Understanding the Electronic Structure Basis for N2 Binding to FeMoco: A Systematic Quantum Mechanics/Molecular Mechanics Investigation.

The FeMo cofactor (FeMoco) of Mo nitrogenase is responsible for reducing dinitrogen to ammonia, but it requires the addition of 3-4 e-/H+ pairs before N2 even binds. A binding site at the Fe2/Fe3/Fe6/Fe7 face of the cofactor has long been suggested based on mutation studies, with Fe2 or Fe6 nowadays being primarily discussed as possibilities. However, the nature of N2 binding to the cofactor is enigmatic as the metal ions are coordinatively saturated in the resting state with no obvious binding site. Furthermore, the cofactor consists of high-spin Fe(II)/Fe(III) ions (antiferromagnetically coupled but also mixed-valence delocalized), which are not known to bind N2. This suggests that an Fe binding site with a different molecular and electronic structure than the resting state must be responsible for the experimentally known N2 binding in the E4 state of FeMoco. We have systematically studied N2 binding to Fe2 and Fe6 sites of FeMoco at the broken-symmetry QM/MM level as a function of the redox-, spin-, and protonation state of the cofactor. The local and global electronic structure changes to the cofactor taking place during redox events, protonation, Fe-S cleavage, hydride formation, and N2 coordination are systematically analyzed. Localized orbital and quasi-restricted orbital analysis via diamagnetic substitution is used to get insights into the local single Fe ion electronic structure in various states of FeMoco. A few factors emerge as essential to N2 binding in the calculations: spin-pairing of dxz and dyz orbitals of the N2-binding Fe ion, a coordination change at the N2-binding Fe ion aided by a hemilabile protonated sulfur, and finally hydride ligation. The results show that N2 binding to E0, E1, and E2 models is generally unfavorable, likely due to the high-energy cost of stabilizing the necessary spin-paired electronic structure of the N2-binding Fe ion in a ligand environment that clearly favors high-spin states. The results for models of E4, however, suggest a feasible model for why N2 binding occurs experimentally in the E4 state. E4 models with two bridging hydrides between Fe2 and Fe6 show much more favorable N2 binding than other models. When two hydrides coordinate to the same Fe ion, an increased ligand-field splitting due to octahedral coordination at either Fe2 or Fe6 is found. This altered ligand field makes it easier for the Fe ion to acquire a spin-paired electronic structure with doubly occupied dxz and dyz orbitals that backbond to a terminal N2 ligand. Within this model for N2 binding, both Fe2 and Fe6 emerge as possible binding site scenarios. Due to steric effects involving the His195 residue, affecting both the N2 ligand and the terminal SH- group, distinguishing between Fe2 and Fe6 is difficult; furthermore, the binding depends on the protonation state of His195.

The E3 state of FeMoco: one hydride, two hydrides or dihydrogen?

Hydrides are present in the reduced states of the iron-molybdenum cofactor (FeMoco) of Mo nitrogenase and are believed to play a key mechanistic role in the dinitrogen reduction reaction catalyzed by the enzyme. Two hydrides are present in the E4 state according to 1H ENDOR and there is likely a single hydride in the E2 redox state. The 2-hydride E4 state has been experimentally observed to bind N2 and it has been speculated that E3 may bind N2 as well. However, the E3 state has not been directly observed and very little is known about its molecular and electronic structure or reactivity. In recent computational studies, we have explored the energy surfaces of the E2 and E4 by QM/MM modelling, and found that the most stable hydride isomers contain bridging or partially bridging hydrides with an open protonated belt sulfide-bridge. In this work we systematically explore the energy surface of the E3 redox state, comparing single hydride and two-hydride isomers with varying coordination and bridging vs. terminal sulfhydryl groups. We also include a model featuring a triply protonated carbide. The results are only mildly dependent on the QM-region size. The three most stable E3 isomers at the r2SCAN level of theory have in common: an open belt sulfide-bridge (terminal sulfhydryl group on Fe6) and either 2 bridging hydrides (between Fe2 and Fe6), 1 bridging-1-terminal hydride (around Fe2 and Fe6) or a dihydrogen ligand bound at the Fe2 site. Analyzing the functional dependency of the results, we find that functionals previously found to predict accurate structures of spin-coupled Fe/Mo dimers and FeMoco (TPSSh, B97-D3, r2SCAN, and B3LYP*) are in generally good agreement about the stability of these 3 E3 isomers. However, B3LYP*, similar to its parent B3LYP method, predicts a triply protonated carbide isomer as the most stable isomer, an unlikely scenario in view of the lack of experimental evidence for carbide protonation occurring in reduced FeMoco states. Distinguishing further between the 3 hydride isomers is difficult and this flexible coordination nature of hydrides suggests that multiple hydride isomers could be present during experimental conditions. N2 binding was explored and resulted in geometries with 2 bridging hydrides and N2 bound to either Fe2 or Fe6 with a local low-spin state on the Fe. N2 binding is predicted to be mildly endothermic, similar to the E2 state, and it seems unlikely that the E3 state is capable of binding N2.

