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Nitrogen activation by nitrogenase is one of the most important enzymatic processes on earth. In spite of the determination of X-ray structures of increasingly higher resolution, the nitrogenase mechanism is still not understood. In the most recent X-ray structures it has been shown that a carbon resides in the center of the MoFe-cofactor. Its role is not known. Recent spectroscopic studies, mainly EPR, have come closest to obtaining a molecular mechanism for activating nitrogen. Two hydrides have been shown to play a key role in this context. In the present study, the mechanism for nitrogenase has been investigated by hybrid DFT using a cluster model. This approach has been shown to be very successful for predicting mechanisms for other redox-active enzymes, such as the one for photosystem II, but has so far not been used in its most recent form for nitrogenase. The mechanism obtained has large similarities to the one suggested by spectroscopy, with a reductive elimination of two hydrides just before nitrogen binding. However, a very surprising finding is that the central carbon becomes protonated and has to move out of the cavity as a methyl group before the hydrides can be formed. This has not been suggested before.

Ring hydroxylation and coupled rearrangement reactions catalyzed by 4-hydroxyphenylpyruvate dioxygenase were studied with the QM/MM method ONIOM(B3LYP:AMBER). For electrophilic attack of the ferryl species on the aromatic ring, five channels were considered: attacks on the three ring atoms closest to the oxo ligand (C1, C2, C6) and insertion of oxygen across two bonds formed by them (C1-C2, C1-C6). For the subsequent migration of the carboxymethyl substituent, two possible directions were tested (C1→C2, C1→C6), and two different mechanisms were sought (stepwise radical, single-step heterolytic). In addition, formation of an epoxide (side)product and benzylic hydroxylation, as catalyzed by the closely related hydroxymandelate synthase, were investigated. From the computed reaction free energy profiles it follows that the most likely mechanism of 4-hydroxyphenylpyruvate dioxygenase involves electrophilic attack on the C1 carbon of the ring and subsequent single-step heterolytic migration of the substituent. Computed values of the kinetic isotope effect for this step are inverse, consistent with available experimental data. Electronic structure arguments for the preferred mechanism of attack on the ring are also presented.

The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Högbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn(III)/Fe(III) dimer linked by a μ-hydroxo/bis-μ-carboxylato bridging network. The Mn(III) ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2loxPhoto) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn(III)/Fe(III) cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn(III) ion, as best evidenced by a reorientation of its unique (55)Mn hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the μ-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform.

Hybrid DFT model calculations have been performed for some cobalt complexes capable of oxidizing water. Since a very plausible mechanism for the oxygen-evolving complex involving the cuboidal Mn4Ca structure in photosystem II (PSII) has recently been established, the most important part of the present study concerns a detailed comparison between cobalt and manganese as water oxidation catalysts. One similarity found is that a M(IV)-O(•) state is the key precursor for O-O bond formation in both cases. This means that simply getting a M(IV) state is not enough; a formal M(V)═O state is required, with two oxidations on one center from M(III). For cobalt, not even that is enough. A singlet coupled state is required at this oxidation level, which is not the ground state. It is shown that there are also more fundamental differences between catalysts based on these metals. The favorable low-barrier direct coupling mechanism found for PSII is not possible for the corresponding cobalt complexes. The origin of this difference is explained. For the only oxygen-evolving cubic Co4O4 complex with a defined structure, described by Dismukes et al., the calculated results are in good agreement with experiments. For the Co4 models of the amorphous cobalt-oxo catalyst found by Nocera et al., higher barriers are found than the one obtained experimentally. The reasons for this are discussed.

Detailed mechanisms for substrate water exchange in the oxygen evolving complex in photosystem II have been determined with DFT methods for large models. Existing interpretations of the experimental water exchange results have been quite different. By many groups, these results have been the main argument against the water oxidation mechanism suggested by DFT, in which the oxygen molecule is formed between a bridging oxo and an oxyl radical ligand in the center of the OEC. That mechanism is otherwise in line with most experiments. The problem has been that the mechanism requires a rather fast exchange of a bridging oxo ligand, which is not a common finding for smaller Mn-containing model systems. However, other groups have actually favored a substrate derived oxo ligand partly based on the same experiments. In the present study, three S-states have been studied, and the rates have been well reproduced by the calculations. The surprising experimental finding that water exchange in S1 is slower than the one in S2 is reproduced and explained. The key to this rate difference is the ease by which one of the manganese centers (Mn3) is reduced. This reduction has to occur to release the substrate water from Mn3. The similar rate of the slow exchange in S2 and S3 has been rationalized on the basis of earlier experiments combined with the present calculations. The results strongly support the previous DFT-suggested water oxidation mechanism.

The Mn 1s near-edge absorption fine structure (NEXAFS) has been computed by means of transition-state gradient-corrected density functional theory (DFT) on four Mn(4)Ca clusters modeling the successive S(0) to S(3) steps of the oxygen-evolving complex (OEC) in photosystem II (PSII). The model clusters were obtained from a previous theoretical study where they were determined by energy minimization. They are composed of Mn(III) and Mn(IV) atoms, progressing from Mn(III)(3)Mn(IV) for S(0) to Mn(III)(2)Mn(IV)(2) for S(1) to Mn(III)Mn(IV)(3) for S(2) to Mn(IV)(4) for S(3), implying an Mn-centered oxidation during each step of the photosynthetic oxygen evolution. The DFT simulations of the Mn 1s absorption edge reproduce the experimentally measured curves quite well. By the half-height method, the theoretical IPEs are shifted by 0.93 eV for the S(0) → S(1) transition, by 1.43 eV for the S(1) → S(2) transition, and by 0.63 eV for the S(2) → S(3) transition. The inflection point energy (IPE) shifts depend strongly on the method used to determine them, and the most interesting result is that the present clusters reproduce the shift in the S(2) → S(3) transition obtained by both the half-height and second-derivative methods, thus giving strong support to the previously suggested structures and assignments.

The mechanism of the chlorination reaction of SyrB2, a representative α-ketoglutarate dependent halogenase, was studied with computational methods. First, a macromolecular model of the Michaelis complex was constructed using molecular docking procedures. Based on this structure, a smaller model comprising the first- and some of the second-shell residues of iron and a model substrate was constructed and used in DFT investigations on the reaction mechanism. Computed relative energies and Mössbauer isomer shifts as well as quadrupole splittings indicate that the two oxoferryl species observed experimentally are two stereoisomers resulting from an exchange of the coordination sites occupied by the oxo and chloro ligands. In principle both Fe(IV)═O species are reactive and decay to Fe(III)Cl (OH)/carbon radical intermediates via C-H bond cleavage. In the final rebound step, which is very fast and thus precluding equilibration between the two forms of the radical intermediate, the ligand (oxo or chloro) placed closest to the carbon radical (trans to His235) is transferred to the carbon. For the native substrate (L-Thr) the lowest barrier for C-H cleavage was found for an isomer of the oxoferryl species favoring chlorination in the rebound step. CASPT2 calculations for the spin state splittings in the oxoferryl species support the conclusion that once the Fe(IV)═O intermediate is formed, the reaction proceeds on the quintet potential energy surface.

The computed total energy from a cluster model DFT calculation is used to discriminate between different suggested models for the oxygen evolving complex of photosystem II. The comparison between different structures rules out several suggestions. Only one suggested structure remains.

The formation of methyl-Ni(F(430)) species in methyl-coenzyme M reductase (MCR) has been investigated using the B3LYP hybrid density functional method and an active-site model built on the basis of X-ray crystal structure. CH(3)-I, CH(3)-Br, CH(3)-Cl, and CH(3)-S-CH(3) were chosen as the substrates, the last one regarded as a model of the native substrate (methyl-coenzyme M, CH(3)-SCoM). The calculations indicate that the formation of CH(3)-Ni(F(430)) in MCR is dependent on the acidity of the substrate leaving group. A CH(3)-Ni(F(430)) species has been observed with methyl halides as substrates, while the formation of CH(3)-Ni(F(430)) from the native substrate is demonstrated to be inaccessible energetically. These results agree well with the current experiments.

