title: Purification and characterization of membrane-associated CooC protein and its functional role in the insertion of nickel into carbon monoxide dehydrogenase from Rhodospirillum rubrum.
abstract: The accessory protein CooC, which contains a nucleotide-binding domain (P-loop) near the N terminus, participates in the maturation of the nickel center of carbon monoxide dehydrogenase (CODH). In this study, CooC was purified from the chromatophore membranes of Rhodospirillum rubrum with a 3,464-fold purification and a 0.8% recovery, and its biochemical properties were characterized. CooC is a homodimer with a molecular mass of 61-63 kDa, contains less than 0.1 atom of Ni(2+) or Fe(2+) per dimer, and has a lambda(max) at 277.5 nm (epsilon(277.5) 32.1 mm(-1) cm(-1)) with no absorption peaks at the visible region. CooC catalyzes the hydrolysis of ATP and GTP with K(m) values of 24.4 and 26.0 microm and V(max) values of 58.7 and 3.7 nmol/min/mg protein for ATP and GTP hydrolysis, respectively. The P-loop mutated form of K13Q CooC was generated by site-specific replacement of lysine by glutamine and was purified according to the protocol for wild-type CooC purification. The K13Q CooC was inactive both in ATP hydrolysis and in vivo nickel insertion. In vitro nickel activation of apoCODH in the cell extracts from UR2 (wild type) and UR871 (K13Q CooC) showed that activation of nickel-deficient CODH was enhanced by CooC and dependent upon ATP hydrolysis. The overall results suggest that CooC couples ATP hydrolysis with nickel insertion into apoCODH. On the basis of our results and models for analogous systems, the functional roles of CooC in nickel processing into the active site of CODH are presented.
authors.affiliation: Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: W B
authors.initials: WB
authors.lastname: Jeon
authors.forename: J
authors.initials: J
authors.lastname: Cheng
authors.forename: P W
authors.initials: PW
authors.lastname: Ludden
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000502:physiology*; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005838:Genotype; D006868:Hydrolysis; D007700:Kinetics; D008565:Membrane Proteins; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012247:Rhodospirillum rubrum / Q000378:metabolism*; D013053:Spectrophotometry; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D001426:Bacterial Proteins; C088906:CooC protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D009532:Nickel; D000255:Adenosine Triphosphate; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases