Open AccessActivity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster
Author(s) -
A C Robinson,
Barbara K. Burgess,
Dennis R. Dean
Publication year - 1986
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.166.1.180-186.1986
Subject(s) - azotobacter vinelandii , nitrogenase , mutant , biology , azotobacteraceae , protein subunit , structural gene , cofactor , biochemistry , gene , enzyme , genetics , bacteria , nitrogen fixation
The Azotobacter vinelandii genes encoding the nitrogenase structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). In this study various A. vinelandii mutant strains which contain defined deletions within the nitrogenase structural genes were isolated and studied. Mutants deleted for the nifD or nifK genes were still able to accumulate significant amounts of the unaltered MoFe protein subunit as well as active Fe protein. Extracts of such nifD or nifK deletion strains had no MoFe protein activity. However, active MoFe protein could be reconstituted by mixing extracts of the mutant strains. These results establish an approach for the purification of the individual MoFe protein subunits. Mutants lacking either or both of the MoFe protein subunits were still able to synthesize the iron-molybdenum cofactor (FeMo-cofactor), indicating that in A. vinelandii the FeMo-cofactor is preassembled and inserted into the MoFe protein. In contrast, a mutant strain lacking both the Fe protein and the MoFe protein failed to accumulate any detectable FeMo-cofactor. The further utility of specifically altered A. vinelandii strains for the study of the assembly, structure, and reactivity of nitrogenase is discussed.