Premium
Evidence that conserved residues Cys‐62 and Cys‐154 within the Azotobacter vinelandii nitrogenase MoFe protein α‐subunit are essential for nitrogenase activity but conserved residues His‐83 and Cys‐88 are not
Author(s) -
Dean D. R.,
Setterquist R. A.,
Brigle K. E.,
Scott D. J.,
Laird N. F.,
Newton W. E.
Publication year - 1990
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1990.tb02061.x
Subject(s) - azotobacter vinelandii , nitrogenase , protein subunit , biology , biochemistry , napa , ligand (biochemistry) , peptide sequence , mutagenesis , mutant , gene , genetics , nitrogen fixation , receptor , bacteria
Summary Metallocluster extrusion requirements, interspecies MoFe‐protein primary sequence comparisons and comparison of the primary sequences of the MoFe‐protein subunits with each other have been used to assign potential P‐cluster (Fe‐S cluster) domains within the MoFe protein. In each β unit of the MoFe protein, subunit domains, which include potential Fe‐S cluster ligands Cys‐62, His‐83, Cys‐88 and Cys‐154, and β‐subunit domains, which include potential Fe‐S cluster ligands Cys‐70, His‐90, Cys‐95 and Cys‐153, are proposed to comprise nearly equivalent P‐cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the α‐ subunit Cys‐62, His‐83, Cys‐88 and Cys‐154 positions by site‐directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates. MoFe—protein acetylene‐reduction activities, and whole‐cell S 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe‐protein α‐submit residues, Cys‐62 and Cys‐154, are probably essential for MoFe‐protein activity but that His‐83 and Cys‐88 residues are not. These results indicate either that His‐83 and Cys‐88 do not provide essential P‐cluster ligand or that a new cluster‐ligand arrangement is formed in their absence.