HOO- and cytochrome P450Fe3+ (+73 kJ/mol). It is shown that the sum of the Gibbs energy of association for cytochrome P450Fe3+ with the hydroperoxo anion and the Gibbs energy for the one-electron reduction of cytochrome P450FeOOH2+, relative to NHE, is constant (–203 kJ/mol). While the estimated E°+ (P450FeO22+, H+/P450FeOOH2+) = +0.93 V at pH 7 is larger than necessary to effect reduction of cytochrome P450FeO22+, the magnitude of this electrode potential implies that the binding constant for cytochrome P450Fe3+ with hydrogen peroxide is ca. 3 × 106 M-1 at pH 7. An association constant of this magnitude ensures that a fraction of cytochrome P450FeOOH2+ is available to form compound I or to react with substrates directly, while a larger one would imply that compound I is too weak an oxidant. In general, the energetics of the reduction of dioxygen to water determines the energetics of catalysis of hydroxylations by cytochrome P450. These results enable calibration of energy levels obtained for intermediates in the cytochome P450 reaction cycle obtained by ab initio calculations and provide insights into the catalytic efficiency of cytochrome P450 and guidelines for the development of competent hydroxylation catalysts. [ABSTRACT FROM AUTHOR]

Peroxynitrite has come into the spotlight in recent years. Its effects on proteins have been implicated in several diseases such as acute lung injury, rheumatoid arthritis, implant rejection, artherosclerosis, Parkinson's disease, and Alzheimer's disease. Peroxynitrite is thought to inactivate a variety of proteins including thiolate-ligated heme proteins such as cytochrome P450 2B1 and PGI2 synthase, through the nitration of tyrosine residues. In previous studies it was reported that thiolate-ligated heme enzymes react with peroxynitrite to form a ferryl intermediate. In an effort to spectroscopically characterize this species in P450BM3, we discovered that the peroxynitrite-generated intermediate is not an FeIVoxo, but rather an iron-nitrosyl {FeNO}6 complex. We present density functional calculations as well as Mössbauer and stopped-flow spectroscopic characterizations of the peroxynitrite-generated intermediate in P450BM3. [ABSTRACT FROM AUTHOR]

Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of 50% of all orally administered drugs which exhibit an intriguing kinetic behavior typified by a sigmoidal dependence of the reaction velocity on the substrate concentration. There is evidence for the binding of two substrates in the active site of the enzyme, but the mechanism of this cooperative binding is unclear. Diazepam is such a drug that undergoes metabolism by CYP3A4 with sigmoidal dependence. Metabolism is initiated by hydrogen atom abstraction from the drug. To understand the factors that determine the cooperative binding and the juxtaposition of the CH bond undergoing abstraction, we carried out molecular dynamics simulations for two enzymatic conformers and examined the differences between the substrate-free and the bound enzymes, with one and two diazepam molecules. Our results indicate that the effector substrate interacts both with the active substrate and with the enzyme, and that this interaction results in side chain reorientation with relatively minor long-range effects. In accord with experiment, we find that F304, in the interface between the active and effector binding sites, is a key residue in the mechanism of cooperative binding. The addition of the effector substrate stabilizes F304 and its environment, especially F213, and induces a favorable orientation of the active substrate, leading to a short distance between the targeted hydrogen for abstraction and the active species of the enzyme. In addition, in one conformer of the enzyme, residue R212 may strongly interact with F304 and counteract the effector's impact on the enzyme. [ABSTRACT FROM AUTHOR]

B3LYP density functional theory calculations are used to unravel the mysterious third step of aromatase catalysis. The feasibility of mechanisms in which the reduced ferrous dioxygen intermediate mediates androgen aromatization is explored and determined to be unlikely. However, proton-assisted homolysis of the peroxo hemiacetal intermediate to produce P450 compound I and the C19 gem-diol likely proceeds with a low energetic barrier. Mechanisms for the aromatization and deformylation sequence which are initiated by 1β-hydrogen atom abstraction by P450 compound I are considered. 1β-Hydrogen atom abstraction from substrates in the presence of the 2,3-enol encounters strikingly low barriers (5.3-7.8 kcal/mol), whereas barriers for this same process rise to 17.0-27.1 kcal/mol in the keto tautomer. Transition states for 1β-hydrogen atom abstraction from enolized substrates in the presence of the 1 9-gem-diol decayed directly to the experimentally observed products. If the C19 aldehyde remains unhydrated, aromatization occurs with concomitant decarbonylation and therefore does not support dehydration of the C19 aldehyde prior to the final catalytic step. On the doublet surface, the transition state connects to a potentially labile 1(10) dehydrogenated product, which may undergo rapid aromatization, as well as formic acid. Ab initio molecular dynamics confirmed that the 1β-hydrogen atom abstraction and deformylation or decarbonylation occur in a nonsynchronous, coordinated manner. These calculations support a dehydrogenase behavior of aromatase in the final catalytic step, which can be summarized by 1β-hydrogen atom abstraction followed by gem-diol deprotonation. [ABSTRACT FROM AUTHOR]

Human steroid hormone biosynthesis is the result of a complex series of chemical transformations operating on cholesterol, with key steps mediated by members of the cytochrome P450 superfamily. In the formation of the male hormone dehydroepiandrosterone, pregnenolone is first hydroxylated by P450 CYP17A1 at the 17-carbon, followed a second round of catalysis by the same enzyme that cleaves the C17–C20 bond, releasing acetic acid and the 17-keto product. In order to explore the mechanism of this C–C “lyase” activity, we investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated CYP17A1. Our experiments revealed the expected small positive (1.3) isotope effect for the hydroxylase chemistry. However, a surprising result was the large inverse isotope effect (0.39) observed for the C–C bond cleavage activity. These results strongly suggest that the P450 reactive intermediate involved in this latter step is an iron-bound ferric peroxoanion. [ABSTRACT FROM AUTHOR]

The article focuses on the effect of heme environment on the hydrogen abstraction reaction of camphor in P450cam catalysis. In bioorganisms, cytochrome P450 monooxygenases catalyze regio- and stereo-selective C-H bond hydroxylation. Compound I is the primary reactive species of P450 enzymes.

In this article, the authors present the first experimental evidence to demonstrate that the NADPH/O[sub2-negative] and iodosobenzene (PhIO)-supported cytochrome P[sub450] N-dealkylations are mechanistically distinct and, thus, the P [sub450]/PhIO system may not be a good mechanistic model for P[sub450]/NADPH/O[sub2]-catalyzed N-dealkylations. Cytochrome P[sub450] is a family of heme-monooxygenases found in most organisms. Due to its central role, especially with respect to drug metabolism and carcinogenesis, the chemical mechanisms of cytochrome P[sub450] reactions have been extensively studied. Recent cryocrystallographic and ENDOR techniques have unequivocally identified the reaction intermediates iron (Fe) II, FeIIIO[sub2], FeIII-OOH, and FeIII-ROH complexes of P[sub450cam].

Hydroxylation by cytochrome P450 enzymes is one of the most important processes in drug metabolism. A detailed understanding of its mechanism is vital for predicting biotransformations of pharmaceuticals and other xenobiotics, and hence for drug development. The rate and selectivity of P450 substrate oxidation is determined by many factors, such as the nature of the rate-limiting step, docking-related steric effects and intrinsic electronic reactivity. There has been intense experimental6 and theoretical work on the mechanism of aliphatic P450-mediated hydroxylation, but aromatic hydroxylation is less well understood.