Fourier transform infrared spectroscopy (FTIR) was used to observe the photolysis and recombination of a new EPR-silent CO-inhibited form of α-H195Q nitrogenase from Azotobacter vinelandii. Photolysis at 4 K reveals 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 was assigned to the “Lo-3” photoproduct. By using an isotopic mixture of ¹²C ¹⁶O and ¹³C ¹⁸O, we show that the Hi-3 bands arise from coupling of two similar CO oscillators with one uncoupled frequency at approximately =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 approximately 6.5 kJ mol⁻¹. The frequencies of the Hi-3 bands suggest terminal CO ligation. This hypothesis was tested 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 about 3.5:1, which is in good agreement with experiment. From the observed IR intensities, Hi-3 was found to be 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.

Fourier-transform infrared-spectroscopy (FT-IR) was used to study the photochemistry of CO-inhibited Azotobacter vinelandii Mo nitrogenase using visible light at cryogenic temperatures. The FT-IR difference spectrum of photolyzed hi-CO at 4 K comprises negative bands at 1973 cm^–1 and 1679 cm^–1 together with positive bands at 1711 cm^–1, 2135 and 2123 cm^–1. The negative bands are assigned to a hi-CO state that comprises 2 metal-bound CO ligands, one terminally bound, and one bridged and/or protonated species. The positive band at 1711 cm^–1 is assigned to a lo-CO product with a single bridged and/or protonated metal-CO group. We term these species “Hi-1” and “Lo-1”, respectively. The high-energy bands are assigned to a liberated CO trapped in the protein pocket. Warming results in CO recombination, and the temperature dependence of the recombination rate yields an activation energy of 4 kJ mol^–1. Two α-H195 variant enzymes yielded additional signals. Asparagine substitution, α-H195N, gives a spectrum containing 2 negative “Hi-2” bands at 1936 and 1858 cm^–1 with a positive “Lo-2” band at 1780 cm^–1, while glutamine substitution, α-H195Q, produces a complex spectrum that includes a third CO species, with negative “Hi-3” bands at 1938 and 1911 cm^–1 and a positive feature “Lo-3” band at 1921 cm^–1. These species can be assigned to a combination of terminal, bridged, and possibly protonated CO groups bound to the FeMo cofactor active site. The proposed structures are discussed in terms of both CO inhibition and the mechanism nitrogenase catalysis. Given the intractability of observing nitrogenase intermediates by crystallographic methods, IR-monitored photolysis appears to be a promising and information-rich probe of nitrogenase structure and chemistry.