Catalytic behavior of the nitrogenase iron-molybdenum cofactor extracted from the enzyme in the reduction of C2H2 under nonenzymatic conditions

Abstract

To compare the catalytic effect of the active center of nitrogenase (iron-molybdenum cofactor (FeMoco)) under nonenzymatic conditions with the behavior of FeMoco incorporated in a protein, the kinetics of C2H2 reduction with Zn and Eu amalgams was examined in the presence of the cofactor extracted from the MoFe protein of nitrogenase (the specific activity of the extracted FeMoco after its integration into the cofactordeficient MoFe protein ofKp 5058 was 200 ± 20 mol of C2H4 (mol of Mo)-1 min-1. It was found that under exposure to reducing agents of different strength—Zn amalgam (I) (−0.84 V with respect to a normal hydrogen electrode (NHE)) and Eu amalgam (II) (−1.4 V with respect to NHE)—different reduction states of FeMoco were produced. They differed in the number and properties of substrateand inhibitor-coordinating active sites. For I, the rate of ethylene formation was described by a hyperbolic function of substrate concentration (K M = 0.045 atm). Carbon monoxide reversibly inhibited the reduction of acetylene(K i - 0.05). For II, a sigmoid relationship between the rate of accumulation of C2H4 or C2H6 and substrate concentration was found. This relationship was explained by the occurrence of three interrelated sites of acetylene coordination and reduction with the apparent constantK M = 0.08 atm in the FeMoco cluster reduced by europium amalgam. In this case, the specific activity was 40–60 mol of C2H4 (mol of Mo)−1 min−1. For the system with Eu (Hg), the CO inhibition constants were 0.004 and 0.009 atm for the formation of ethylene and ethane, respectively. The behavior of FeMoco as a catalyst for acetylene reduction and the inhibition of this reaction by carbon monoxide in various reducing protein and nonprotein media were compared. This comparison demonstrated that typical features of the catalytic behavior of FeMoco depend primarily on its composition and structure and only secondarily on the type of the reducing agent and on the reaction medium.