THE IDENTIFICATION, PURIFICATION, AND CHARACTERIZATION OF COOJ - A NICKEL-BINDING PROTEIN THAT IS COREGULATED WITH THE NI-CONTAINING CO DEHYDROGENASE FROM RHODOSPIRILLUM-RUBRUM

CooJ, a nickel-binding protein from the CO dehydrogenase system of Rho dospirillum rubrum, was purified by immobilized metal affinity chromat ography. CooJ is a CO-induced protein predicted to contain a nickel bi nding motif composed of 16 histidine residues in the final 34 amino ac ids of the 12.5-kDa protein. When cells grown in the presence of CO we re fractionated on an immobilized metal affinity chromatography column and analyzed by SDS-polyacrylamide gel electrophoresis, the major pro tein observed in the effluent migrated at an apparent molecular mass o f 19 kDa. The 19-kDa protein was absent in extracts of cells grown in the absence of CO and the mutant strain, UR294, which lacks a function al cooJ gene. N-terminal sequence analysis confirmed that the 19-kDa p rotein is the product of the cooJ gene. Purified CooJ was shown to bin d four nickel atoms per CooJ monomer with a K-d of 4.3 mu M. Other div alent metals competed with the following order of affinity and corresp onding K-i:Zn2+ (5 mu M) > Cd2+ (19 mu M) > Co2+ (23 mu M) > Cu2+ (122 mu M). CooJ chromatographed on a calibrated Superose 12 gel filtratio n column eluted at 39 kDa, a position consistent with a multimeric nat ive molecular mass for CooJ.