The Fe–V Cofactor of Vanadium Nitrogenase Contains an Interstitial Carbon Atom

Abstract

The first direct evidence is provided for the presence of an interstitial carbide in the FeÀVc ofactor of Azotobacter vinelandii vanadium nitrogenase.A sf or our identification of the central carbide in the FeÀMo cofactor,weemployed Fe Kb valence-to-core X-ray emission spectroscopyand density functional theory calculations,and herein report the highly similar spectra of both variants of the cofactor-containing protein. The identification of an analogous carbide,and thus an atomically homologous active site in vanadium nitrogenase,highlights the importance and influence of both the interstitial carbide and the identity of the heteroatom on the electronic structure and catalytic activity of the enzyme.The biological fixation of atmospheric dinitrogen (N 2 )t o ammonium ions is exclusively promoted by nitrogenases (N 2 ases), multicomponent metalloenzymes that occur in diazotrophic bacteria and archaea.[1, 2] These enzymes utilize highly complex Fe-S clusters to effect N 2 reduction, and in the case of the more widely studied Mo-dependent N 2 ase,t he redox centers of the protein have been characterized by X-ray crystallography.[3] Thea ctive-site-containing MoFep rotein, an a 2 b 2 heterotetramer,uses an [8Fe-7S] cluster (P-cluster) as an electron-transfer relay,a nd the

QM/MM Study of the Nitrogenase MoFe Protein Resting State: Broken-Symmetry States, Protonation States, and QM Region Convergence in the FeMoco Active Site

“…Due to resolution limitations and the fact that homocitrate was severely truncated and approximated as a glycolate group in their study, the validity of this assignment was unclear. In previous studies 28,29,30,31,32 by one of us (RB) we have assigned the alkoxide group of the homocitrate as protonated but have not until now given a complete justification for this as the question of how well the protein environment is described surrounding the homocitrate remained unclear. The only exception to this is the 4ND8 crystal structure, recently published by Rees & Howard et al 85 which is a crystal structure solved under basic (pH 9.5) conditions, where the O-O distance is on average 2.77 Å as shown in Figure 6d.…”

“…Recently Adamo 26 et al used QM/QM' calculations to propose new mechanisms and Ryde et al studied protonation states by QM/MM 27 . In previous articles by one of us (RB) we utilized large 225-atom cluster models in our studies of the spectroscopic properties of the FeMo cofactor 28,29,30,31,32 . Large cluster models become difficult to work with and require many constraints to keep the correct active site geometry and may not describe correctly the structural flexibility of the cofactor in the enzyme active site pocket.…”

Towards theoretical spectroscopy with error bars: systematic quantification of the structural sensitivity of calculated spectra

“…For the calculation of XES spectra, we employed the DDFT approach of Lee et al, 38 in which excitation energies are calculated as occupied orbital energy differences. This DDFT approach is a rather simple approximation, but it has been shown to be reliable for valence-to-core XES spectra of diverse transition metal complexes, [63][64][65][66][67][68][69][70][71] including the ones considered here. 39 XES intensities are obtained from transition moments between occupied orbitals, including contributions beyond the electric dipole approximation.…”

Uncertainty quantification in theoretical spectroscopy: The structural sensitivity of X‐ray emission spectra

“…Even though this method constitutes the simplest possible approach for the DFT calculation of XES spectra, it has been shown to be reliable for VtC‐XES spectra of diverse transition metal complexes, including the ones considered here . More sophisticated methods for the calculation of XES spectra, such as ΔSCF‐DFT or transition potential DFT (for reviews, see Refs.…”

“…In combination with quantum‐chemical calculations, VtC‐XES can be used to obtain partial structural information, in particular in the vicinity of transition metal centers. VtC‐XES makes it possible to identify which ligands are coordinated to a transition metal center, for instance in molecular transition metal complexes, for catalytic metal centers in zeolites, and for metal clusters in enzymes . The ability of VtC‐XES spectroscopy to unravel further structural details, such as coordination number or bond angles, as well as insights into the electronic structure has also been explored …”