The identification, purification, and characterization of CooJ. A nickel-binding protein that is co-regulated with the Ni-containing CO dehydrogenase from Rhodospirillum rubrum.
CooJ, a nickel-binding protein from the CO dehydrogenase system of Rhodospirillum rubrum, was purified by immobilized metal affinity chromatography. CooJ is a CO-induced protein predicted to contain a nickel binding motif composed of 16 histidine residues in the final 34 amino acids of the 12.5-kDa protein. When cells grown in the presence of CO were fractionated on an immobilized metal affinity chromatography column and analyzed by SDS-polyacrylamide gel electrophoresis, the major protein observed in the effluent migrated at an apparent molecular mass of 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 functional cooJ gene. N-terminal sequence analysis confirmed that the 19-kDa protein is the product of the cooJ gene. Purified CooJ was shown to bind four nickel atoms per CooJ monomer with a Kd of 4.3 microM. Other divalent metals competed with the following order of affinity and corresponding Ki: Zn2+ (5 microM) > Cd2+ (19 microM) > Co2+ (23 microM) > Cu2+ (122 microM). CooJ chromatographed on a calibrated Superose 12 gel filtration column eluted at 39 kDa, a position consistent with a multimeric native molecular mass for CooJ.
Watt
R K
RK
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Ludden
P W
PW
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D056511:Metallochaperones
D000445:Aldehyde Oxidoreductases; D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D009532:Nickel; C105283:CooJ protein, Rhodospirillum rubrum; D056511:Metallochaperones; C019823:carbon monoxide dehydrogenase