In Silico Design of a Mutant of Cytochrome P450 Containing Selenocysteine

A mutant of P450cam, in which the cysteine ligand was replaced by selenocysteine, was designed theoretically using hybrid QM/MM (quantum mechanical/molecular mechanical) calculations. The calculations of the active species, Se−CpdI (selenocysteine−Compound I), of the mutant enzyme indicate that Se−Cpd I will be formed faster than the wild-type species and be consumed more slowly in C−H hydroxylation. As such, our calculations suggest that Se−Cpd I can be observed unlike the elusive species of the wild-type enzyme (Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I. Chem. Rev. 2005, 105, 2253−2277). Spectral features of Se−Cpd I were calculated and may assist such eventual characterization. The observation of Se−Cpd I will resolve the major puzzle in the catalytic cycle of a key enzyme in nature

Theory Favors a Stepwise Mechanism of Porphyrin Degradation by a Ferric Hydroperoxide Model of the Active Species of Heme Oxygenase

The report uses density functional theory to address the mechanism of heme degradation by the enzyme heme oxygenase (HO) using a model ferric hydroperoxide complex. HO is known to trap heme molecules and degrade them to maintain iron homeostasis in the biosystem (Ortiz de Montellano, P. R. Acc. Chem. Res. 1998, 31, 543). The degradation is initiated by complexation of the heme, then formation of the iron−hydroperoxo species, which subsequently oxidizes the meso position of the porphyrin by hydroxylation, thereby enabling eventually the cleavage of the porphyrin ring. Kinetic isotope effect studies (Davydov, R.; Matsui, T.; Fujii, H.; Ikeda-Saito, M.; Hoffman, B. M. J. Am. Chem. Soc. 2003, 125, 16208) indicate that the mechanism is assisted by general acid catalysis, via a chain of water molecules, and that all the events occur in concert. However, previous theoretical treatments indicated that the concerted mechanism has a high barrier, much higher than an alternative mechanism that is initiated by O−O bond homolysis of iron−hydroperoxide (Sharma, P. K.; Kevorkiants, R.; de Visser, S. P.; Kumar, D.; Shaik, S. Angew. Chem. Int. Ed. 2004, 43, 1129). The present contribution studies the stepwise and concerted acid-catalyzed mechanisms using H3O+(H2O)n, n = 0−2. The effect of the acid strength is tested using the H4N+(H2O)2 cluster and a fully protonated ferric hydroperoxide. All the calculations show that a stepwise mechanism that involves proton relay and O−O homolysis, in the rate-determining step, has a much lower barrier (>10 kcal/mol) than the corresponding fully concerted mechanism. The best fit of the calculated solvent kinetic isotope effect, to the experimental data, is obtained for the H3O+(H2O)2 cluster. The calculated α-deuterium secondary kinetic isotope effect is inverse (0.95−0.98), but much less so than the experimental value (0.7). Possible reasons for this quantitative difference are discussed. Some probes are suggested that may enable experiment to distinguish the stepwise from the concerted mechanism

Oxygen Economy of Cytochrome P450: What Is the Origin of the Mixed Functionality as a Dehydrogenase−Oxidase Enzyme Compared with Its Normal Function?

The economy of dioxygen consumption by enzymes constitutes a fundamental problem in enzymatic chemistry (ref ). Sometimes, the enzyme converts ALL the oxygen into water, without affecting the organic substrate, thereby acting as an “oxidase” (ref ). Other times, the enzyme converts all the oxygen into water and causes desaturation in the substrate, thus exhibiting a mixed function as both “oxidase” and “dehydrogenase” (refs −5). The present paper describes density functional calculations demonstrating that the oxidase−dehydrogenase mixed activity occurs from the cationic intermediate species and requires electro−steric inhibition of the rebound process. Furthermore, the calculations reveal that the carbocation is formally nascent from an excited state of the active species of the enzyme (2Cpd I), in which the FeO moiety is singlet coupled as in the Δg state of dioxygen! Thus, our results resolve an important mechanism and reveal the factors that underlie its observability

How Does Product Isotope Effect Prove the Operation of a Two-State “Rebound” Mechanism in C−H Hydroxylation by Cytochrome P450?

