The Alcaligenes eutrophus membrane-bound hydrogenase gene locus encodes functions involved in maturation and electron transport coupling

Alcaligenes eutrophus H16 produces two [NiFe] hydrogenases which catalyze the oxidation of hydrogen and enable the organism to utilize H-2 as the sole energy source, The genes (hoxK and hoxG) for the heterodimeric, membrane-bound hydrogenase (MBH) are located adjacent to a series of eight accessory genes (hoxZ, hoxM, hoxL, hoxO, hoxQ, hoxR, hoxT, and hoxV). In the present study, we generated a set of isogenic mutants with in-frame deletions in the two structural genes and in each of the eight accessory genes. The resulting mutants can be grouped into two classes on the basis of the H-2-oxidizing activity of the MEW. Class I mutants (hoxK Delta, hoxG Delta, hoxM Delta, hoxO Delta, and hoxQ Delta) were totally devoid of MBH-mediated, H-2-oxidizing activity. The hoxM deletion strain was the only mutant in our collection which was completely blocked in carboxy-terminal processing of large subunit HoxG, indicating that hoxM encodes a specific protease, Class II mutants (hoxZ Delta, hoxL Delta, hoxR Delta, hoxT Delta, and hoxV Delta) contained residual amounts of MBH activity in the membrane fraction of the extracts, Immunochemical analysis and Ni-63 incorporation experiments revealed that the mutations affect various steps in MBH maturation, A lesion in hoxZ led to the production of a soluble MBH which was highly active with redox dye.