Photolysis Of Hi-Co Nitrogenase - Observation Of A Plethora Of Distinct Co Species Using Infrared Spectroscopy

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 kJmol(-1). Two alpha-H195 variant enzymes yielded additional signals. Asparagine substitution, alpha-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, alpha-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.