The mechanism of the catalytic reaction of protocatechuate 3,4-dioxygenase (3,4-PCD), a representative intradiol dioxygenase, was studied with the hybrid density functional method B3LYP. First, a smaller model involving only the iron first-shell ligands (His460, His462, and Tyr408) and the substrates (catechol and dioxygen) was used to probe various a priori plausible reaction mechanisms. Then, an extended model involving also the most important second-shell groups (Arg457, Gln477, and Tyr479) was used for the refinement of the preselected mechanisms. The computational results suggest that the chemical reactions constituting the catalytic cycle of intradiol dioxygenases involve: (1) binding of the substrate as a dianion, in agreement with experimental suggestions, (2) binding of dioxygen to the metal aided by an electron transfer from the substrate to O(2), (3) formation of a bridging peroxo intermediate and its conformational change, which opens the coordination site trans to His462, (4) binding of a neutral XOH ligand (H(2)O or Tyr447) at the open site, (5) proton transfer from XOH to the neighboring peroxo ligand yielding the hydroperoxo intermediate, (6) a Criegee rearrangement leading to the anhydride intermediate, and (7) hydrolysis of the anhydride to the final acyclic product. One of the most important results obtained is that the Criegee mechanism requires an in-plane orientation of the four atoms (two oxygen and two carbon atoms) mainly involved in the reaction. This orientation yields a good overlap between the two sigma orbitals involved, C-C sigma and O-O sigma, allowing an efficient electron flow between them. Another interesting result is that under some conditions, a homolytic O-O bond cleavage might compete with the Criegee rearrangement. The role of the second-shell residues and the substituent effects are also discussed.

The reaction mechanism for the disproportionation of the toxic superoxide radical to molecular oxygen and hydrogen peroxide by the nickel-dependent superoxide dismutase (NiSOD) has been studied using the B3LYP hybrid DFT method. Based on the recent X-ray structures of the enzyme in the resting oxidized Ni(III) and X-ray-reduced Ni(II) states, the model investigated includes the backbone spacer of six residues (sequence numbers 1-6) as a structural framework. The side chains of residues His1, Cys2, and Cys6, which are essential for nickel binding and catalysis, were modeled explicitly. The catalytic cycle consists of two half-reactions, each initiated by the successive substrate approach to the metal center. The two protons necessary for the dismutation are postulated to be delivered concertedly with the superoxide radical anions. The first (reductive) phase involves Ni(III) reduction to Ni(II), and the second (oxidative) phase involves the metal reoxidation back to its resting state. The Cys2 thiolate sulfur serves as a transient protonation site in the interim between the two half-reactions, allowing for the dioxygen and hydrogen peroxide molecules to be released in the reductive and oxidative phases, respectively. The His1 side chain nitrogen and backbone amides of the active site channel are shown to be less favorable transient proton locations, as compared to the Cys2 sulfur. Comparisons are made to the Cu- and Zn-dependent SOD, studied previously using similar models.

Human homogentisate dioxygenase is an Fe(II)-dependent enzyme responsible for aromatic ring cleavage. The mechanism of its catalytic reaction has been investigated with the hybrid density functional method B3LYP. A relatively big model of the active site was first used to determine the substrate binding mode. It was found that binding of the substrate dianion with a vacant position trans to Glu341 is most favorable. The model was then truncated to include only the most relevant parts of the active-site residues involved in iron coordination and substrate binding. Thus, methylimidazole was used to model His292, His335, His365, and His371, while propionate modeled Glu341. The computational results suggest that the catalytic reaction of homogentisate dioxygenases involves three major chemical steps: formation of the peroxo intermediate, homolytic cleavage of the O-O bond leading to an arene oxide radical, and finally, cleavage of the six-membered ring. Calculated barriers for alternative reaction paths are markedly higher than for the proposed mechanism, and thus the computational results successfully explain the product specificity of the enzyme. Interestingly, the results indicate that the type of ring scission, intra or extra with respect to the substituents coordinating to iron, is controlled by the barrier heights for the decay of the arene oxide radical intermediate.

