Abstract
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.
Highlights
The purple, nonsulfur bacterium Rhodospirillum rubrum can grow using carbon monoxide (CO) as the sole source of energy during anaerobic growth in the dark [1]
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 not observed when the identical procedure was followed with R. rubrum grown under conditions where the cooJ gene product would not be expressed, i.e. cells grown anaerobically in the absence of CO (Fig. 1, lane 1), and cells from a mutant strain (UR294) that contains an insertion into the cooC gene that is polar onto cooTJ (Fig. 1, lane 5)
Summary
Anaerobic Conditions—All work was performed under anaerobic conditions unless otherwise stated because of the oxygen lability of several of the coo gene products. The supernatant from the centrifugation was loaded onto a 2-ml IMAC column (His-bind, Novagen), which was equilibrated with 25 ml of binding buffer. The supernatant was loaded onto a 1 ϫ 8-cm IMAC column equilibrated in wash buffer. CooJ was loaded onto an HR10 Source Q fast protein liquid chromatography column equilibrated with anaerobic 50 mM Tris, 100 mM NaCl, pH 8.0. The column was washed with 50 ml of 50 mM Tris, pH 8.0, and eluted with a linear gradient from 50 to 500 mM NaCl. CooJ eluted in fractions containing 200 –500 mM NaCl. The purest fractions (as determined by SDS-PAGEs) were pooled for use. Amino Acid Analysis and Protein Concentration Determinations— Samples of CooJ were prepared for N-terminal sequencing by electrophoresis on a 15% SDS gel. The elution of the proteins was determined by SDS-PAGE
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