Investigation of CO Bound to Inhibited Forms of Nitrogenase MoFe Protein by ~(13)C ENDOR

摘要

Nitrogenase catalyzes one of the most remarkable biological reactions, the reduction of dinitrogen to ammonia at ambient temperature and pressure. N_2 + 8H~+ + 8e~- + 16MgATP → 2NH_3 + H_2 + 16 MgADP + 16P_i (1) The MoFe protein of nitrogenase contains two metal centers, the P-cluster (Fe_8S_(7-8)) and the FeMo-cofactor (Fe_7S_9Mo: homocitrate), which is believed to be the substrate binding site. This protein has been the subject of numerous biophysical studies, culminating in the disclosure of its beautiful and unexpected structure. Current X-ray crystal-lographic modeling shows that the P-cluster is composed of two ferredoxin-like Fe_4S_4 cubane clusters linked by two cysteine thiols and one disulfide; the cofactor can be viewed as Fe_4S_3 and MoFe_3S_3 clusters linked by three sulfides. Surprisingly, knowledge of the structure gives no obvious insights into the nature of substrate or inhibitor binding. We report here a first step in addressing this problem: Q-band CW and pulsed electron-nuclear double resonance (ENDOR) spectroscopy gives the first direct observations of a diatomic molecule, CO, bound to a metal center of the MoFe protein.