Generation of active [NiFe] hydrogenase in vitro from a nickel-free precursor form

The maturation process of [NiFe] hydrogenases includes formation of the nickel metallocenter, proteolytic processing of the large subunit, and assembly with the other hydrogenase subunit(s). An in vitro system for the maturation of the large subunit (HycE) of hydrogenase 3 of Escherichia coli leading to an active enzyme was established. The system is based on extracts of an E. coli mutant lacking the nickel-specific transport system (nik). HycE was present in these extracts in the C-terminally extended precursor form devoid of nickel. Addition of nickel led to nickel incorporation and proteolytic processing of HycE. Under anaerobic conditions, hydrogenase 3 activity was subsequently generated. The maximal rate of the processing reaction was reached at a nickel concentration of 400 microM. The accessory proteins known to be involved in the maturation of HycE in vivo, namely HypB, HypC, HypD, HypE, HypF, and the protease HycI, are required for the in vitro reaction, since processing of HycE did not occur in extracts of double mutants affected in the nik system and in one of the accessory genes. Processing of HycE and generation of hydrogenase 3 activity were achieved in extracts of the nik- delta hycI mutant by addition of both nickel and purified HycI protease.