C‐Terminal Extension of the H 2 ‐Activating Subunit, HoxH, Directs Maturation of the NAD‐Reducing Hydrogenase in Alcaligenes Eutrophus

Formation of enzymatically active [NiFe] hydrogenases is dependent on a number of posttranslational steps, including metal attachment to a precursor of the catalytic subunit, truncation of a small C‐terminal peptide from the precursor, and oligomerisation of the subunits. Two amino acid replacements were introduced by site‐directed mutagenesis at the C‐terminal proteolytic cleavage site of HoxH, the Ni‐containing subunit of the cytoplasmic NAD‐reducing hydrogenase of Alcaligenes eutrophus H16. Replacement of Ala465, the first residue of the 24‐amino‐acid cleaved polypeptide, by Pro yielded a form of HoxH that was blocked in C‐terminal proteolysis. This HoxH subunit, although capable of binding Ni, was blocked in formation of a stable tetrameric holoenzyme. In the second mutant, the C‐terminal extension of HoxH was eliminated by substituting the Ala codon for a translational stop codon. Although this mutant subunit was able to form the oligomeric holoenzyme, it was devoid of Ni. Both mutant proteins contained only traces of H 2 ‐activating functions. H 2 ‐dependent reduction of NAD and benzylviologen, and D 2 /H + ‐exchange activity were almost completely abolished, while the NADH oxidoreductase activity, mediated by the diaphorase moiety of the hydrogenase, was retained. These results allow the following conclusions: the C‐terminal extension of HoxH is neccessary to direct specific Ni insertion into the hydrogenase; subunit assembly to the holoenzyme is not dependent on Ni insertion; and a precursor with the C‐terminal peptide is not competent for assembly.