CO Binding to and Metal-Ion Valencies of the Femo-Cofactor in CO-Inhibited Nitrogenase

Abstract

When Mo-nitrogenase undergoes enzymatic turnover in the presence of CO, the S = 3/2 cofactor signal is replaced by two different S = 1/2 signals, one (lo-CO; g = 2.09, 1.97, 1.93) at low pressure (0.08 atm) and the other (hi-CO; g = 2.06, 2.06, 2.17) at high pressure (0.5 atm). Using ¹³C and ⁵⁷Fe Q-band ENDOR spectroscopy with Azotobacter vinelandii nitrogenase, we recently showed (Pollock et al, 1995; Christie et al, 1996) that these signals arise from CO bound to the cofactor, where lo-CO corresponds to one CO molecule and hi-CO contains two CO’s. The mechanistic relationship between the two CO molecules has also been revealed through pulse-chase experiments showing that one of the two CO molecules bound to the cofactor in hi-CO arises from the single CO in lo-CO. We now extend those measurements to probe the mode and region of CO binding to the cofactor as well as determine the metal-ion valencies of the cofactor in both the resting state and the CO-bound state.