Mechanisms for Methane and Ammonia Oxidation by Particulate Methane Monooxygenase.

Particulate MMO (pMMO) catalyzes the oxidation of methane to methanol and also ammonia to hydroxylamine. Experimental characterization of the active site has been very difficult partly because the enzyme is membrane-bound. However, recently, there has been major progress mainly through the use of cryogenic electron microscopy (cryoEM). Electron paramagnetic resonance (EPR) and X-ray spectroscopy have also been employed. Surprisingly, the active site has only one copper. There are two histidine ligands and one asparagine ligand, and the active site is surrounded by phenyl alanines but no charged amino acids in the close surrounding. The present study is the first quantum chemical study using a model of that active site (CuD). Low barrier mechanisms have been found, where an important part is that there are two initial proton-coupled electron transfer steps to a bound O2 ligand before the substrate enters. Surprisingly, this leads to large radical character for the oxygens even though they are protonated. That result is very important for the ability to accept a proton from the substrates. Methods have been used which have been thoroughly tested for redox enzyme mechanisms.

Computational Model Study of the Experimentally Suggested Mechanism for Nitrogenase.

The mechanism for N2 activation in the E4 state of nitrogenase was investigated by model calculations. In the experimentally suggested mechanism, the E4 state is obtained after four reductions to the ground state. In a recent theoretical study, results for a different mechanism have been found in excellent agreement with available Electron Paramagnetic Resonance (EPR) experiments for E4. The two hydrides in E4 leave as H2 concertedly with the binding of N2. The mechanism suggested differs from the experimentally suggested one by a requirement for four activation steps prior to catalysis. In the present study, the experimentally suggested mechanism is studied using the same methods as those used in the previous study on the theoretical mechanism. The computed results make it very unlikely that a structure obtained after four reductions from the ground state has two hydrides, and the experimentally suggested mechanism does therefore not agree with the EPR experiments for E4. Another structure with only one hydride is here suggested to be the one that has been observed to bind N2 after only four reductions of the ground state.

The energetics of N2 reduction by vanadium containing nitrogenase.

The main class of nitrogenases has a molybdenum in its cofactor. A mechanism for Mo-nitrogenase has recently been described. In the present study, another class of nitrogenases has been studied, the one with a vanadium instead of a molybdenum in its cofactor. It is generally believed that these classes use the same general mechanism to activate nitrogen. The same methodology has been used here as the one used for Mo-nitrogenase. N2 activation is known to occur after four reductions in the catalytic cycle, in the E4 state. The main features of the mechanism for Mo-nitrogenase found in the previous study are an activation process in four steps prior to catalysis, the release of a sulfide during the activation steps and the formation of H2 from two hydrides in E4, just before N2 is activated. The same features have been found here for V-nitrogenase. A difference is that five steps are needed in the activation process, which explains why the ground state of V-nitrogenase is a triplet (even number) and the one for Mo-nitrogenase is a quartet (odd number). The reason an additional step is needed for V-nitrogenase is that V3+ can be reduced to V2+, in contrast to the case for Mo3+ in Mo-nitrogenase. The fact that V3+ is Jahn-Teller active has important consequences. N2H2 is formed in E4 with reasonably small barriers.

The mechanism for N2 activation in the E4 - state of nitrogenase.

Nitrogenases take nitrogen from the air and reduce it to ammonia. It has long been known that N2 becomes activated after four reductions in the catalytic cycle, in the E4 state. Several mechanisms for the activation have been suggested. In the present study a previous mechanism has been revised based on recent experimental findings. In the present mechanism N2H2 is formed in E4. As in the previously suggested mechanism, there are four initial reductions before catalysis (the A-states), after which a sulfide is released and the first state in catalysis (E0) is formed. In E4, N2 becomes bound and protonated in the Fe1, Fe2, Fe4 region, in which the hydrides have left two electrons. The rate-limiting step is the formation of N2H by a hydrogen atom transfer from Cys275 to N2 bound to Fe4, concerted with an additional electron transfer from the cofactor. The mechanism fulfills all requirements set by experiments. The activation of N2 is preceded by a formation of H2 from two hydrides, the carbide is kinetically hindered from being protonated, the E4 state is reversible. An important aspect is the presence of a water molecule in the Fe2, Fe6 region. The non-allowed formations of H2 from a hydride and a proton have been investigated and found to have higher barriers than the allowed formation of H2 from two hydrides.

Is There a Different Mechanism for Water Oxidation in Higher Plants?

