title: In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii.
abstract: H2 oxidation in Azotobacter vinelandii is catalyzed by a membrane-bound, alpha beta dimeric [NiFe] hydrogenase. Maturation of the enzyme involves cleavage of a putative N-terminal signal sequence in the beta subunit and removal of 15 amino acids from the C terminus of the alpha subunit. Cells limited for nickel exhibited low hydrogenase activities and contained an apparently large form of the alpha subunit. Addition of nickel to such cells increased hydrogenase activities fivefold over 2 h. The increase in the first hour did not require transcription and translation and correlated with processing of the large form of the alpha subunit (pre-alpha) to the small form (alpha) resembling the alpha subunit from the purified enzyme. In vivo, pre-alpha appeared soluble whereas the majority of alpha was membrane bound. Processing of pre-alpha to alpha was reproduced in vitro in membrane-depleted extracts of nickel-limited cells. Processing specifically required the addition of Ni2+, whereas Co2+, Cu2+, Ca2+, Fe2+, Mn2+, and Zn2+ were ineffective. However, Zn2+, Co2+, and Cu2+ inhibited nickel-dependent processing. Mg-ATP and Mg-GTP stimulated processing, whereas anaerobic conditions and/or the addition of dithiothreitol and sodium dithionite was unnecessary. Processing was not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, E64, and pepstatin.
authors.affiliation: Department of Biochemistry, University of Georgia, Athens 30602-7229.
authors.forename: A L
authors.initials: AL
authors.lastname: Menon
authors.forename: R L
authors.initials: RL
authors.lastname: Robson
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002701:Chloramphenicol / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006864:Hydrogenase / Q000096:biosynthesis* / Q000187:drug effects; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009532:Nickel / Q000494:pharmacology*; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D012293:Rifampin / Q000494:pharmacology
chemical_list: D008670:Metals; D011480:Protease Inhibitors; C036852:magnesium GTP; D002701:Chloramphenicol; D009532:Nickel; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D012293:Rifampin