Hydroxylation by the hydroperoxy-iron species in cytochrome P450 enzymes

Intramolecular and intermolecular kinetic isotope effects (KIEs) were determined for hydroxylation of the enantiomers of trans-2-(p-trifluoromethylphenyl)cyclopropylmethane (1) by hepatic cytochrome P450 enzymes, P450s 2B1, Delta2B4, Delta2B4 T302A, Delta2E1, and Delta2E1 T303A. Two products from oxidation of the methyl group were obtained, unrearranged trans-2-(p-trifluoromethylphenyl)cyclopropylmethanol (2) and rearranged 1-(p-trifluoromethylphenyl)but-3-en-1-ol (3). In intramolecular KIE studies with dideuteriomethyl substrates (1-d(2)) and in intermolecular KIE studies with mixtures of undeuterated (1-d(0)) and trideuteriomethyl (1-d(3)) substrates, the apparent KIE for product 2 was consistently larger than the apparent KIE for product 3 by a factor of ca. 1.2. Large intramolecular KIEs found with 1-d(2) (k(H)/k(D) = 9-11 at 10 degrees C) were shown not to be complicated by tunneling effects by variable temperature studies with two P450 enzymes. The results require two independent isotope-sensitive processes in the overall hydroxylation reactions that are either competitive or sequential. Intermolecular KIEs were partially masked in all cases and largely masked for some P450s. The intra- and intermolecular KIE results were combined to determine the relative rate constants for the unmasking and hydroxylation reactions, and a qualitative correlation was found for the unmasking reaction and release of hydrogen peroxide from four of the P450 enzymes in the absence of substrate. The results are consistent with the two-oxidants model for P450 (Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555), which postulates that a hydroperoxy-iron species (or a protonated analogue of this species) is a viable electrophilic oxidant in addition to the consensus oxidant, iron-oxo.     

Formation of the active species of cytochrome p450 by using iodosylbenzene: a case for spin-selective reactivity

The generation of the active species for the enzyme cytochrome P450 by using the highly versatile oxygen surrogate iodosylbenzene (PhIO) often produces different results compared with the native route, in which the active species is generated through O(2) uptake and reduction by NADPH. One of these differences that is addressed here is the deuterium kinetic isotope effect (KIE) jump observed during N-dealkylation of N,N-dimethylaniline (DMA) by P450, when the reaction conditions change from the native to the PhIO route. The paper presents a theoretical analysis targeted to elucidate the mechanism of the reaction of PhIO with heme, to form the high-valent iron-oxo species Compound I (Cpd I), and define the origins of the KIE jump in the reaction of Cpd I with DMA. It is concluded that the likely origin of the KIE jump is the spin-selective chemistry of the enzyme cytochrome P450 under different preparation procedures. In the native route, the reaction proceeds via the doublet spin state of Cpd I and leads to a low KIE value. PhIO, however, diverts the reaction to the quartet spin state of Cpd I, which leads to the observed high KIE values. The KIE jump is reproduced here experimentally for the dealkylation of N,N-dimethyl-4-(methylthio)aniline, by using intra-molecular KIE measurements that avoid kinetic complexities. The effect of PhIO is compared with N,N-dimethylaniline-N-oxide (DMAO), which acts both as the oxygen donor and the substrate and leads to the same KIE values as the native route.     

Probing the oxyferrous and catalytically active ferryl states of Amphitrite ornata dehaloperoxidase by cryoreduction and EPR/ENDOR spectroscopy. Detection of compound I

Dehaloperoxidase (DHP) from Amphitrite ornata is a heme protein that can function both as a hemoglobin and as a peroxidase. This report describes the use of 77 K cryoreduction EPR/ENDOR techniques to study both functions of DHP. Cryoreduced oxyferrous [Fe(II)-O(2)] DHP exhibits two EPR signals characteristic of a peroxoferric [Fe(III)-O(2)(2-)] heme species, reflecting the presence of conformational substates in the oxyferrous precursor. (1)H ENDOR spectroscopy of the cryogenerated substates shows that H-bonding interactions between His N(ε)H and heme-bound O(2) in these conformers are similar to those in the β-chain of oxyferrous hemoglobin A (HbA) and oxyferrous myoglobin, respectively. Decay of cryogenerated peroxoferric heme DHP intermediates upon annealing at temperatures above 180 K is accompanied by the appearance of a new paramagnetic species with an axial EPR signal with g(⊥) = 3.75 and g(∥) = 1.96, characteristic of an S = 3/2 spin state. This species is assigned to Compound I (Cpd I), in which a porphyrin π-cation radical is ferromagnetically coupled with an S = 1 ferryl [Fe(IV)═O] ion. This species was also trapped by rapid freeze-quench of the ambient-temperature reaction mixture of ferric [Fe(III)] DHP and H(2)O(2). However, in the latter case Cpd I is reduced very rapidly by a nearby tyrosine to form Cpd ES [(Fe(IV)═O)(porphyrin)/Tyr(?)]. Addition of the substrate analogue 2,4,6-trifluorophenol (F(3)PhOH) suppresses formation of the Cpd I intermediate during annealing of cryoreduced oxyferrous DHP at 190 K but has no effect on the spectroscopic properties of the remaining cryoreduced oxyferrous DHP intermediates and kinetics of their decay. These observations indicate that substrate (i) binds to oxyferrous DHP outside of the distal pocket and (ii) can reduce Cpd I to Cpd II [Fe(IV)═O]. These assumptions are also supported by the observation that F(3)PhOH has only a small effect on the EPR properties of radiolytically cryooxidized and cryoreduced ferrous [Fe(II)] DHP. EPR spectra of cryoreduced ferrous DHP disclose the multiconformational nature of the ferrous DHP precursor. The observation and characterization of Cpds I, II, and ES in the absence and in the presence of F(3)PhOH provides definitive evidence of a mechanism involving consecutive one-electron steps and clarifies the role of all intermediates formed during turnover.     

Combined Experimental and Theoretical Study on the Reactivity of Compounds I and II in Horseradish Peroxidase Biomimetics

For the exploration of the intrinsic reactivity of two key active species in the catalytic cycle of horseradish peroxidase (HRP), Compound I (HRP‐I) and Compound II (HRP‐II), we generated in situ [Fe^IV?O(TMP^+.)(2‐MeIm)]⁺ and [Fe^IV?O(TMP)(2‐MeIm)]⁰ (TMP=5,10,15,20‐tetramesitylporphyrin; 2‐MeIm=2‐methylimidazole) as biomimetics for HRP‐I and HRP‐II, respectively. Their catalytic activities in epoxidation, hydrogen abstraction, and heteroatom oxidation reactions were studied in acetonitrile at ?15 °C by utilizing rapid‐scan UV/Vis spectroscopy. Comparison of the second‐order rate constants measured for the direct reactions of the HRP‐I and HRP‐II mimics with the selected substrates clearly confirmed the outstanding oxidizing capability of the HRP‐I mimic, which is significantly higher than that of HRP‐II. The experimental study was supported by computational modeling (DFT calculations) of the oxidation mechanism of the selected substrates with the involvement of quartet and doublet HRP‐I mimics (^2,4Cpd I) and the closed‐shell triplet spin HRP‐II model (³Cpd II) as oxidizing species. The significantly lower activation barriers calculated for the oxidation systems involving ^2,4Cpd I than those found for ³Cpd II are in line with the much higher oxidizing efficiency of the HRP‐I mimic proven in the experimental part of the study. In addition, the DFT calculations show that all three reaction types catalyzed by HRP‐I occur on the doublet spin surface in an effectively concerted manner, whereas these reactions may proceed in a stepwise mechanism with the HRP‐II mimic as oxidant. However, the high desaturation or oxygen rebound barriers during C?H bond activation processes by the HRP‐II mimic predict a sufficient lifetime for the substrate radical formed through hydrogen abstraction. Thus, the theoretical calculations suggest that the dissociation of the substrate radical may be a more favorable pathway than desaturation or oxygen rebound processes. Importantly, depending on the electronic nature of the oxidizing species, that is, ^2,4Cpd I or ³Cpd II, an interesting region‐selective conversion phenomenon between sulfoxidation and H‐atom abstraction was revealed in the course of the oxidation reaction of dimethylsulfide. The combined experimental and theoretical study on the elucidation of the intrinsic reactivity patterns of the HRP‐I and HRP‐II mimics provides a valuable tool for evaluating the particular role of the HRP active species in biological systems.     

Kinetic isotope effect is a sensitive probe of spin state reactivity in C-H hydroxylation of N,N-dimethylaniline by cytochrome P450

This paper presents DFT calculations of C-H hydroxylation of N,N-dimethylaniline by Compound I (Cpd I) of cytochrome P450. The reaction involves two processes nascent from the two spin states of Cpd I, the low-spin (LS) and high-spin (HS) states. The calculations demonstrate that the kinetic isotope effects (KIEs) of the two processes are very different, and only KIELS fits the experimental datum. As such, KIE can be a sensitive probe of spin state reactivity.     

Heme compound II models in chemoselectivity and disproportionation reactions

Heme compound II models bearing electron-deficient and -rich porphyrins, [Fe^IV(O)(TPFPP)(Cl)]⁻ (1a) and [Fe^IV(O)(TMP)(Cl)]⁻ (2a), respectively, are synthesized, spectroscopically characterized, and investigated in chemoselectivity and disproportionation reactions using cyclohexene as a mechanistic probe. Interestingly, cyclohexene oxidation by 1a occurs at the allylic C–H bonds with a high kinetic isotope effect (KIE) of 41, yielding 2-cyclohexen-1-ol product; this chemoselectivity is the same as that of nonheme iron(iv)-oxo intermediates. In contrast, as observed in heme compound I models, 2a yields cyclohexene oxide product with a KIE of 1, demonstrating a preference for C C epoxidation. The latter result is interpreted as 2a disproportionating to form [Fe^IV(O)(TMP⁺˙)]⁺ (2b) and Fe^III(OH)(TMP), and 2b becoming the active oxidant to conduct the cyclohexene epoxidation. In contrast to 2a, 1a does not disproportionate under the present reaction conditions. DFT calculations confirm that compound II models prefer C–H bond hydroxylation and that disproportionation of compound II models is controlled thermodynamically by the porphyrin ligands. Other aspects, such as acid and base effects on the disproportionation of compound II models, have been discussed as well.

Disproportionation of Cpd II models depends on the electron-richness of the porphyrin ligand; Cpd II with an electron-deficient ligand is difficult to disproportionate, whereas Cpd II with an electron-rich ligand readily disproportionates to form Cpd I as a true oxidant.     

A high-valent iron-oxo corrolazine activates C-H bonds via hydrogen-atom transfer

Oxidation of the Fe(III) complex (TBP(8)Cz)Fe(III) [TBP(8)Cz = octakis(4-tert-butylphenyl)corrolazinate] with O-atom transfer oxidants under a variety of conditions gives the reactive high-valent Fe(O) complex (TBP(8)Cz(+?))Fe(IV)(O) (2). The solution state structure of 2 was characterized by XAS [d(Fe-O) = 1.64 ?]. This complex is competent to oxidize a range of C-H substrates. Product analyses and kinetic data show that these reactions occur via rate-determining hydrogen-atom transfer (HAT), with a linear correlation for log k versus BDE(C-H), and the following activation parameters for xanthene (Xn) substrate: ΔH(++) = 12.7 ± 0.8 kcal mol(-1), ΔS(++) = -9 ± 3 cal K(-1) mol(-1), and KIE = 5.7. Rebound hydroxylation versus radical dimerization for Xn is favored by lowering the reaction temperature. These findings provide insights into the factors that control the intrinsic reactivity of Compound I heme analogues.     

Mechanistic studies of the oxidative N-dealkylation of a substrate tethered to carboxylate-bridged diiron(II) complexes, [Fe2(mu-O2CAr(Tol))2(O2CAr(Tol))2(N,N-Bn2en)2

Carboxylate-bridged diiron(II) centers activate dioxygen for the selective oxidation of hydrocarbon substrates in bacterial multicomponent monooxygenases. Synthetic analogues of these systems exist in which substrate fragments tethered to the diiron(II) core through attachment to an N-donor ligand are oxidized by transient species that arise following the introduction of O2 into the system. The present study describes the results of experiments designed to probe mechanistic details of these oxidative N-dealkylation reactions. A series of diiron(II) complexes with ligands N,N-(4-R-Bn)Bnen, where en is ethylenediamine, Bn is benzyl, and R-Bn is benzyl with a para-directing group R = Cl, F, CH3, t-Bu, or OCH3, were prepared. A Hammett plot of the oxygenation product distributions of these complexes, determined by gas chromatographic analysis, reveals a small positive slope of rho = +0.48. Kinetic isotope effect (KIE(intra)) values for oxygenation of [Fe2(mu-O2CAr(Tol))2(O2CAr(Tol))2(N,N-(C6H5CDH)2en)2] and [Fe2(mu-O2CAr(Tol))2(O2CAr(Tol))2(N,N-(C6H5CD2)(C6H5CH2)en)2] are 1.3(1) and 2.2(2) at 23 degrees C, respectively. The positive slope rho and low KIE(intra) values are consistent with a mechanism involving one-electron transfer from the dangling nitrogen atom in N,N-Bn2en to a transient electrophilic diiron intermediate, followed by proton transfer and rearrangement to eliminate benzaldehyde.     

On the functional role of a water molecule in clade 3 catalases: a proposal for the mechanism by which NADPH prevents the formation of compound II

X-ray structures of the 13 different monofunctional heme catalases published to date were scrutinized in order to gain insight in the mechanism by which NADPH in Clade 3 catalases may protect the reactive ferryloxo intermediate Compound I (Cpd I; por (*+)Fe (IV)O) against deactivation to the catalytically inactive intermediate Compound II (Cpd II; porFe (IV)O). Striking similarities in the molecular network of the protein subunits encompassing the heme center and the surface-bound NADPH were found for all of the Clade 3 catalases. Unique features in this region are the presence of a water molecule (W1) adjacent to the 4-vinyl group of heme and a serine residue or a second water molecule hydrogen-bonded to both W1 and the carbonyl group of a threonine-proline linkage, with the proline in van der Waals contact with the dihydronicotinamide group of NADPH. A mechanism is proposed in which a hydroxyl anion released from W1 undergoes reversible nucleophilic addition to the terminal carbon of the 4-vinyl group of Cpd I, thereby producing a neutral porphyrin pi-radical ferryloxo (HO-por (*)Fe (IV)O) species of reduced reactivity. This structure is suggested to be the elusive Cpd II' intermediate proposed in previous studies. An accompanying proton-shifting process along the hydrogen-bonded network is believed to facilitate the NADPH-mediated reduction of Cpd I to ferricatalase and to serve as a funnel for electron transfer from NADPH to the heme center to restore the catalase Fe (III) resting state. The proposed reaction paths were fully supported as chemically reasonable and energetically feasible by means of density functional theory calculations at the (U)B3LYP/6-31G* level. A particularly attractive feature of the present mechanism is that the previously discussed formation of protein-derived radicals is avoided.     

The Poulos-Kraut mechanism of Compound I formation in horseradish peroxidase: a QM/MM study

QM/MM calculations are used to elucidate the Poulos-Kraut (Poulos, T. L.; Kraut, J. J. Biol. Chem. 1980, 255, 8199-8205) mechanism of O-O bond activation and Compound I (Cpd I) formation in HRP, in conditions corresponding to neutral to basic pH. Attempts to generate Compound I directly from the Fe(H2O2) complex by migrating the proton from the proximal oxygen to the distal one (1,2- proton shift) result in high barriers. The lowest energy mechanism was found to involve initial deprotonation of ferric hydrogen peroxide complex (involving spin crossover from the quartet to the doublet state) by His42 to form ferric-hydroperoxide (Cpd 0). Subsequently, the distal OH group of Cpd 0 is pulled by Arg38 and reprotonated by His42(H+) to form Cpd I and a water molecule that bridges the two residues. The structures of the intermediate and the transition state reveal the manner by which the Arg-His residues promote cooperatively the electronic reorganization that is required to attend the heterolytic O-O cleavage.     

