Identification of an Fe protein residue (Glu146) of Azotobacter vinelandii nitrogenase that is specifically involved in FeMo cofactor insertion.
The Fe protein of nitrogenase has three separate functions. Much is known about the regions of the protein that are critical to its function as an electron donor to the MoFe protein, but almost nothing is known about the regions of the protein that are critical to its functions in either FeMo cofactor biosynthesis or FeMo cofactor insertion. Using computer modeling and information obtained from Fe protein mutants that were made decades ago by chemical mutagenesis, we targeted a surface residue Glu(146) as potentially being involved in FeMo cofactor biosynthesis and/or insertion. The Azotobacter vinelandii strain expressing an E146D Fe protein variant grows at approximately 50% of the wild type rate. The purified E146D Fe protein is fully functional as an electron donor to the MoFe protein, but the MoFe protein synthesized by that strain is partially ( approximately 50%) FeMo cofactor-deficient. The E146D Fe protein is fully functional in an in vitro FeMo cofactor biosynthesis assay, and the strain expressing this protein accumulates "free" FeMo cofactor. Assays that compared the ability of wild type and E146D Fe proteins to participate in FeMo cofactor insertion demonstrate, however, that the mutant is severely altered in this last reaction. This is the first known mutation that only influences the insertion reaction.
Ribbe
M W
MW
Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA.
Bursey
E H
EH
Burgess
B K
BK
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000254:growth & development; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D018698:Glutamic Acid; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D008983:Molybdoferredoxin; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D007501:Iron; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase