The molybdenum atom in FeMoco is imperative to the high activity of the enzyme and has been proposed to be Mo(iv). We demonstrate that only Mo(iii) fits Mo HERFD XAS data, the first example of Mo(iii) in biology. Theoretical calculations further reveal an unusual spin-coupled Mo(iii).

Despite decades of research, the structure-activity relationship of nitrogenase is still not understood. Only recently was the full molecular structure of the FeMo cofactor (FeMoco) revealed, but the charge and metal oxidation states of FeMoco have been controversial. With the recent identification of the interstitial atom as a carbide and the more recent revised oxidation-state assignment of the molybdenum atom as Mo(III), here we revisit the Mössbauer properties of FeMoco. By a detailed error analysis of density functional theory-computed isomer shifts and computing isomer shifts relative to the P-cluster, we find that only the charge of [MoFeSC] fits the experimental data. In view of the recent Mo(III) identification, the charge of [MoFeSC] corresponds to a formal oxidation-state assignment of Mo(III)3Fe(II)4Fe(III), although due to spin delocalization, the physical oxidation state distribution might also be interpreted as Mo(III)1Fe(II)4Fe(2.5)2Fe(III), according to a localized orbital analysis of the M = 3/2 broken symmetry solution. These results can be reconciled with the recent spatially resolved anomalous dispersion study by Einsle et al. that suggests the Mo(III)3Fe(II)4Fe(III) distribution, if some spin localization (either through interactions with the protein environment or through vibronic coupling) were to take place.

Nitrogenase is one of the most fascinating enzymes in nature, being responsible for all biological nitrogen reduction. Despite decades of research it is among the enzymes in bioinorganic chemistry whose mechanism is the most poorly understood. The MoFe protein of nitrogenase contains an iron-molybdenum-sulfur cluster, FeMoco, where N 2 reduction takes place. The resting state of FeMoco has been characterized by crystallography, multiple spectroscopic techniques and theory (broken-symmetry density functional theory) and all heavy atoms are now characterized. The cofactor charge, however, has been controversial, the electronic structure has proved enigmatic and little is known about the mechanism. While many computational studies have been performed on FeMoco, few have taken the protein environment properly into account. In this study, we put forward QM/MM models of the MoFe protein from Azotobacter vinelandii, centered on FeMoco. By a detailed analysis of the FeMoco geometry and comparing to the atomic resolution crystal structure we conclude that only the [MoFe 7 S 9 C] 1-charge is a possible resting state charge. Further we find that, of the 3 lowest energy broken-symmetry solutions of FeMoco the BS7-235 spin isomer (where 235 refers to Fe atoms that are "spin-down") is the only one that can be reconciled with experiment. This is revealed by a comparison of the metal-metal distances in the experimental crystal structure, a rare case of spin-coupling phenomena being visible through the molecular structure. This could be interpreted as the enzyme deliberately stabilizing a specific electronic state of the cofactor, possibly for tuning specific reactivity on specific metal atoms. Finally, we show that the alkoxide group on the Mo-bound homocitrate must be protonated under resting state conditions; the presence of which has implications regarding the nature of FeMoco redox states as well as for potential substrate reduction mechanisms.3

Nitrogenases are responsible for biological nitrogen fixation, a crucial step in the biogeochemical nitrogen cycle. These enzymes utilize a two-component protein system and a series of iron–sulfur clusters to perform this reaction, culminating at the FeMco active site (M = Mo, V, Fe), which is capable of binding and reducing N2 to 2NH3. In this review, we summarize how different spectroscopic approaches have shed light on various aspects of these enzymes, including their structure, mechanism, alternative reactivity, and maturation. Synthetic model chemistry and theory have also played significant roles in developing our present understanding of these systems and are discussed in the context of their contributions to interpreting the nature of nitrogenases. Despite years of significant progress, there is still much to be learned from these enzymes through spectroscopic means, and we highlight where further spectroscopic investigations are needed.