QM/MM study of the active species of the human cytochrome P450 3A4, and the influence thereof of the multiple substrate binding

Cytochrome P450 3A4 is involved in the metabolism of 50% of all swallowed drugs. The enzyme functions by means of a high-valent iron-oxo species, called compound I (Cpd I), which is formed after entrance of the substrate to the active site. We explored the features of Cpd I using hybrid quantum mechanical/molecular mechanical calculations on various models that are either substrate-free or containing one and two molecules of diazepam as a substrate. M?ssbauer parameters of Cpd I were computed. Our major finding shows that without the substrate, Cpd I tends to elongate its Fe-S bond, localize the radical on the sulfur, and form hydrogen bonds with A305 and T309, which may hypothetically lead to Cpd I consumption by H-abstraction. However, the positioning of diazepam close to Cpd I, as enforced by the effector molecule, was found to strengthen the NH...S interactions of the conserved I443 and G444 residues with the proximal cysteinate ligand. These interactions are known to stabilize the Fe-S bond, and as such, the presence of the substrate leads to a shorter Fe-S bond and it prevents the localization of the radical on the sulfur. This diazepam-Cpd I stabilization was manifested in the 1W0E conformer. The effector substrate did not influence Cpd I directly but rather by positioning the active substrate close to Cpd I, thus displacing the hydrogen bonds with A305 and T309, and thereby giving preference to substrate oxidation. It is hypothesized that these effects on Cpd I, promoted by the restrained substrate, may be behind the special metabolic behavior observed in cases of multiple substrate binding (also called cooperative binding). This restraint constitutes a mechanism whereby substrates stabilize Cpd I sufficiently long to affect monooxygenation by P450s at the expense of Cpd I destruction by the protein residues.     

Can a single oxidant with two spin states masquerade as two different oxidants? A study of the sulfoxidation mechanism by cytochrome p450

Density functional calculations were performed on the sulfoxidation reaction by a model compound I (Cpd I) of cytochrome P450. By contrast to previous alkane hydroxylation studies, which exhibit a dominant low-spin (LS) pathway, the sulfoxidation follows a dominant high-spin (HS) reaction. Thus, competing hydroxylation and sulfoxidation processes as observed for instance by Jones et al. (Volz, T. J.; Rock, D. A.; Jones, J. P. J. Am. Chem. Soc. 2002, 124, 9724) are the result of a two-state reactivity scenario, whereby the hydroxylation originates from the LS pathway and the sulfoxidation from the HS pathway. In this manner, two spin states of a single oxidant (Cpd I) can be disguised as two different oxidants. The calculations rule out the possibility that a second oxidant (the ferric peroxide, Cpd 0 species) interferes in the observed results of Jones et al.     

Compound I of nitric oxide synthase: the active site protonation state

A quantum mechanical/molecular mechanical (QM/MM) study of the formation of the elusive active species Compound I (Cpd I) of nitric oxide synthase (NOS) from the oxyferrous intermediate shows that two protons have to be provided to produce a reaction that is reasonably exothermic and that leads to the appearance of a radical on the tetrahydrobiopterin cofactor. Molecular dynamics and energy considerations show that a possible source of proton is the water H-bond chain formed from the surface to the active site, but that a water molecule by itself cannot be the source of the proton; an H3O+ species that is propagated along the chain is more likely. The QM/MM calculations demonstrate that Cpd I and H2O are formed from the ferric-hydrogen peroxide complex in a unique heterolytic O-O cleavage mechanism. The properties of the so-formed Cpd I are compared with those of the known species of chloroperoxidase, and the geometry and spin densities are found to be compatible. The M?ssbauer parameters are calculated and may serve as experimental probes in attempts to characterize NOS Cpd I.     

Can ferric-superoxide act as a potential oxidant in P450(cam)? QM/MM investigation of hydroxylation, epoxidation, and sulfoxidation