C−H hydroxylation is a fundamental process. In Nature it is catalyzed by the enzyme cytochrome P450, in a still-debated mechanism that poses a major intellectual challenge for both experiment and theory; currently, the opinions keep swaying between the original single-state rebound mechanism, a two-oxidant mechanism (where ferric peroxide participates as a second oxidant, in addition to the primary active species, the high-valent iron−oxo species), and two-state reactivity (TSR) mechanism (where two spin states are involved). Recent product isotope effect (PIE) measurements for the trans-2-phenyl-methyl cyclopropane probe (1), led Newcomb and co-workers (Newcomb, M.; Aebisher, D.; Shen, R.; Esala, R.; Chandrasena, P.; Hollenberg, P.; Coon, M. J. J. Am. Chem. Soc. 2003, 125, 6064−6065) to rule out TSR in favor of the two-oxidant scenario, since the direction of the PIE was at odds with the one predicted from calculations on methane hydroxylation. The present report describes a density functional theoretical study of C−H hydroxylation of the Newcomb probe, 1, leading to rearranged (3) and unrearranged (2) products. Our study shows that the reaction occurs via TSR in which the high-spin pathway gives dominant rearranged products, whereas the low-spin pathway favors unrearranged products. The calculated PIE(2/3) values based on TSR are found to be in excellent agreement with the experimental data of Newcomb and co-workers. This match between experiment and theory makes a strong case that the reaction occurs via TSR mechanism

A Valence Bond Modeling of Trends in Hydrogen Abstraction Barriers and Transition States of Hydroxylation Reactions Catalyzed by Cytochrome P450 Enzymes

The paper outlines the fundamental factors that govern the mechanisms of alkane hydroxylation by cytochrome P450 and the corresponding barrier heights during the hydrogen abstraction and radical rebound steps of the process. This is done by a combination of density functional theory calculations for 11 alkanes and valence bond (VB) modeling of the results. The energy profiles and transition states for the various steps are reconstructed using VB diagrams (Shaik, S. S. J. Am. Chem. Soc. 1981, 103, 3692–3701. Shaik, S.; Shurki, A. Angew. Chem. Int. Ed. 1999, 38, 586–625.) and the DFT barriers are reproduced by the VB model from raw data based on C−H bond energies. The model explains a variety of other features of P450 hydroxylations: (a) the nature of the polar effect during hydrogen abstraction, (b) the difference between the activation mechanisms leading to the FeIV vs the FeIII electromers, (c) the difference between the gas phase and the enzymatic reaction, and (d) the dependence of the rebound barrier on the spin state. The VB mechanism shows that the active species of the enzyme performs a complex reaction that involves multiple bond making and breakage mechanisms by utilizing an intermediate VB structure that cuts through the high barrier of the principal transformation between reactants and products, thereby mediating the process at a low energy cost. The correlations derived in this paper create order and organize the data for a process of a complex and important enzyme. This treatment can be generalized to the reactivity patterns of nonheme systems and synthetic iron−oxo porphyrin reagents

Axial Ligand Effect On The Rate Constant of Aromatic Hydroxylation By Iron(IV)–Oxo Complexes Mimicking Cytochrome P450 Enzymes

The cytochromes P450 are important iron-heme based monoxygenases that catalyze a range of different oxygen atom transfer reactions in nature. One of the key bioprocesses catalyzed by these enzymes is the aromatic hydroxylation of unactivated arenes. To gain insight into axial ligand effects and, in particular, how it affects aromatic hydroxylation processes by P450 model complexes, we studied the effects of the axial ligand on spectroscopic parameters (trans-influence) as well as on aromatic hydroxylation kinetics (trans-effect) using a range of [FeIV(O)(Por+•)X] oxidants with X = SH–, Cl–, F–, OH–, acetonitrile, GlyGlyCys–, CH3COO–, and CF3COO–. These systems give red-shifted Fe–O vibrations that are dependent on the strength of the axial ligand. Despite structural changes, however, the electron affinities of these oxidants are very close in energy, but sharp differences in pKa values are found. The aromatic hydroxylation of the para-position of ethylbenzene was tested with these oxidants, and they all show two-state-reactivity patterns although the initial low-spin C–O bond formation barrier is rate determining. We show, for the first time, that the rate determining barrier for aromatic hydroxylation is proportional to the strength of the O–H bond in the corresponding iron(IV)–hydroxo complex, i.e., BDEOH, hence this thermochemical property of the oxidant drives the reaction and represents the axial ligand effect. We have rationalized our observed barrier heights for these axially ligated systems using thermochemical cycles and a valence bond curve crossing diagram to explain the origins of the rate constants

Theoretical Investigation of C−H Hydroxylation by (N4Py)Fe^IVO²⁺: An Oxidant More Powerful than P450?

DFT calculations of C−H hydroxylation by a synthetic nonheme oxoiron(IV) oxidant supported by a neutral pentadentate N5 ligand show that this reagent is intrinsically more reactive than compound I of P450. This nonheme iron oxidant is predicted to exhibit stereoselective reactions, strong solvent effect, and involve multistate reactivity with spin-state crossing

External Electric Field Will Control the Selectivity of Enzymatic-Like Bond Activations

Controlling the selectivity of a chemical reaction is a Holy Grail in chemistry. This paper reports theoretical results of unprecedented effects induced by moderately strong electric fields on the selectivity of two competing nonpolar bond activation processes, C−H hydroxylation vs CC epoxidation, promoted by an active species that is common to heme-enzymes and to metallo-organic catalysts. The molecular system by itself shows no selectivity whatsoever. However, the presence of an electric field induces absolute selectivity that can be controlled at will. Thus, the choice of the orientation and direction of the field vis-à-vis the molecular axes drives the reaction in the direction of complete C−H hydroxylation or complete CC epoxidation

Theoretical Study on the Mechanism of the Oxygen Activation Process in Cysteine Dioxygenase Enzymes

Cysteine dioxygenase (CDO) is a vital enzyme for human health involved in the biodegradation of toxic cysteine and thereby regulation of the cysteine concentration in the body. The enzyme belongs to the group of nonheme iron dioxygenases and utilizes molecular oxygen to transfer two oxygen atoms to cysteinate to form cysteine sulfinic acid products. The mechanism for this reaction is currently disputed, with crystallographic studies implicating a persulfenate intermediate in the catalytic cycle. To resolve the dispute we have performed quantum mechanics/molecular mechanics (QM/MM) calculations on substrate activation by CDO enzymes using an enzyme monomer and a large QM active region. We find a stepwise mechanism, whereby the distal oxygen atom of the iron(II)-superoxo complex attacks the sulfur atom of cysteinate to form a ring structure, followed by dioxygen bond breaking and the formation of a sulfoxide bound to an iron(IV)-oxo complex. A sulfoxide rotation precedes the second oxygen atom transfer to the substrate to give cysteine sulfinic acid products. The reaction takes place on several low-lying spin-state surfaces via multistate reactivity patterns. It starts in the singlet ground state of the iron(II)-superoxo reactant and then proceeds mainly on the quintet and triplet surfaces. The initial and rate-determining attack of the superoxo group on the cysteinate sulfur atom involves a spin-state crossing from singlet to quintet. We have also investigated an alternative mechanism via a persulfenate intermediate, with a realignment of hydrogen bonding interactions in the substrate binding pocket. However, this alternative mechanism of proximal oxygen atom attack on the sulfur atom of cysteinate is computed to be a high-energy pathway, and therefore, the persulfenate intermediate is unlikely to participate in the catalytic cycle of CDO enzymes

Catalysts for Monooxygenations Made from Polyoxometalate: An Iron(V)−Oxo Derivative of the Lindqvist Anion

This work uses density functional calculations to design a new high-valent Fe(V)O catalyst [Mo5O18FeO]3-, which is based on the Lindqvist polyoxometalate (Mo6O192-). Because the parent species is stable to oxidative conditions, one may assume that the newly proposed iron−oxo species will be stable, too. The calculated Mössbauer spectroscopic data may be helpful toward an eventual identification of the species. The calculations of C−H hydroxylation and CC epoxidation of propene show that, if made, [Mo5O18FeO]3- should be a potent oxidant that will be subject to strong solvent effect. Moreover, the Lindqvist catalyst leads to an intriguing result; the reaction that starts along an epoxidation pathway with CC activation ends with a C−H hydroxylation product (46) due to rearrangement on the catalyst. The origins of this result are analyzed in terms of the structure of the catalyst and the electronic requirements for conversion of an epoxidation intermediate to a hydroxylation product. Thus, if made, the [Mo5O18FeO]3 will be a selective C−H hydroxylation reagent