Molybdenum nitrogenase is one of the most intriguing metalloenzymes in nature, featuring an exotic ironmolybdenum-sulfur cofactor, FeMoco, whose mode of action remains elusive. In particular, the molecular and electronic structure of the N 2 -binding E 4 state is not known. In this study we present theoretical QM/ MM calculations of new structural models of the E 4 state of molybdenum-dependent nitrogenase and compare to previously suggested models for this enigmatic redox state. We propose two models as possible candidates for the E 4 state. Both models feature two hydrides on the FeMo cofactor, bridging atoms Fe 2 and Fe 6 with a terminal sulfhydryl group on either Fe 2 or Fe 6 (derived from the S2B bridge) and the change in coordination results in local lower-spin electronic structure at Fe 2 and Fe 6 . These structures appear consistent with the bridging hydride proposal put forward from ENDOR studies and are calculated to be lower in energy than other proposed models for E 4 at the TPSSh-QM/MM level of theory. We critically analyze the DFT method dependency in calculations of FeMoco that has resulted in strikingly different proposals for this state. Importantly, dinitrogen binds exothermically to either Fe 2 or Fe 6 in our models, contrary to others, an effect rationalized via the unique ligand field (from the hydrides) at the Fe with an empty coordination site. A low-spin Fe site is proposed as being important to N 2 binding. Furthermore, the geometries of these states suggest a feasible reductive elimination step that could follow, as experiments indicate. Via this step, two electrons are released, reducing the cofactor to yield a distorted 4-coordinate Fe 2 or Fe 6 that partially activates N 2 . We speculate that stabilization of an N 2 -bound Fe(I) at Fe 6 (not found for Fe 2 model) via reductive elimination is a crucial part of N 2 activation in nitrogenases, possibly aided by the apical heterometal ion (Mo or V). By using protons from the sulfhydryl group (to regenerate the sulfide bridge between Fe 2 and Fe 6 ) and the nearby homocitrate hydroxy group, we calculate a plausible route to yield a diazene intermediate. This is found to be more favorable with the Fe 6 -bound model than the Fe 2 -bound model; however, this protonation is uphill in energy, suggesting protonation of N 2 might occur later in the catalytic cycle or via another mechanism. Electronic supplementary information (ESI) available: Further information on all broken-symmetry solutions calculated for all models. Details of the QM/MM model preparation. Spin populations of all models. Localized orbital analysis of selected models. Geometry analysis of E 0 state calculated with different functionals and electronic structure analysis. Cartesian coordinates for the QM regions of all optimized structures available as XYZ les. See

The climbing image nudged elastic band method (CI-NEB) is used to identify reaction coordinates and to find saddle points representing transition states of reactions. It can make efficient use of parallel computing as the calculations of the discretization points, the so-called images, can be carried out simultaneously. In typical implementations, the images are distributed evenly along the path by connecting adjacent images with equally stiff springs. However, for systems with a high degree of flexibility, this can lead to poor resolution near the saddle point. By making the spring constants increase with energy, the resolution near the saddle point is improved. To assess the performance of this energy-weighted CI-NEB method, calculations are carried out for a benchmark set of 121 molecular reactions. The performance of the method is analyzed with respect to the input parameters. Energy-weighted springs are found to greatly improve performance and result in successful location of the saddle points in less than a thousand energy and force evaluations on average (about a hundred per image) using the same set of parameter values for all of the reactions. Even better performance is obtained by stopping the calculation before full convergence and complete the saddle point search using an eigenvector following method starting from the location of the climbing image. This combination of methods, referred to as NEB-TS, turns out to be robust and highly efficient as it reduces the average number of energy and force evaluations down to a third, to 305. An efficient and flexible implementation of these methods has been made available in the ORCA software.

High-resolution X-ray spectroscopy provides insights into the electronic structural differences between the nitrogenase FeMoco and FeVco clusters.

One bond makes all the difference: Three suitably positioned amino acid side chains (see picture) and a hydrophobic environment are all that is required for an amidation reaction with remarkable consequences. An emerging central building block of bacterial surface proteins owes its stability to a spontaneously formed isopeptide bond. The impact of this bond on protein structure and dynamics and the mechanism of its formation are scrutinized in detail.