Theoretical investigation on the reaction mechanism of UTP cyclohydrolase.

Nucleoside triphosphate cyclohydrolase (UrcA) is a critical enzyme of the uracil catabolism pathway that catalyses the two-step hydrolysis of uridine triphosphate (UTP). Although the recently resolved X-ray structure of UrcA in complex with substrate analogue dUTP provided insights into the structural characteristics of the enzyme, the detailed catalytic mechanism, including how the reaction intermediate accomplishes conformational conversion in the active centre, remains unclear. In this study, extensive DFT calculations and MD simulations were performed to investigate the catalytic reaction process of UrcA. This study shows that the first hydrolytic reactions in UrcA follow a three-step mechanism, while the second hydrolytic reaction follows a two-step mechanism. Glu392 plays a critical role in deprotonating the lytic water in both hydrolytic reactions. The rate-limiting step of the first hydrolytic reaction lies in the cleavage of the uracil ring, in which an extraneous water molecule bridges the proton transfer from C6-OH to N1 to enable the reaction to go through a six-membered transition state with relatively low steric tension. In the second hydrolytic reaction, Glu392 abstracts protons from the lytic water and directly transfers them to the nitrogen atom of the cleaved C4-N3 bond so that the hydrolytic reaction is no longer rate-limited by the C-N bond cleavage step. MD simulations show that the reaction intermediate experiences spontaneous conformation overturn in the active site of UrcA under the assistance of the hydrogen bond interaction from Tyr307 to place its C4-N3 bond alongside the Zn2+ centre of the enzyme to trigger the second hydrolytic reaction.

Reorienting Mechanism of Harderoheme in Coproheme Decarboxylase-A Computational Study.

Coproheme decarboxylase (ChdC) is an important enzyme in the coproporphyrin-dependent pathway (CPD) of Gram-positive bacteria that decarboxylates coproheme on two propionates at position 2 and position 4 sequentially to generate heme b by using H2O2 as an oxidant. This work focused on the ChdC from Geobacillus stearothermophilus (GsChdC) to elucidate the mechanism of its sequential two-step decarboxylation of coproheme. The models of GsChdC in a complex with substrate and reaction intermediate were built to investigate the reorienting mechanism of harderoheme. Targeted molecular dynamics simulations on these models validated that harderoheme is able to rotate in the active site of GsChdC with a 19.06-kcal·mol-1 energy barrier after the first step of decarboxylation to bring the propionate at position 4 in proximity of Tyr145 to continue the second decarboxylation step. The harderoheme rotation mechanism is confirmed to be much easier than the release-rebinding mechanism. In the active site of GsChdC, Trp157 and Trp198 comprise a "gate" construction to regulate the clockwise rotation of the harderoheme. Lys149 plays a critical role in the rotation mechanism, which not only keeps the Trp157-Trp198 "gate" from being closed but also guides the propionate at position 4 through the gap between Trp157 and Trp198 through a salt bridge interaction.

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.

Binding of exogenous cyanide reveals new active-site states in [FeFe] hydrogenases.

[FeFe] hydrogenases are highly efficient metalloenyzmes for hydrogen conversion. Their active site cofactor (the H-cluster) is composed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) linked to a unique organometallic di-iron subcluster ([2Fe]H). In [2Fe]H the two Fe ions are coordinated by a bridging 2-azapropane-1,3-dithiolate (ADT) ligand, three CO and two CN- ligands, leaving an open coordination site on one Fe where substrates (H2 and H+) as well as inhibitors (e.g. O2, CO, H2S) may bind. Here, we investigate two new active site states that accumulate in [FeFe] hydrogenase variants where the cysteine (Cys) in the proton transfer pathway is mutated to alanine (Ala). Our experimental data, including atomic resolution crystal structures and supported by calculations, suggest that in these two states a third CN- ligand is bound to the apical position of [2Fe]H. These states can be generated both by "cannibalization" of CN- from damaged [2Fe]H subclusters as well as by addition of exogenous CN-. This is the first detailed spectroscopic and computational characterisation of the interaction of exogenous CN- with [FeFe] hydrogenases. Similar CN--bound states can also be generated in wild-type hydrogenases, but do not form as readily as with the Cys to Ala variants. These results highlight how the interaction between the first amino acid in the proton transfer pathway and the active site tunes ligand binding to the open coordination site and affects the electronic structure of the H-cluster.

Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli.

The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive.