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. 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 weight of 61-63 kDa, contains less than 0.1 atom of Ni2+ or Fe2+ per dimer, and has a lmax at 277.5 nm (e277.5 32.1 mM-1cm-1) with no absorption peaks at the visible region. CooC catalyzes the hydrolysis of ATP and GTP with Km values of 24.4 and 26.0 mM, and Vmax 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 apo-CODH 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 apo-CODH. 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.