Mutual Effects of Substrates and Inhibitors in Reactions Catalyzed by Isolated Iron–Molybdenum Cofactor of Nitrogenase

Abstract

The inhibiting effects of CO and N2 on the ability of the nitrogenase iron–molybdenum cofactor (FeMoco) to catalyze acetylene reduction outside the protein were studied to obtain data on the mechanism of substrate reduction at the active center of the enzyme nitrogenase. It was found that CO and N2 reacted with FeMoco that was separated from the enzyme and reduced by zinc amalgam (E = –0.84 V relative to a normal hydrogen electrode (NHE)) (I) or europium amalgam (E = –1.4 V relative to NHE) (II). In system I, CO reversibly inhibited the reaction of acetylene reduction to ethylene with K i = 0.05 atm CO. In system II, CO inhibited the formation of the two products of C2H2 reduction in different manners: the mixed-type or competitive inhibition was found for ethylene formation with K i = 0.003 atm CO and the incomplete competitive inhibition was found for ethane formation with K i = 0.006 atm CO. The fraction of C2H6 in the reaction products was greater than 50% at a CO pressure of 0.05 atm because of the stronger inhibiting effect of CO on the formation of C2H4. The change in the product specificity of acetylene-reduction centers under influence of CO was explained by some stabilization of the intermediate complex [FeMoco · C2H2] upon the simultaneous coordination of CO to the catalytic cluster. Because of this, the fraction value of ethane as a multielectron reduction product increased. The experimental results suggest that several active sites at the FeMoco cluster reduced outside the protein can be simultaneously occupied by substrates and (or) inhibitors. The inhibition of both ethane and ethylene formation by molecular nitrogen in system II is competitive with K i = 0.5 atm N2 for either product. That is, N2 and C2H2 as ligands compete for the same coordination site at the reduced FeMoco cluster. The inhibiting effects of CO and N2 on the catalytic behaviors of both isolated FeMoco and that in the enzyme were compared.