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