Identification of distinct NAD‐linked hydrogenase protein species in mutants and nickel‐deficient wild‐type cells of Alcaligenes eutrophus H16

By crossed immunoelectrophoresis with antibodies against the NAD‐linked hydrogenase the presence of three hydrogenase protein species was demonstrated in crude extracts of Alcaligenes eutrophus H16. Protein 1 (antigen 1) exhibited NAD‐reducing activity and was shown to be identical with the native heterotetrameric enzyme. Protein 2 (antigen 2) was catalytically inactive in the antibody‐precipitated form and corresponded to the β subunit (56 kDa) of the holoenzyme. Protein 3 (antigen 3) was serologically distinct from antigen 2 and catalyzed NADH‐oxidizing (diaphorase) activity, suggesting that it either consists of the α peptide or of the α and γ subunits of the diaphorase dimer. Tandem immunoelectrophoresis revealed that antigen 2 was the predominant protein species in cells cultivated under nickel deficiency. Low concentrations of the diaphorase‐active antigen 3 were also detected under these conditions. Extracts from mutants defective in the catalytic activity of NAD‐reducing hydrogenase under these conditions. Extracts from mutants defective in the catalytic activity of NAD‐reducing hydrogenase still contained the four polypeptides. This was shown by immunodiffusion and immunoblotting with antibodies raised against the individual subunits. However, as observed with nickel‐deficient cells, no complete tetrameric protein could be identified, and the dominant subunit species (70–80%) was the β peptide.