IR‐Monitored Photolysis of CO‐Inhibited Nitrogenase: A Major EPR‐Silent Species with Coupled Terminal CO Ligands

Abstract

We have used Fourier transform infrared spectroscopy (FT-IR) to observe the photolysis and recombination of a novel EPR-silent CO-inhibited form of α-H195Q nitrogenase from Azotobacter vinelandii. Photolysis at 4 K yields a strong negative IR difference band at 1938 cm−1, along with a weaker negative feature at 1911 cm−1. These bands and the associated chemical species have both been assigned the label ‘Hi-3’. A positive band at 1921 cm−1 is assigned to the ‘Lo-3’ photoproduct. By using an isotopic mixture of 12C16O and 13C18O, we show that the Hi-3 bands arise from coupling of two similar CO oscillators with one uncoupled frequency at ~1917 cm−1. Although in previous studies Lo-3 was not observed to recombine, by extending the observation range to 200–240 K we found that recombination to Hi-3 does indeed occur, with an activation energy of ~6.5 kJ mol−1. The frequencies of the Hi-3 bands suggest terminal CO ligation. We tested this hypothesis with DFT calculations on models with terminal CO ligands on Fe2 and Fe6 of the FeMo-cofactor. An S = 0 model with both CO ligands in exo positions predicts symmetric and asymmetric stretches at 1938 and 1909 cm−1 respectively, with relative band intensities of ~3.5:1, in good agreement with experiment. From the observed IR intensities, we find that Hi-3 is present at a concentration about equal to that of the EPR-active Hi-1 species. The relevance of Hi-3 to the nitrogenase catalytic mechanism and its recently discovered Fischer-Tropsch chemistry is discussed.

Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy — Observation of a candidate promoting vibration

“…The geometries, optimized at the PBE/LACV3P** level using GAUSSIAN 03 [54] software, were used for the analytic Hessian calculations, resulting in the DFT Raman intensities and other vibrational properties discussed in the text. We found the present and essentially equivalent setups to perform well for the vibrational dynamics of several iron-sulfur systems [4, 33, 55–58]. The analysis of the computed normal modes was done using an in-house Q-SPECTOR Python tool to model the Raman spectra.…”

“…Using stopped-flow IR spectroscopy, variations in the rate and type of CO binding have been observed for variant MoFe proteins with amino-acid substitutions at the α-70Val side chain position [31]. IR-monitored photolysis of frozen CO-inhibited N 2 ase solutions has revealed evidence for both terminal and ‘formyl-like’ CO ligands, in some cases, bound to EPR-silent FeMo-cofactor forms [32, 33]. The combination of NRVS and EXAFS, together with DFT calculations, have offered evidence for significant structural changes upon binding of CO [4] or nitrogenous ligands [34].…”

Ligand binding to the FeMo-cofactor: Structures of CO-bound and reactivated nitrogenase

“…Like the structure presented here, the spectroscopically identified “lo-CO” state has been proposed to involve one molecule of CO bound to the active site in a bridging mode (22, 26). A state with two CO bound to Fe2 and Fe6 could correspond to the “high-CO” form (22, 23) and might represent an intermediate relevant to the C-C coupling reaction.…”

NifA- and CooA-Coordinated cowN Expression Sustains Nitrogen Fixation by Rhodobacter capsulatus in the Presence of Carbon Monoxide

“…N 2 reduction by Mo-and V-nitrogenases is strongly inhibited by carbon monoxide (CO) (5)(6)(7)(8)(9)(10). It is assumed that CO blocks electron flow in Mo-and V-nitrogenases at different stages, because hydrogen production by Mo-nitrogenase is largely unaffected by CO, whereas V-nitrogenase-catalyzed H 2 evolution decreases gradually with increasing CO concentrations (6).…”