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.