Abstract
Nitrogenase is composed of two separately purified proteins called the Fe protein and the MoFe protein. In Azotobacter vinelandii the genes encoding these structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). The MoFe protein contains an ironmolybdenum cofactor (FeMo cofactor) whose biosynthesis involves the participation of at least five gene products, nifQ, nifB, nifN, nifE, and nifV. In this study an A. vinelandii mutant strain, which contains a defined deletion within the nifH (Fe protein) gene, was isolated and studied. This mutant is still able to accumulate significant amounts of MoFe protein subunits. However, extracts of this nifH deletion strain have only very low levels of MoFe protein acetylene reduction activity. Fully active MoFe protein can be reconstituted by simply adding isolated FeMo cofactor to the extracts. Fe protein is not necessary to stabilize or insert this preformed FeMo cofactor into the FeMo cofactor-deficient MoFe protein synthesized by the nifH deletion strain. Extracts of the nifH deletion strain can carry out molybdate and ATP-dependent in vitro FeMo cofactor biosynthesis provided Fe protein is added, demonstrating that they contain the products encoded by the FeMo cofactor biosynthetic genes. These data demonstrate that the Fe protein is physically required for the biosynthesis of FeMo cofactor in A. vinelandii.
Highlights
Nitrogenase is composed of two separately purified attributed, at leastin part,tothe fact thatit binds two proteins called the Fe protein andMthoFee protein
Active MoFe protein can be recon- FeMo cofactor can be separated from purified MoFe protein stitutedby adding isolateFdeMo cofactor to the by anaerobic acid treatment followedby extraction into N
Feprotein is notnecessaryto stabilize or methylformamide (8) and it is often studied in that form. Insert this preformed FeMo cofactorintothe FeMo Isolated FeMo cofactor can reconstitute MoFe protein activcofactor-deficient MoFe proteinsynthesized by the ities inextracts of mutant strainsof Klebsiella pneumoniae or nifH deletion strain
Summary
Material.-MoFe protein and Fe protein ofA. uinelandii were purified according to our published method (19). Construction of nifH Deletion Strain-Growth of Escherichia coli strains carrying hybrid nif-containing plasmids and thepreparation, restriction enzyme digestion, and ligation of hybrid plasmid DNAs were all performed as described previously (24). Assays for in vitro synthesis of FeMo cofactor were performed essentially as described by Shah et al (30).For internal consistency, we have reported our activities (Fig. 5) in units of nanomoles of acetylene reduced/ min/mg of totalprotein. Construction of nifH Deletion Strain DJ54"The A. uinelundii nitrogenase structural genes have been previously isolated andtheir complete nucleotide sequences have been determined (24). These genes are clustered and arranged as promoter-nifH (Fe protein)-niD (MoFe protein a subunit)-. The resultant mutant strai(nDJ54,AnifH) has a 535-base pair deletion within the nifH gene, does not pro-
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