The catalytic mechanism of the non-heme iron extradiol dioxygenases has been studied using hybrid density functional theory. These enzymes cleave a C-C bond outside the two hydroxyl groups of catechols, in contrast to the intradiol enzymes which cleave the C-C bond between these two groups. The chemical models used comprise about 70 atoms and include the first-shell ligands, two histidines, one glutamate, and one water, as well as some second-shell ligands, two histidines, one aspartate, and one tyrosine. Catechol is found to bind as a monoanion in agreement with experiments, while dioxygen is found to replace the water ligand. A spin-transition from the initial septet to a quintet state prepares the system for formation of a bridging peroxide with the catechol substrate. When the O-O bond is cleaved in the suggested rate-limiting step, a key substrate intermediate with partly radical and partly anionic character is formed. The partly anionic character is found to determine the selectivity of the enzyme. The results are compared to available experimental information and to previous studies.

In the present quantum chemical study, the biogenesis of the cofactor topaquinone (TPQ) has been studied using hybrid density functional theory (B3LYP). The suggested mechanism is divided into six steps and incorporates the observation of four crystallized intermediates. The experimental suggestion that the formation of the Cu(II)-peroxy species is the rate-limiting step is consistent with the results of the present study. Before the formation of the Cu(II)-peroxy species, dioxygen is suggested to first bind at the equatorial position on the copper metal center. A mechanism for the critical O-O bond cleavage is suggested, and this step is found to be driven by an unusually large exothermicity. A complex, spin-forbidden formation of H(2)O(2) with and without the involvement of the copper metal center is discussed. The results are discussed in detail, and comparisons are made to experimental findings and suggestions.

Elucidating the role of nuclear quantum mechanical (NQM) effects in enzyme catalysis is a topic of significant current interest. Despite the great experimental progress in this field it is important to have theoretical approaches capable of evaluating and analyzing nuclear quantum mechanical contributions to catalysis. In this study, we use the catalytic reaction of lipoxygenase, which is characterized by an extremely large kinetic isotope effect, as a challenging test case for our simulation approach. This is done by applying the quantum classical path (QCP) method with an empirical valence bond potential energy surface. Our computational strategy evaluates the relevant NQM corrections and reproduces the large observed kinetic isotope effect and the temperature dependence of the H atom transfer reaction while being less successful with the D atom transfer reaction. However, the main point of our study is not so much to explore the temperature dependence of the isotope effect but rather to develop and validate an approach for calculations of nuclear quantum mechanical contributions to activation free energies. Here, we find that the deviation between the calculated and observed activation free energies is small for both H and D at all investigated temperatures. The present study also explores the nature of the reorganization energy in the enzyme and solution reactions. It is found that the outer-sphere reorganization energy is extremely small. This reflects the fact that the considered reaction involves a very small charge transfer. The implication of this finding is discussed in the framework of the qualitative vibronic model. The main point of the present study is, however, that the rigorous QCP approach provides a reliable computational tool for evaluating NQM contributions to catalysis even when the given reaction includes large tunneling contributions. Interestingly, our results indicate that the NQM effects in the lipoxygenase reaction are similar in the enzyme and in the reference solution reactions, and thus do not contribute to catalysis. We also reached similar conclusions in studies of other enzymes.

Density functional theory using the B3LYP hybrid functional has been employed to investigate the reactivity of Fe(TPA) complexes (TPA = tris(2-pyridylmethyl)amine), which are known to catalyze stereospecific hydrocarbon oxidation when H(2)O(2) is used as oxidant. The reaction pathway leading to O-O bond heterolysis in the active catalytic species Fe(III)(TPA)-OOH has been explored, and it is shown that a high-valent iron-oxo intermediate is formed, where an Fe(V) oxidation state is attained, in agreement with previous suggestions based on experiments. In contrast to the analogous intermediate [(Por.)Fe(IV)=O](+1) in P450, the TPA ligand is not oxidized, and the electrons are extracted almost exclusively from the mononuclear iron center. The corresponding homolytic O-O bond cleavage, yielding the two oxidants Fe(IV)=O and the OH. radical, has also been considered, and it is shown that this pathway is inaccessible in the hydrocarbon oxidation reaction with Fe(TPA) and hydrogen peroxide. Investigations have also been performed for the O-O cleavage in the Fe(III)(TPA)-alkylperoxide species. In this case, the barrier for O-O homolysis is found to be slightly lower, leading to loss of stereospecificity and supporting the experimental conclusion that this is the preferred pathway for alkylperoxide oxidants. The difference between hydroperoxide and alkylperoxide as oxidant derives from the higher O-O bond strength for hydrogen peroxide (by 8.0 kcal/mol).