In view of recent reports of high reactivity of ferric-superoxide species in heme and nonheme systems (Morokuma et al. J. Am. Chem. Soc. 2010, 132, 11993-12005; Que et al. Inorg. Chem. 2010, 49, 3618-3628; Nam et al. J. Am. Chem. Soc. 2010, 132, 5958-5959; J. Am. Chem. Soc. 2010, 132, 10668-10670), we use herein combined quantum mechanics/molecular mechanics (QM/MM) methods to explore the potential reactivity of P450(cam) ferric-superoxide toward hydroxylation, epoxidation, and sulfoxidation. The calculations demonstrate that P450 ferric-superoxide is a sluggish oxidant compared with the high-valent oxoiron porphyrin cation-radical species. As such, unlike heme enzymes with a histidine axial ligand, the P450 superoxo species does not function as an oxidant in P450(cam). The origin of this different behavior of the superoxo species of P450 vis-a?-vis other heme enzymes like tryptophan 2, 3-dioxygenase (TDO) is traced to the ability of the latter superoxo species to make a stronger FeOO-X (X = H,C) bond and to stabilize the corresponding bond-activation transition states by resonance with charge-transfer configurations. By contrast, the negatively charged thiolate ligand in the P450 superoxo species minimizes the mixing of charge transfer configurations in the transition state and raises the reaction barrier. However, as we demonstrate, an external electric field oriented along the Fe-O axis with a direction pointing from Fe toward O will quench Cpd I formation by slowing the reduction of ferric-superoxide and will simultaneously lower the barriers for oxidation by the latter species, thereby enabling observation of superoxo chemistry in P450. Other options for nascent superoxo reactivity in P450 are discussed.     

New features in the catalytic cycle of cytochrome P450 during the formation of compound I from compound 0

Density functional theory (DFT) is applied to the dark section of the catalytic cycle of the enzyme cytochrome P450, namely, the formation of the active species, Compound I (Cpd I), from the ferric-hydroperoxide species (Cpd 0) by a protonation-assisted mechanism. The chosen 96-atom model includes the key functionalities deduced from experiment: Asp(251), Thr(252), Glu(366), and the water channels that relay the protons. The DFT model calculations show that (a) Cpd I is not formed spontaneously from Cpd 0 by direct protonation, nor is the process very exothermic. The process is virtually thermoneutral and involves a significant barrier such that formation of Cpd I is not facile on this route. (b) Along the protonation pathway, there exists an intermediate, a protonated Cpd 0, which is a potent oxidant since it is a ferric complex of water oxide. Preliminary quantum mechanical/molecular mechanical calculations confirm that Cpd 0 and Cpd I are of similar energy for the chosen model and that protonated Cpd 0 may exist as an unstable intermediate. The paper also addresses the essential role of Thr(252) as a hydrogen-bond acceptor (in accord with mutation studies of the OH group to OMe).     

Spectroscopic and Kinetic Evidence for the Crucial Role of Compound 0 in the P450cam‐Catalyzed Hydroxylation of Camphor by Hydrogen Peroxide

The hydroperoxo iron(III) intermediate P450_camFe^III–OOH, being the true Compound 0 (Cpd 0) involved in the natural catalytic cycle of P450_cam, could be transiently observed in the peroxo‐shunt oxidation of the substrate‐free enzyme by hydrogen peroxide under mild basic conditions and low temperature. The prolonged lifetime of Cpd 0 enabled us to kinetically examine the formation and reactivity of P450_camFe^III–OOH species as a function of varying reaction conditions, such as pH, and concentration of H₂O₂, camphor, and potassium ions. The mechanism of hydrogen peroxide binding to the substrate‐free form of P450_cam differs completely from that observed for other heme proteins possessing the distal histidine as a general acid–base catalyst and is mainly governed by the ability of H₂O₂ to undergo deprotonation at the hydroxo ligand coordinated to the iron(III) center under conditions of pH≥p${K{{{\rm P450}\hfill \atop {\rm a}\hfill}}}$ . Notably, no spectroscopic evidence for the formation of either Cpd I or Cpd II as products of heterolytic or homolytic O?O bond cleavage, respectively, in Cpd 0 could be observed under the selected reaction conditions. The kinetic data obtained from the reactivity studies involving (1R)‐camphor, provide, for the first time, experimental evidence for the catalytic activity of the P450Fe^III–OOH intermediate in the oxidation of the natural substrate of P450_cam.     

