CARBOXYL-TERMINAL PROCESSING MAY BE ESSENTIAL FOR PRODUCTION OF ACTIVE NIFE HYDROGENASE IN AZOTOBACTER-VINELANDII

The NiFe hydrogenase from Azotobacter vinelandii is a membrane-bound alpha-beta heterodimer that can oxidize H-2 to protons and electrons and thereby provide energy. Genes encoding the alpha and beta-subunits, hoxG and hoxK respectively, followed by thirteen contiguous accessory genes potentially involved in H-2 oxidation, have been previously sequenced. Mutations in some of these accessory genes give rise to inactive enzyme containing an alpha-subunit with decreased electrophoretic mobility. Mass spectral analysis of the subunits demonstrated that the alpha-subunit had a molecular weight 1,663 Da less than that predicted from hoxG. Since the N-terminal sequence of the purified alpha-subunit matches the sequence predicted from hoxG we suggest this difference is due to removal of the C-terminus of the alpha-subunit which may be an important step linked to metal insertion, localization, and formation of active hydrogenase.