The mechanism for methane formation in methyl-coenzyme M reductase (MCR) has been investigated using the B3LYP hybrid density functional method and chemical models consisting of 107 atoms. The experimental X-ray crystal structure of the enzyme in the inactive MCR(ox1)(-)(silent) state was used to set up the initial model structure. The calculations suggest a mechanism not previously proposed, in which the most remarkable feature is the formation of an essentially free methyl radical at the transition state. The reaction cycle suggested starts from a Michaelis complex with CoB and methyl-CoM coenzymes bound and with a squareplanar coordination of the Ni(I) center in the tetrapyrrole F(430) prosthetic group. In the rate-limiting step the methyl radical is released from methyl-CoM, induced by the attack of Ni(I) on the methyl-CoM thioether sulfur. In this step, the metal center is oxidized from Ni(I) to Ni(II). The resulting methyl radical is rapidly quenched by hydrogen-atom transfer from the CoB thiol group, yielding the methane molecule and the CoB radical. The estimated activation energy is around 20 kcal/mol, which includes a significant contribution from entropy due to the formation of the free methyl. The mechanism implies an inversion of configuration at the reactive carbon. The size of the inversion barrier is used to explain the fact that CF(3)-S-CoM is an inactive substrate. Heterodisulfide CoB-S-S-CoM formation is proposed in the final step in which nickel is reduced back to Ni(I). The suggested mechanism agrees well with experimental observations.

Hybrid density functional theory is used to study the catalytic mechanism of human glyoxalase I (GlxI). This zinc enzyme catalyzes the conversion of the hemithioacetal of toxic methylglyoxal and glutathione to nontoxic (S)-D-lactoylglutathione. GlxI can process both diastereomeric forms of the substrate, yielding the same form of the product. As a starting point for the calculations, we use a recent crystal structure of the enzyme in complex with a transition-state analogue, where it was found that the inhibitor is bound directly to the zinc by its hydroxycarbamoyl functions. It is shown that the Zn ligand Glu172 can abstract the substrate C1 proton from the S enantiomer of the substrate, without being displaced from the Zn ion. The calculated activation barrier is in excellent agreement with experimental rates. Analogously, the Zn ligand Glu99 can abstract the proton from the R form of the substrate. To account for the stereochemical findings, it is argued that the S and R reactions cannot be fully symmetric. A detailed mechanistic scheme is proposed.

Density functional calculations using hybrid functionals (B3LYP) have been performed to study the mechanism of the autocatalytic posttranslational cyclization observed in histidine ammonia-lyase. Two mechanisms were analyzed, the commonly accepted mechanism in which cyclization precedes dehydrogenation (reduced mechanism) and a mechanism in which dehydrogenation precedes cyclization (oxidized mechanism). The reduced pathway is not supported by the calculations, while the alternative oxidized mechanism where a dehydration occurs prior to the formation of the ring yields reasonable energetics for the system. Database searches showed that the oxidative mechanism in which the formation of the dehydro amino acids in residue i + 1 precedes the cyclization is also structurally advantageous as it results in shorter distances between the carbonyl carbon of residue i and the amide nitrogen of residue i + 2 and, therefore, preorganizes the protein for cyclization. Conformational searches showed that these distances were also unusually short and exhibited very little variation in the Delta-Ala143 HAL tetramer, indicating that like GFP the tetrameric form of HAL is rigidly preorganized for cyclization. The monomeric form of HAL is less preorganized than the tetrameric form of HAL. Dehydro amino acids aid in the preorganization, but the main driving force in the rigid tight turn formation is the influence of the surrounding protein.