The leading mechanism for the formation of O2 in photosystem II (PSII) has, during the past decade, been established as the so-called oxyl-oxo mechanism. In that mechanism, O2 is formed from a binding between an oxygen radical (oxyl) and a bridging oxo group. For the case of higher plants, that mechanism has recently been criticized. Instead, a nucleophilic attack of an oxo group on a five-coordinated Mn(V)═O group forming O2 has been suggested in a so-called water-unbound (WU) mechanism. In the present study, the WU mechanism has been investigated. It is found that the WU mechanism is just a variant of a previously suggested mechanism but with a reactant and a transition state that have much higher energies. The addition of a water molecule on the empty site of the Mn(V)═O center is very exergonic and leads back to the previously suggested oxyl-oxo mechanism.

Theoretical Study of the Catalytic Mechanism of the Cu-Only Superoxide Dismutase.

The catalytic mechanisms for the wild-type and the mutated Cu-only superoxide dismutase were studied using the hybrid density functional B3LYP and a quantum chemical cluster approach. Optimal protonation states of the active site were examined for each stage of the catalytic cycle. For both the reductive and the oxidative half-reactions, the arrival of the substrate O2•- was found to be accompanied by a charge-compensating H+ with exergonicities of -15.4 kcal·mol and -4.7 kcal·mol, respectively. The second-sphere Glu-110 and first-sphere His-93 were suggested to be the transient protonation site for the reductive and the oxidative half-reactions, respectively, which collaborates with the hydrogen bonding water chain to position the substrate near the redox-active copper center. For the reductive half-reaction, the rate-limiting step was found to be the inner-sphere electron transfer from the partially coordinated O2•- to CuII with a barrier of 8.1 kcal·mol. The formed O2 is released from the active site with an exergonicity of -14.9 kcal·mol. For the oxidative half-reaction, the inner-sphere electron transfer from CuI to the partially coordinated O2•- was found to be accompanied by the proton transfer from the protonated His-93 and barrierless. The rate-limiting step was found to be the second proton transfer from the protonated Glu-110 to HO2- with a barrier of 7.3 kcal·mol. The barriers are reasonably consistent with experimental activities, and a proton-transfer rate-limiting step in the oxidative half-reaction could explain the experimentally observed pH-dependence. For the E110Q CuSOD, Asp-113 was suggested to be likely to serve as the transient protonation site in the reductive half-reaction. The rate-limiting barriers were found to be 8.0 and 8.6 kcal·mol, respectively, which could explain the slightly lower performance of E110X mutants. The results were found to be stable, with respect to the percentage of exact exchange in B3LYP.

How Protons Move in Enzymes─The Case of Nitrogenase.

When moving protons in enzymes, water molecules are often used as intermediates. The water molecules used are not necessarily seen in the crystal structures if they move around at high rates. In a different situation, for metal containing cofactors in enzymes, it is sometimes necessary to move protons on the cofactor from the position they enter the cofactor to another position where the energy is lower. That is, for example, the situation in nitrogenase. In recent studies on that enzyme, prohibitively high barriers were sometimes found for transferring protons, and that was used as a strong argument against mechanisms where a sulfide is lost in the mechanism. A high barrier could be due to nonoptimal distances and angles at the transition state. In the present study, possibilities are investigated to use water molecules to reduce these barriers. The study is very general and could have been done for many other enzymes. The effect of water was found to be very large in the case of nitrogenase with a lowering of one barrier from 15.6 kcal/mol down to essentially zero. It is concluded that the effect of water molecules must be taken into account for meaningful results.

Can the E1 state in nitrogenase tell if there is an activation process prior to catalysis?

Model calculations have been performed for the singly reduced ground state of Mo-nitrogenase, usually termed E1. Contradictory conclusions have been reached in two recent experimental studies. In a study based on EPR, it was concluded that there is a bridging hydride in E1, while in an X-ray study it was concluded that there is no hydride in E1. Therefore, the EPR study implies that there is an oxidation of the cofactor going from E0 to E1, the X-ray study implies a reduction. DFT methods have here been used, which have previously been benchmarked on a set of redox enzymes that led to the conclusion that the accuracy is about 3 kcal mol-1 in all cases, even for redox transitions. The methodology should therefore be adequate for resolving the question of the hydride presence in E1. As a comparison, calculations are performed on both Mo- and V-nitrogenase with the same conclusion. The conclusion from the calculations has far reaching consequences for the mechanism of nitrogenase.

Computational modeling of redox enzymes.

A computational methodology is briefly described, which appears to be able to accurately describe the mechanisms of redox active enzymes. The method is built on hybrid density functional theory where the inclusion of a fraction of exact exchange is critical. Two examples of where the methodology has been applied are described. The first example is the mechanism for water oxidation in photosystem II, and the second one is the mechanism for N2 activation by nitrogenase. The mechanism for PSII has obtained very strong support from subsequent experiments. For nitrogenase, the calculations suggest that there should be an activation process prior to catalysis, which is still strongly debated.

Energetics for CO2 Reduction by Molybdenum-Containing Formate Dehydrogenase.

The level of carbon dioxide in the atmosphere has increased in a dangerous way during the past century. Methods to decrease this level are therefore of high interest at present. Inspiration to do so in an efficient way could come from biological systems. Molybdenum-containing formate dehydrogenase (Mo-FDH) is one of the most interesting enzymes in this respect. For example, the reduction potential required is not very low. The normal reaction catalyzed by Mo-FDH is actually the opposite one of oxidizing formate to CO2. However, recent electrochemical studies have shown that the reaction can be reversed by a moderate lowering of the reduction potential. The goal of the present study has been to study the full mechanism of Mo-FDH, particularly in the most interesting direction of reducing CO2, which has not been done before. The methods used are the same as those that have been shown to give excellent results for redox enzymes in all cases they have been tested. The results obtained for Mo-FDH are also in excellent agreement with the experimental results.

A Mechanism for Nitrogenase Including Loss of a Sulfide.

Nitrogenase is the only enzyme in nature that can fix N2 from the air. The active cofactor of the leading form of this enzyme contains seven irons and one molybdenum connected by sulfide bridges. In several recent experimental studies, it has been suggested that the cofactor is very flexible, and might lose one of its sulfides during catalysis. In this study, the possible loss of a sulfide has been investigated by model calculations. In previous studies, we have shown that there should be four activation steps before catalysis starts, and this study is based on that finding. It was found here that, after the four reductions in the activation steps, a sulfide will become very loosely bound and can be released in a quite exergonic step with a low barrier. The binding of N2 has no part in that release. In our previous studies, we suggested that the central carbide should be protonated three times after the four activation steps. With the new finding, there will instead be a loss of a sulfide, as the barrier for the loss is much lower than the ones for protonating the carbide. Still, it is suggested here that the carbide will be protonated anyway, but only with one proton, in the E3 to E4 step. A very complicated transition state for H2 formation involving a large structural change was obtained. The combined step, with a loss of H2 and binding of N2 , is calculated to be endergonic by +2.3 kcal mol-1 ; this is in excellent agreement with experiments in which an easily reversible step has been found.

The active E4 structure of nitrogenase studied with different DFT functionals.

The present study concerns the technical aspects of obtaining the energetics for the E4 state of nitrogenase, the enzyme that fixes N2 in nature. EPR experiments have shown that the critical E4 structure that activates N2 should contain two bridging hydrides in the FeMo-cofactor. It is furthermore in equilibrium with a structure where the two hydrides have been released and N2 binds. These observations led to the suggestion that E4 should have two bridging hydrides and two protonated sulfides. It is important to note that the structure for E4 has not been determined, but only suggested. For a long time, no DFT study led to the suggested structure, independent of which functional was used. However, in two recent DFT studies a good agreement with the experimental suggestion was claimed to have been obtained. In one of them the TPSS functional was used. That was the first out of 11 functionals tried that led to the experimentally suggested structure. In the second of the recent DFT studies, a similar conclusion was reached using the TPSSh functional. The conclusions in the recent studies have here been studied in detail, by calculating a critical energetic value strongly implied by the same EPR experiments. Both the TPSS and TPSSh functionals have been used. The present calculations suggest that those DFT functionals would not lead to agreement with the experimental EPR results either.

A quantum chemical approach for the mechanisms of redox-active metalloenzymes.

During the past 20 years, quantum chemistry has grown to be a significant part in the investigation of mechanisms for redox-active enzymes. In our group we have developed an approach that has been applied to a large number of such systems. Hybrid density functional theory (hybrid DFT) has from the start of these investigations been the leading electronic structure tool. An understanding of how the method works in practice has significantly improved the accuracy and applicability. During the past ten years, it has been found that the results for redox enzymes mainly depend on the chosen fraction of exact exchange in the functional, and that a choice of 15% has worked best. The idea has therefore been to vary that fraction over a reasonable range and study the relative energy dependence. For modeling the enzymes, a cluster approach has been developed. In the present review the development of the method we used is described from its start in work on photosystem II, fifteen years ago. Examples from a few recent applications are described, where the metals have been iron, nickel, copper, cobalt or manganese. The results are in excellent agreement with available experiments, and a large number of new predictions have been made.

