NADH reduction of nitroaromatics as a probe for residual ferric form high-spin in a cytochrome P450 (original) (raw)
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Journal of Biological Chemistry, 2009
NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR.
NADPH-Cytochrome P450 Oxidoreductase
Journal of Biological …, 2001
NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP ؉ has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the reface of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. The first mutant, W677X, has the last two C-terminal residues, Trp-677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, the nicotinamide moiety of NADP ؉ lies against the FAD isoalloxazine ring with a tilt of ϳ30°b etween the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding andinteractions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis. . The abbreviations used are: CYPOR, NADPH-cytochrome P450 oxidoreductase; NOS, nitric-oxide synthase; FNR, ferredoxin-NADP ϩ reductase; r.m.s., root mean square.
Biochemistry, 2006
Structural perturbations in cytochrome P450 cam (CYP101) induced by the soluble fragment of rate cytochrome b 5 , a non-physiological effector of CYP101, were investigated by NMR spectroscopy and compared with the perturbations induced by the physiological reductant and effector, putidaredoxin (Pdx). Chemical shifts of perdeuterated [U-15 N] CYP101 backbone amide (NH) resonances were monitored as a function of cytochrome b 5 concentration by 1 H, 15 N TROSY-HSQC experiments. The association of cytochrome b 5 with the reduced CYP101-camphor-carbon monoxide complex (CYP-S-CO) perturbs many of the same resonances that Pdx does, including regions of the CYP101 molecule implicated in substrate access and orientation. The perturbations are smaller in magnitude than those observed with Pdx r due to a lower binding affinity (K d = 13 ± 3 mM, for reduced cytochrome b 5 -CYP-S-CO complex compared to K d = 26 ± 12 μM for the Pdx-CYP-S-CO complex). The results are in accord with our previous suggestion that the observed perturbations are related to effector activity and support the proposal that the primary role of the effector is to populate the active conformation of CYP101 to prevent uncoupling [Pochapsky et al. Biochemistry 42, 5649-5656 (2003)]. A titratable perturbation is observed at the 1 H resonance of the 8-CH 3 of CYP101-bound camphor upon addition of cytochrome b 5 , a phenomenon also associated with the formation of CYP101·Pdx complex albeit with larger perturbations [Wei et al., J. Am. Chem. Soc. 127, 6974-6 976 (2005)]. The effector activity of the particular rat cytochrome b 5 construct used for NMR studies was confirmed by monitoring the enzymatic turnover to yield 5-exo-hydroxy camphor using gas chromatography/mass spectrometry. Finally, the common features of the perturbations observed in the NMR spectra of the two complexes are discussed and their relevance to effector activity considered.
MECHANISTIC ENZYMOLOGY OF OXYGEN ACTIVATION BY THE CYTOCHROMES P450
Drug Metabolism Reviews, 2002
The P450 cytochromes represent a universal class of heme-monooxygenases. The detailed mechanistic understanding of their oxidative prowess is a critical theme in the studies of metabolism of a wide range of organic compounds including xenobiotics. Integral to the O 2 bond cleavage mechanism by P450 is the enzyme's concerted use of protein and solvent-mediated proton transfer events to transform reduced dioxygen to a species capable of oxidative chemistry. To this end, a wide range of kinetic, structural, and mutagenesis data has been accrued. A critical role of conserved acid-alcohol residues in the P450 distal pocket, as well as stabilized waters, enables the enzyme to catalyze effective monooxygenation chemistry. In this review, we discuss the detailed mechanism of P450 dioxygen scission utilizing the CYP101 hydroxylation of camphor as a model system. The application of low-temperature radiolytic techniques has enabled a structural and spectroscopic analysis of the nature of critical intermediate states in the reaction.
One small step for cytochrome P450 in its catalytic cycle, one giant leap for enzymology
The intermediates operating in the cytochrome P450 catalytic cycle have been investigated for more than half a century, fascinating many enzymologists. Each intermediate has its unique role to carry out diverse oxidations. Natural time course of the catalytic cycle is quite fast, hence, not all of the reactive intermediates could be isolated during physiological catalysis. Different high-valent iron intermediates have been proposed as primary oxidants: the candidates are compound 0 (Cpd 0, [FeOOH] 2+ P450) and compound I (Cpd I, Fe(IV)=O por •+ P450). Among them, the role of Cpd I in hydroxylation is fairly well understood due the discovery of the peroxide shunt. This review endeavors to put the outstanding research efforts conducted to isolate and characterize the intermediates together. In addition to spectral features of each intermediate in the catalytic cycle, the oxidizing powers of Cpd 0 and Cpd I will be discussed along with most recent scientific findings.
Journal of Biological Chemistry, 2005
The single turnover of (1R)(؉)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3°C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, ϳ140 and ϳ85 s ؊1 , were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM⅐Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substratefree P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron. . 3 The abbreviations used are: P450-CAM, cytochrome P450 CYP101 isolated from Pseudomonas putida; Pdx, putidaredoxin; MOPS, 4-morpholinepropanesulfonic acid.
Redox-Dependent Dynamics in Cytochrome P450 cam
Biochemistry, 2009
Local protein backbone dynamics of the camphor hydroxylase cytochrome P450 cam (CYP101) depend upon the oxidation and ligation state of the heme iron. 1 H, 15 N correlation nuclear magnetic resonance experiments were used to compare backbone dynamics of oxidized and reduced forms of this 414-residue metalloenzyme via hydrogen-deuterium exchange kinetics (H/D exchange) and 15 N relaxation measurements, and these results are compared with previously published results obtained by H/D exchange mass spectrometry. In general, the reduced enzyme shows lower amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible to these experiments, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B′ helix, β3 and β5 sheets) and binding of putidaredoxin (C and L helices) the iron-sulfur protein that acts as effector and reductant of CYP101 in vivo. These results are interpreted in terms of local structural effects of changes in the heme oxidation state, and relevance of the observed effects to the enzyme mechanism is discussed.