Photolysis of Hi‐CO Nitrogenase – Observation of a Plethora of Distinct CO Species Using Infrared Spectroscopy

Abstract

Fourier transform infrared spectroscopy (FT-IR) was used to study the photochemistry of CO-inhibited Azotobacter vinelandii 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.

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

“…(In these and later descriptions, the first value refers to results with 12 C 16 O whereas the second and third values in parentheses, if present, refer to results with 13 C 16 O and 13 C 18 O, respectively.) As noted before [15] , for α -H195Q N 2 ase the spectra are complicated by the presence of other photolyzable species, including bands for Hi-1 at 1969 (1925, 1879) cm −1 and for Hi-2 at 1932 (1888, 1844) cm −1 .…”

“…At long times, the largest spectral changes come from the Hi-3 species, [15] with a strong negative band at 1938 (1894, 1849) cm −1 and a weaker negative band at 1911 (1867, 1824) cm −1 . A positive product band from Lo-3 is seen in between these two bands at 1921 (1877, 1833) cm −1 .…”

“…[15] Accordingly, we warmed the photolyzed samples to 120 K for 10–20 min and then photolyzed a second time. As shown in Figure 1, there was almost no trace of the Hi-3 or Lo-3 features in the second photolysis spectrum, consistent with the absence of Hi-3.…”

“…[15] (In more recent studies we have found that with brief irradiation, the Hi-2 recombination can occur with a much smaller activation energy). [17] The Hi-3 value is similar to the barriers of 8–10 kJ mol −1 seen for Ni-SI a →NiSCO CO recombination in [NiFe] hydrogenases [18] and for photolyzed MbCO.…”

“…[15] Three distinct types of photolyzable CO complexes were found under hi-CO conditions. We labeled these stable inhibited forms ‘Hi-1’, ‘Hi-2’, and ‘Hi-3’.…”