Probing Catalytic Function through Amino-Acid Substitutions in Azotobacter Vinelandii Molybdenum-Dependent Nitrogenase

Abstract

As part of our studies to elucidate the functions of both types of prosthetic group in the MoFe protein of molybdenum-dependent nitrogenase (Mo-nitrogenase), we are attempting to map the catalytic surface of both the FeMo-cofactor (FeMoco) and the P-cluster pair by individually substituting amino-acid residues within their polypeptide environments and analyzing the catalytic, redox and spectroscopic consequences for the resulting altered MoFe proteins (Newton, Dean, 1993). There is substantial evidence that implicates FeMoco as the site of substrate reduction (Shah, Brill, 1977; Hawkes et al., 1984; Scott et al., 1990, Kim et al., 1995), which occurs through a series of events that starts with the sequential association and dissociation of the two nitrogenase component proteins (Hageman, Burris, 1978; Lowe, Thorneley, 1984). During this first stage, two molecules of MgATP are hydrolyzed and a single electron is transferred from the Fe-protein component (a homodimer of ca. 63kDa, encoded by nifH) to the MoFe-protein component (an a2ß2 tetramer of ca. 230kDa, encoded by nifDK). The transferred electron is generally believed to be accepted by the P-cluster pair before intramolecular transfer to first FeMoco and then bound substrate (for a review, see Newton, 1993). The native MoFe protein exhibits a S=3/2 electron paramagnetic resonance (EPR) signal that is unique among metallobiomolecules. This EPR signal arises in FeMoco.