Energetics for Proton Reduction in FeFe Hydrogenase.

The energetics for proton reduction in FeFe-hydrogenase has been reinvestigated by theoretical modeling, in light of recent experiments. Two different mechanisms have been considered. In the first one, the bridging hydride position was blocked by the enzyme, which is the mechanism that has been supported by a recent spectroscopic study by Cramer et al. A major difficulty in the present study to agree with experimental energetics was to find the right position for the added proton in the first reduction step. It was eventually found that the best position was as a terminal hydride on the distal iron, which has not been suggested in any of the recent, experimentally based mechanisms. The lowest transition state was surprisingly found to be a bond formation between a proton on a cysteine and the terminal hydride. This type of TS is similar to the one for heterolytic H2 cleavage in NiFe hydrogenase. The second mechanism investigated here is not supported by the present calculations or the recent experiments by Cramer et al., but was still studied as an interesting comparison. In that mechanism, the formation of the bridging hydride was allowed. The H-H formation barrier is only 3.6 kcal/mol higher than for the first mechanism, but there are severe problems concerning the motion of the protons.

Insights into the Chemical Reactivity in Acetyl-CoA Synthase.

The biological synthesis of acetyl-coenzyme A (acetyl-CoA), catalyzed by acetyl-CoA synthase (ACS), is of biological significance and chemical interest acting as a source of energy and carbon. The catalyst contains an unusual hexa-metal cluster with two nickel ions and a [Fe4S4] cluster. DFT calculations have been performed to investigate the ACS reaction mechanism starting from three different oxidation states (+2, +1, and 0) of Nip, the nickel proximal to [Fe4S4]. The results indicate that the ACS reaction proceeds first through a methyl radical transfer from cobalamin (Cbl) to Nip randomly accompanying with the CO binding. After that, C-C bond formation occurs between the Nip-bound methyl and CO, forming Nip-acetyl. The substrate CoA-S- then binds to Nip, allowing C-S bond formation between the Nip-bound acetyl and CoA-S-. Methyl transfer is rate-limiting with a barrier of ∼14 kcal/mol, which does not depend on the presence or absence of CO. Both the Nip2+ and Nip1+ states are chemically capable of catalyzing the ACS reaction independent of the state (+2 or +1) of the [Fe4S4] cluster. The [Fe4S4] cluster is not found to affect the steps of methyl transfer and C-C bond formation but may be involved in the C-S bond formation depending on the detailed mechanism chosen. An ACS active site containing a Nip(0) state could not be obtained. Optimizations always led to a Nip1+ state coupled with [Fe4S4]1+. The calculations show a comparable activity for Nip1+/[Fe4S4]1+, Nip1+/[Fe4S4]2+, and Nip2+/[Fe4S4]2+. The results here give significant insights into the chemistry of the important ACS reaction.

A Theoretical Study of the Recently Suggested MnVII Mechanism for O-O Bond Formation in Photosystem II.

The mechanism for water oxidation in photosystem II has been a major topic for several decades. The active catalyst has four manganese atoms connected by bridging oxo bonds, in a complex termed the oxygen-evolving complex (OEC), which also includes a calcium atom. The O-O bond of oxygen is formed after absorption of four photons in a state of the OEC termed S4. There has been essential consensus that in the S4 state, all manganese atoms are in the Mn(IV) oxidation state. However, recently there has been a suggestion that one of the atoms is in the Mn(VII) state. In the present computational study, the feasibility of that proposal has been investigated. It is here shown that the mechanism involving Mn(VII) has a much higher barrier for forming O2 than the previous proposal with four Mn(IV) atoms.

Theoretical Study of O2 Reduction and Water Oxidation in Multicopper Oxidases.

Recent electrochemical experiments have shown that the reduction of O2 can be driven backward to water oxidation, which is the first case that has been successfully demonstrated for an enzyme. To understand this ability of the enzyme, both the forward reduction and backward oxidation have been studied here. For the forward reaction, a mechanism similar to earlier studies was obtained. All steps of the full catalytic cycle were obtained for the first time, and it was shown that the explicit reduction steps contribute significantly to the rate-limiting step of the O-O bond cleavage. For the backward oxidation reaction, it was found that the mechanism of the O-O bond formation is not just the reverse of the reduction step where the O-O bond is cleaved for a protonated peroxide. The formation of two fully deprotonated oxo groups was found to be important, which leads to a large radical character for one of the oxo groups. For this possibility, it is important that the pKa of the water bound to the cofactor is quite high.

The mechanism for nitrogenase including all steps.

The catalytic cofactor of the most common form of nitrogenase contains seven irons and one molybdenum bound together by sulfide bonds. Surprisingly, a central carbide has been demonstrated by experiments. Another noteworthy structural component is a large homocitrate ligand. In recent theoretical studies it has been shown that the central carbide is needed as a place for the incoming protons that are necessary parts of a reduction process. It has also been shown that a role for the homocitrate ligand could be that it may be rotated to release one bond to molybdenum. In the present study, the carbide protonation steps are reinvestigated with similar results to those reported before. The actual activation of N2 in the E4 state is an extremely complicated process. It has been found experimentally that two hydrides should leave as H2, in a reductive elimination process, to allow N2 activation in E4 in an easily reversible step. It is here suggested that after H2 is released, it is necessary for the metal cofactor to get rid of one proton. This is achieved by protonating the homocitrate and then rotating it to release one of the bonds to Mo. After this rotation, N2 can bind. In the E5 step, the homocitrate is rotated back to its original position and remains that way until the end of the catalytic process. The N2 protonation steps are energetically easy. Since a protonated carbide has never been observed experimentally, it is necessary to also have a mechanism for deprotonating the carbon at the end of the catalytic cycles. Such a mechanism is suggested here.

Energetics for the Mechanism of Nickel-Containing Carbon Monoxide Dehydrogenase.

Nickel-containing carbon monoxide (CO) dehydrogenase is an enzyme that catalyzes the important reversible carbon dioxide reduction. Several high-resolution structures have been determined at various stages of the reduction, which can be used as good starting points for the present computational study. The cluster model is used in combination with a systematic application of the density functional theory as recently described. The results are in very good agreement with experimental evidence. There are a few important results. To explain why the X-ray structure for the reduced Cred1 state has an empty site on nickel, it is here suggested that the cluster has been over-reduced by X-rays and is therefore not the desired reduced state, which instead contains a bound CO on nickel. After an additional reduction, a hydride bound to nickel is suggested to play a role. In order to obtain energetics in agreement with experiments, it is concluded that one sulfide bridge in the Ni-Fe cluster should be protonated. The best test of the accuracy obtained is to compare the computed rate for reduction using -0.6 V with that for oxidation using -0.3 V, where good agreement was obtained. Obtaining a mechanism that is easily reversible is another demanding aspect of the modeling. Nickel oscillates between nickel(II) and nickel(I), while nickel(0) never comes in.

Quantum Chemical Study of the Mechanism of Water Oxidation Catalyzed by a Heterotrinuclear Ru2 Mn Complex.

The heterotrinuclear complex A {[RuII (H2 O)(tpy)]2 (µ-[MnII (H2 O)2 (bpp)2 ])}4+ [tpy=2,2':6',2''-terpyridine, bpp=3,5-bis(2-pyridyl)pyrazolate] was found to catalyze water oxidation both electrochemically and photochemically with [Ru(bpy)3 ]3+ (bpy=2,2'-bipyridine) as the photosensitizer and Na2 S2 O8 as the electron acceptor in neutral phosphate buffer. The mechanism of water oxidation catalyzed by this unprecedented trinuclear complex was studied by density functional calculations. The calculations showed that a series of oxidation and deprotonation events take place from A, leading to the formation of complex 1 (formal oxidation state of Ru1IV MnIII Ru2III ), which is the starting species for the catalytic cycle. Three sequential oxidations of 1 result in the generation of the catalytically competing species 4 (formal oxidation state of Ru1IV MnV Ru2IV ), which triggers the O-O bond formation. The direct coupling of two adjacent oxo ligands bound to Ru and Mn leads to the production of a superoxide intermediate Int1. This step was calculated to have a barrier of 7.2 kcal mol-1 at the B3LYP*-D3 level. Subsequent O2 release from Int1 turns out to be quite facile. Other possible pathways were found to be much less favorable, including water nucleophilic attack, the coupling of an oxo and a hydroxide, and the direct coupling pathway at a lower oxidation state (RuIV MnIV RuIV ).