不同催化剂对苯酚H2O2羟基化反应的比较

用苯酚、双氧水经羟基化反应合成邻、对苯二酚,在Fe2O3为催化剂的反应系统中,反应诱导期长,且反应剧烈;当以杂多酸盐或Fe/ZrO2为催化剂时,诱导期消失,且羟基化反应平稳。 反应的诱导期可能是由铁的氧化物转化为活性物种Fe3+和Fe2+的过程引起的;在杂多酸盐或Fe/ZrO2催化剂的反应体系中,羟基化反应可能发生在催化剂表面而使反应变得缓慢平稳,反应诱导期消失。     

QM/MM study of mechanisms for compound I formation in the catalytic cycle of cytochrome P450cam

In the catalytic cycle of cytochrome P450cam, after molecular oxygen binds as a ligand to the heme iron atom to yield a ferrous dioxygen complex, there are fast proton transfers that lead to the formation of the active species, Compound I (Cpd I), which are not well understood because they occur so rapidly. In the present work, the conversion of the ferric hydroperoxo complex (Cpd 0) to Cpd I has been investigated by combined quantum-mechanical/molecular-mechanical (QM/MM) calculations. The residues Asp(251) and Glu(366) are considered as proton sources. In mechanism I, a proton is transported to the distal oxygen atom of the hydroperoxo group via a hydrogen bonding network to form protonated Cpd 0 (prot-Cpd0: FeOOH(2)), followed by heterolytic O-O bond cleavage that generates Cpd I and water. Although a local minimum is found for prot-Cpd0 in the Glu(366) channel, it is very high in energy (more than 20 kcal/mol above Cpd 0) and the barriers for its decay are only 3-4 kcal/mol (both toward Cpd 0 and Cpd I). In mechanism II, an initial O-O bond cleavage followed by a concomitant proton and electron transfer yields Cpd I and water. The rate-limiting step in mechanism II is O-O cleavage with a barrier of about 13-14 kcal/mol. According to the QM/MM calculations, the favored low-energy pathway to Cpd I is provided by mechanism II in the Asp(251) channel. Cpd 0 and Cpd I are of similar energies, with a slight preference for Cpd I.     

What is the active species of cytochrome P450 during camphor hydroxylation? QM/MM studies of different electronic states of compound I and of reduced and oxidized iron-oxo intermediates

We have investigated C-H hydroxylation of camphor by Compound I (Cpd I) of cytochrome P450cam in different electronic states and by its one-electron reduced and oxidized forms, using QM/MM calculations in the native protein/solvent environment. Cpd I species with five unpaired electrons (pentaradicaloids) are ca. 12 kcal/mol higher in energy than the ground state Cpd I species with three unpaired electrons (triradicaloids). The H-abstraction transition states of pentaradicaloids lie ca. 21 (9) kcal/mol above the triradicaloid (pentaradicaloid) reactants. Hydroxylation via pentaradicaloids is thus facile provided that they can react before relaxing to the ground-state triradicaloids. Excited states of Cpd I with an Fe(V)-oxo moiety lie more than 20 kcal/mol above the triradicaloid ground state in single-point gas-phase calculations, but these electronic configurations are not stable upon including the point-charge protein environment which causes SCF convergence to the triradicaloid ground state. One-electron reduced species (Cpd II) show sluggish reactivity compared with Cpd I in agreement with experimental model studies. One-electron oxidized species are more reactive than Cpd I but seem too high in energy to be accessible. The barriers to hydrogen abstraction for the various forms of Cpd I are generally not affected much by the chosen protonation states of the Asp297 and His355 residues near the propionate side chains of the heme or by the appearance of radical character at Asp297, His355, or the propionates.     

Electronic state of the molecular oxygen released by catalase

In catalases, the high redox intermediate known as compound I (Cpd I) is reduced back to the resting state by means of hydrogen peroxide in a 2-electron reaction [Cpd I (Por(*+)-Fe(IV)O) + H(2)O(2) --> Enz (Por-Fe(III)) + H(2)O + O(2)]. It has been proposed that this reaction takes place via proton transfer toward the distal His and hydride transfer toward the oxoferryl oxygen (H(+)/H(-) scheme) and some authors have related it to singlet oxygen generation. Here, we consider the possible reaction schemes and qualitatively analyze the electronic state of the species involved to show that the commonly used association of the H(+)/H(-) scheme with singlet oxygen production is not justified. The analysis is complemented with density functional theory (DFT) calculations for a gas-phase active site model of the reactants and products.