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Molybdenum nitrogenase plays a crucial role in the biological nitrogen cycle by catalyzing the reduction of dinitrogen (N 2 ) to ammonia (NH 3 ) under ambient conditions. However, the underlying mechanisms of nitrogenase catalysis, including electron and proton transfer dynamics, remain only partially understood. In this study, we covalently attached molybdenum nitrogenase (MoFe) to gold electrodes and utilized surface-enhanced infrared absorption spectroscopy (SEIRA) coupled with electrochemistry techniques to investigate its catalytic mechanism. Our biohybrid system enabled electron transfer via a mild mediator, likely mimicking the natural electron flow through the P-cluster to FeMoco, the enzyme's active site. For the first time, we experimentally observed both terminal and bridging S-H stretching frequencies, resulting from the protonation of bridging sulfides in FeMoco during turnover conditions providing direct evidence of their role in catalysis. These experimental observations are further supported by QM/MM calculations. Additionally, we investigated CO inhibition, demonstrating both CO binding and unbinding dynamics under electrochemical conditions. These insights not only advance our understanding of the mechanistic cycle of molybdenum nitrogenase but also establish a foundation for studying alternative nitrogenases, including vanadium and iron nitrogenases.

Multiply-bonded main group compounds of groups 13-15 are attracting significant interest not only because they provide fundamental insight into the nature of metal-metal bonding, but also for their potential in small molecule bond activation and catalysis. This includes dialumenes, neutral Al(I) compounds that contain AlAl double bonds, which display high reactivity owing to their intrinsic diradicaloid character. The electronic structure of the simplest dialumene, Al 2 H 2 , is here analyzed up to a practical Full-CI limit using DMRG and selected CI methods for the bond dissociation energy (BDE), geometry and properties of the electron density (difference density, ELF). Acquiring Full-CI reference values for the simplest dialumene (but possessing the highest diradical character) allows for a rigorous benchmarking of simpler correlated wavefunction theory (WFT) methods and density functional methods in treating the electronic structure of such systems. Single-reference coupled cluster theory using a RHF reference is found to reliably converge to the Full-CI limit and CCSD(T) is fully capable of capturing the diradical character, while multi-reference methods offer no clear advantages. Density functional methods struggle to fully describe the electronic structure complexity although non-hybrid functionals such as TPSS come close. Solving the inverse Kohn-Sham problem for a Full-CI-quality density revealed minimal density-driven errors in the TPSS-calculated BDE unlike high-percentage hybrids such as M06-2X. No density functional, however, predicts accurate relative energies. Fractional-occupation density plots at the TPSS level correlate well with WFT-based diradical character metrics, a useful result for determining diradical character in larger systems.

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 1 H ENDOR and there is likely a single hydride in the E2 redox state. The 2-hydride E4 state has been experimentally observed to bind N 2 and it has been speculated that E3 may bind N 2 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 r 2 SCAN 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, r 2 SCAN, 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. N 2 binding was explored and resulted in geometries with 2 bridging hydrides and N 2 bound to either Fe 2 or Fe 6 with a local low-spin state on the Fe. N 2 binding is predicted to be mildly endothermic, similar to the E2 state, and it seems unlikely that the E3 state is capable of binding N 2 .

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 H 2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H 2 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 H 2 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., Competing Interests: The authors report no conflict of interests., (This journal is © The Royal Society of Chemistry.)

The biological conversion of N 2 to NH 3 is accomplished by the nitrogenase family, which is collectively comprised of three closely related but unique metalloenzymes. In the present study, we have employed a combination of the synchrotron-based technique of 57 Fe nuclear resonance vibrational spectroscopy together with DFT-based quantum mechanics/molecular mechanics (QM/MM) calculations to probe the electronic structure and dynamics of the catalytic components of each of the three unique M N 2 ase enzymes (M = Mo, V, Fe) in both the presence (holo-) and absence (apo-) of the catalytic FeMco clusters (FeMoco, FeVco and FeFeco). The results described herein provide vibrational mode assignments for important fingerprint regions of the FeMco clusters, and demonstrate the sensitivity of the calculated partial vibrational density of states (PVDOS) to the geometric and electronic structures of these clusters. Furthermore, we discuss the challenges that are faced when employing NRVS to investigate large, multi-component metalloenzymatic systems, and outline the scope and limitations of current state-of-the-art theory in reproducing complex spectra.

Orange protein (Orp) is a small bacterial metalloprotein of unknown function that harbors a unique molybdenum/copper (Mo/Cu) heterometallic cluster, [S 2 MoS 2 CuS 2 MoS 2 ] 3- . In this paper, the performance of Orp as a catalyst for the photocatalytic reduction of protons into H 2 has been investigated under visible light irradiation. We report the complete biochemical and spectroscopic characterization of holo -Orp containing the [S 2 MoS 2 CuS 2 MoS 2 ] 3- cluster, with docking and molecular dynamics simulations suggesting a positively charged Arg, Lys-containing pocket as the binding site. Holo -Orp exhibits excellent photocatalytic activity, in the presence of ascorbate as the sacrificial electron donor and [Ru(bpy) 3 ]Cl 2 as the photosensitizer, for hydrogen evolution with a maximum turnover number of 890 after 4 h irradiation. Density functional theory (DFT) calculations were used to propose a consistent reaction mechanism in which the terminal sulfur atoms are playing a key role in promoting H 2 formation. A series of dinuclear [S 2 MS 2 M'S 2 MS 2 ] (4 n )- clusters, with M = Mo VI , W VI and M' ( n +) = Cu I , Fe I , Ni I , Co I, Zn II , Cd II were assembled in Orp, leading to different M/M'-Orp versions which are shown to display catalytic activity, with the Mo/Fe-Orp catalyst giving a remarkable turnover number (TON) of 1150 after 2.5 h reaction and an initial turnover frequency (TOF°) of 800 h -1 establishing a record among previously reported artificial hydrogenases.

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 N 2 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 N 2 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 N 2 . 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 N 2 binding in the E 4 state of FeMoco. We have systematically studied N 2 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 N 2 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 N 2 binding in the calculations: spin-pairing of d xz and d yz orbitals of the N 2 -binding Fe ion, a coordination change at the N 2 -binding Fe ion aided by a hemilabile protonated sulfur, and finally hydride ligation. The results show that N 2 binding to E 0 , E 1 , and E 2 models is generally unfavorable, likely due to the high-energy cost of stabilizing the necessary spin-paired electronic structure of the N 2 -binding Fe ion in a ligand environment that clearly favors high-spin states. The results for models of E 4 , however, suggest a feasible model for why N 2 binding occurs experimentally in the E 4 state. E 4 models with two bridging hydrides between Fe2 and Fe6 show much more favorable N 2 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 d xz and d yz orbitals that backbond to a terminal N 2 ligand. Within this model for N 2 binding, both Fe2 and Fe6 emerge as possible binding site scenarios. Due to steric effects involving the His195 residue, affecting both the N 2 ligand and the terminal SH - group, distinguishing between Fe2 and Fe6 is difficult; furthermore, the binding depends on the protonation state of His195.

Open-shell transition metal chemistry presents challenges to contemporary electronic structure methods, based on either density functional or wavefunction theory. While CCSD(T) is the well-trusted gold standard for maingroup thermochemistry, the accuracy and robustness of the method is less clear for open-shell transition metal chemistry, requiring benchmarking of CCSD(T)-based protocols against either higher-level theory or experiment. Ionization energies (IEs) of metallocenes provide an interesting test case with metallocenes being common redox reagents as well as playing roles as redox mediators and cocatalysts in redox catalysis. Using highly accurate ZEKE-MATI experimental measurements of gas phase adiabatic (5.3275 ± 0.0006 eV) and vertical (5.4424 ± 0.0006 eV) ionization energies of cobaltocene, we systematically assessed the accuracy of the local coupled-cluster method DLPNO-CCSD(T) with respect to geometry, reference determinant, basis set size and extrapolation schemes, PNO cut-off and extrapolation, local triples approximation, relativistic effects and core-valence correlation. We show that PNO errors are controllable via the recently introduced PNO extrapolation schemes and that the expensive iterative triples (T 1 ) contribution can be made more manageable by calculating it as a smaller-basis/smaller PNO-cutoff correction. The reference determinant turns out to be a critical aspect in these calculations with the HF determinant resulting in large DLPNO-CCSD(T) errors, likely due to the qualitatively flawed molecular orbital spectrum. The BP86 functional on the other hand was found to provide reference orbitals giving small DLPNO-CCSD(T) errors, likely due to more realistic orbitals as suggested by the more consistent MO spectrum compared to HF. A protocol including complete basis set extrapolations with correlation-consistent basis sets, complete PNO space extrapolations, iterative triples- and core-valence correlation corrections was found to give errors of -0.07 eV and -0.03 eV for adiabatic- and vertical-IE of cobaltocene, respectively, giving close to chemical accuracy for both properties. A computationally efficient DLPNO-CCSD(T) protocol was devised and tested against adiabatic ionization energies of 6 different metallocenes (V, Cr, Mn, Fe, Co, Ni). For the other metallocenes, the iterative triples (T 1 ) and PNO extrapolation contributions turn out to be even more important. The results give errors close to the experimental uncertainty, similar to recent auxiliary-field quantum Monte Carlo results. The quality of the reference determinant orbitals is identified as the main source of uncertainty in CCSD(T) calculations of metallocenes.

[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 (H 2 and H + ) as well as inhibitors ( e.g. O 2 , CO, H 2 S) 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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The open-shell electronic structure of iron-sulfur clusters presents considerable challenges to quantum chemistry, with the complex iron-molybdenum cofactor (FeMoco) of nitrogenase representing perhaps the ultimate challenge for either wavefunction or density functional theory. While broken-symmetry density functional theory has seen some success in describing the electronic structure of such cofactors, there is a large exchange-correlation functional dependence in calculations that is not fully understood. In this work, we present a geometric benchmarking test set, FeMoD11, of synthetic spin-coupled Fe-Fe and Mo-Fe dimers, with relevance to the molecular and electronic structure of the Mo-nitrogenase FeMo cofactor. The reference data consists of high-resolution crystal structures of metal dimer compounds in different oxidation states. Multiple density functionals are tested on their ability to reproduce the local geometry, specifically the Fe-Fe/Mo-Fe distance, for both antiferromagnetically coupled and ferromagnetically coupled dimers via the broken-symmetry approach. The metal-metal distance is revealed not only to be highly sensitive to the amount of exact exchange in the functional but also to the specific exchange and correlation functionals. For the antiferromagnetically coupled dimers, the calculated metal-metal distance correlates well with the covalency of the bridging metal-ligand bonds, as revealed via the corresponding orbital analysis, Hirshfeld S/Fe charges, and Fe-S Mayer bond order. Superexchange via bridging ligands is expected to be the dominant interaction in these dimers, and our results suggest that functionals that predict accurate Fe-Fe and Mo-Fe distances describe the overall metal-ligand covalency more accurately and in turn the superexchange of these systems. The best performing density functionals of the 16 tested for the FeMoD11 test set are revealed to be either the nonhybrid functionals r 2 SCAN and B97-D3 or hybrid functionals with 10-15% exact exchange: TPSSh and B3LYP*. These same four functionals are furthermore found to reproduce the high-resolution X-ray structure of FeMoco well according to quantum mechanics/molecular mechanics (QM/MM) calculations. Almost all nonhybrid functionals systematically underestimate Fe-Fe and Mo-Fe distances (with r 2 SCAN and B97-D3 being the sole exceptions), while hybrid functionals with >15% exact exchange (including range-separated hybrid functionals) overestimate them. The results overall suggest r 2 SCAN, B97-D3, TPSSh, and B3LYP* as accurate density functionals for describing the electronic structure of iron-sulfur clusters in general, including the complex FeMoco cluster of nitrogenase.