The Identification, Purification, and Characterization of CooJ
A NICKEL-BINDING PROTEIN THAT IS CO-REGULATED WITH THE Ni-CONTAINING CO DEHYDROGENASE FROM RHODOSPIRILLUM RUBRUM*

Richard K. Watt and Paul W. LuddenDagger

From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 µM. Other divalent metals competed with the following order of affinity and corresponding Ki: Zn2+ (5 µM) > Cd2+ (19 µM) > Co2+ (23 µM) > Cu2+ (122 µM). 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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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). In the presence of CO, the CooA regulatory protein recognizes CO and binds to the promoter regions of the cooFSCTJ and cooMKLXUH operons, initiating the expression of the CO oxidation system (2-4). CO dehydrogenase (CODH)1 is encoded by the cooS gene (5) and is a redox enzyme that catalyzes the oxidation of CO to CO2. CODH contains two metal clusters: an oxygen-labile, nickel-iron-sulfur cluster referred to as the C cluster and a Fe4S4 cluster referred to as the B cluster (6, 7). The electrons liberated by the oxidation of CO are transferred to a ferredoxin-like protein, the cooF product (CooF), and then through an undefined path to the CO-tolerant hydrogenase (CooH), which uses the electrons to evolve H2 (8-10).

The cooFSCTJ operon contains the genes encoding CODH (cooS) and CooF (cooF), as well as three downstream genes, cooCTJ, that show sequence similarity to some of the nickel-processing genes required for metallocluster assembly in urease and hydrogenase (11). Physiological characterization of mutants containing polar or nonpolar insertions into the cooCTJ genes produced results consistent with a role in nickel processing for these gene products (11). The CooC gene product is predicted to contain a nucleotide binding domain (P-loop) and is similar to the UreG and HypB proteins that are involved in processing nickel for urease and hydrogenase (11). CooJ contains a nickel binding motif in the C terminus with 16 histidine residues in the final 34 amino acids. Nickel binding motifs have been found in the predicted amino acid sequences of most HypB and UreE proteins (12-14). CooT shows no significant similarity to proteins in the sequence data base, and its role in nickel cluster assembly is unknown. Although the active sites of urease, hydrogenase, and CODH are not similar (6, 15, 16), the processing of nickel for each of these enzymes appears to have a common requirement for proteins containing a P-loop and a histidine-rich region.

The CooJ protein shares sequence similarity to UreE both in the histidine-rich C terminus and in other domains of the protein (11). UreE has been purified by IMAC from several organisms (17, 18), and it has been implicated in the accumulation of nickel for urease (19). Interestingly, Brayman and Hausinger (19) have shown that a truncated version of UreE (H144* UreE), which lacks the the histidine-rich C-terminal region, still binds approximately 2 Ni2+ atoms/dimer and appears to function in vivo. Organisms that have high affinity uptake systems for nickel have UreE-like proteins that lack the histidine-rich region, leading to the suggestion that the histidine-rich region functions to store nickel ions (19).

The HypB protein is involved in maturation of hydrogenase, and it contains domains with sequence similarity to CooC (P-loop) and CooJ (histidine-rich region) (11), with the exception of the Escherichia coli hypB gene, which lacks the histidine-rich region (20). HypB proteins have been purified and characterized from E. coli (21), Rhizobium leguminosarum (22), and Bradyrhizobium japonicum (23). HypB is required for in vivo (24) and in vitro (25) nickel insertion because deletions of HypB yield inactive hydrogenase (24, 25). The histidine-rich region of HypB has been shown to store nickel in B. japonicum (26).

In this work, we report the identification, purification, and characterization of the nickel-binding protein, CooJ, from R. rubrum. The purification of wild type CooJ was complicated by the co-purification of CODH and several other proteins until detergent and chaotropic agents separated CooJ from these proteins. The characteristics of CooJ are compared with the analogous nickel-processing proteins from the hydrogenase and urease nickel-processing systems. CooJ binds nickel and other divalent metals with comparable affinity and selectivity as reported for HypB and UreE. The native molecular composition of CooJ shows some similarity to HypB from R. leguminosarum because it appears to multimerize (22), and this observation differs from the dimeric stiochiometry reported for most HypB and UreE proteins.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Anaerobic Conditions-- All work was performed under anaerobic conditions unless otherwise stated because of the oxygen lability of several of the coo gene products.

Buffers-- The following buffers were used for the IMAC column: binding buffer, 20 mM Tris with 1.0 M NaCl and 5 mM imidazole, pH 8.0; wash buffer, 20 mM Tris with 1.0 M NaCl and 60 mM imidazole, pH 8.0; elute buffer, 20 mM Tris with 1.0 M NaCl and 500 mM imidazole, pH 8.0; strip buffer, 20 mM Tris with 1.0 M NaCl and 100 mM EDTA, pH 8.0; chaotropic buffer, 0.5% CHAPS and 1.0 M NaBr in 85 mM imidazole buffer, pH 8.0. The 1.0 M NaCl was omitted from the elute buffer when the fraction was to be loaded onto the Source 15Q ion exchange column.

Strains-- The R. rubrum strain UR2 (a spontaneous streptomycin-resistant mutant of R. rubrum UR1 (ATCC 11170)) was used as the wild type strain. R. rubrum strain UR294 contains a kanamycin resistance cassette in cooC that is polar onto cooT and cooJ (1).

Identification of CooJ-- The nickel binding motif of CooJ (11) made it a candidate for binding to an IMAC column (27). Cultures of wild type R. rubrum (strain UR2) or mutant strain UR294 were cultured in 0.5 liter of Ormerod's medium (28) with NH4Cl as the nitrogen source in the presence or absence of CO in the head-space. Cultures were harvested by centrifugation at 6000 × g. The cell paste was frozen in liquid nitrogen until cells were used. Cell paste was thawed anaerobically in 0.1 M MOPS buffer, pH 8.0, and broken in a French pressure cell. The crude extract was fractionated by centrifugation at 42,000 × g for 2 h. 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. After loading, the IMAC column was washed with 25 ml of binding buffer, 15 ml of wash buffer, 15 ml of elute buffer, and 15 ml of strip buffer. Fractions from each wash were collected and analyzed by SDS-PAGE on a 15% acrylamide gel (29). A protein band that appeared under conditions consistent with cooJ expression was submitted for N-terminal sequencing to confirm the identification.

Cell Growth and Purification of CooJ-- R. rubrum (strain UR2) was grown in 20-liter cultures under a CO atmosphere in either the presence or absence of nickel as described previously (30). Cell paste (100 g) was thawed anaerobically in 100 ml of 0.1 M MOPS, pH 8.0, and cells were broken in a French pressure cell. The crude extract was fractionated by centrifugation at 15,000 × g for 15 h. The supernatant was loaded onto a 1 × 8-cm IMAC column equilibrated in wash buffer. The column was washed with the following buffers: 40 ml of wash buffer, 50 ml of chaotropic buffer, 50 ml of 0.1 M Tris, pH 8.0, and 15 ml of elute buffer. CooJ eluted in the elute buffer wash. EDTA was added to the fraction containing CooJ to a final concentration of 1 mM. 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 then 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.

Molecular Mass Determination-- The apparent subunit molecular mass of CooJ in SDS-PAGE gels was calculated by comparison to standard proteins bovine serum albumin (67 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), myoglobin (17 kDa), and cytochrome c (12.5 kDa).

The native molecular mass was determined using an Amersham Pharmacia Biotech Superose 12 gel filtration column on an LKB HPLC. The column was standardized by recording the elution volumes of the standard proteins listed above.

Mass Spectrometry-- CooJ was prepared for mass spectrometry by exchanging the buffer with 0.010 M Tris, pH 8.0, using an Amersham Pharmacia Biotech PD-10 column. The samples were analyzed by matrix-assisted laser desorption using a Shimadzu Kratos Kompact MALDI 4, Version 5.0.1.

Equilibrium Dialysis Measurements-- Nickel binding to CooJ was determined by equilibrium dialysis using the dialysis chambers described by Reinard and Jacobsen (31), except that Sarstadt microcentrifuge tubes were used in place of Eppendorf tubes. Spectropore 4 membranes, with a nominal 12,000-14,000 molecular weight cutoff, were prepared by boiling in 0.1 M NaHCO3 and 1 mM EDTA to remove traces of metal and sulfur from the membrane. CooJ did not pass through the membrane. Each tube contained 600 µl of total volume, with 300 µl each in the upper and lower chambers. The lower chamber contained CooJ (5 µM), and the upper chamber contained 0.4 µCi of 63Ni (788 mCi/mmol; NEN Life Science Products) with the indicated concentration of unlabeled nickel. All solutions were prepared in 0.05 M Tris, pH 8.0. The samples were allowed to equilibrate for 24 h, after which the solution from each side of the membrane was collected, and the amount of 63Ni in each was determined by liquid scintillation counting using the 14C setting in a Packard Minaxi Tricarb 4000 series scintillation counter. The values obtained for nickel binding to CooJ were the same when equilibrium dialysis was performed aerobically or anaerobically; therefore, the majority of the equilibrium dialysis measurements were performed aerobically. Competition dialysis was performed by adding MnCl2, CoCl2, ZnCl2, CuCl2, MgSO4, or CaCl2 as indicated to the upper chamber containing 0.4 µCi of 63Ni and 4 µM unlabeled NiCl2. The lower chamber contained CooJ. The competition dialysis samples were allowed to equilibrate and were analyzed as described above. The binding constants and inhibition constants were calculated using the Cheng-Prusoff method (32) using the Prism program (Graphpad Graphics, Inc).

Amino Acid Analysis and Protein Concentration Determinations-- Samples of CooJ were prepared for N-terminal sequencing by electrophoresis on a 15% SDS gel. The protein was then transferred to a polyvinylidene difluoride membrane. N-terminal sequencing was performed at the University of California-Riverside Biotechnology Instrumentation Facility.

Total amino acid analysis was done at the W. M. Keck Foundation Biotechnology Resource Laboratory, Protein and Nucleic Acid Chemistry Facility, Yale University. The protein concentration from the amino acid analysis was standardized to the Bradford protein assay (33) using carbonic anhydrase as a standard. The Bradford protein assay was chosen due to the observation that CooJ appeared to react with the Cu2+ in the Lowry and BCA protein assays (34, 35).

Antibodies-- Purified fractions of CooJ were electrophoresed on a preparative 15% SDS gel (17 cm × 15 cm × 2 mm) by pouring a stacking gel with only one well over the entire resolving gel and loading 400 µg of total protein. After electrophoresis, the gel was soaked in ice-cold 0.25 M KCl to visualize the location of the protein. The protein band was cut from the gel and eluted from the polyacrylamide by grinding the acrylamide in PBS buffer. The supernatant was collected, and a sample was reanalyzed by SDS-PAGE to confirm that pure CooJ was obtained. This sample was used for antibody preparation at the University of Wisconsin-Madison Animal Care Unit Polyclonal Anitbody Service.

Purification Table-- Immunoblot analysis was used to estimate the amount of CooJ present in each fraction of the purification. A CooJ standard curve was prepared in each gel by loading 50, 65, 80, 95, and 110 µg of purified CooJ. The remaining wells were filled with fractions from a purification. An immunoblot was performed and developed using the ECL Western blotting protocol (Amersham Pharmacia Biotech). The immunoblot was analyzed using a Molecular Dynamics Personal Densitometer SI, and data analysis was performed using the ImageQuant program (Molecular Dynamics, Inc.). The amount of CooJ in the purification fractions was estimated by comparison to the standard curve of CooJ.

Metal Analysis-- The metal content of CooJ was determined by atomic absorption spectrometry using a Perkin-Elmer 3030 graphite-furnace atomic absorption spectrometer with an HGA-300 programmer and an AS-40 auto sampler. A separate analysis was performed at the University of Wisconsin Plant and Soil Analysis Lab using inductively coupled plasma mass spectrometry. Iron analysis was done by the addition of Na2S2O4 to the CooJ sample followed by the addition of alpha ,alpha '-dipyridyl and monitoring the absorbance at 520 nm.

UV Visible Spectra of CooJ-- The spectrum of purified CooJ was recorded on a Shimadzu 1601PC spectrophotometer. Purified cooJ contained less than 0.1 Ni2+ atom/CooJ monomer, and all other metals were below the level of detection.

IMAC under Denaturing Conditions-- CooJ was tested for binding to IMAC when denatured by urea. Solid urea was added to a final concentration of 8 M to a CooJ solution purified from the IMAC column. A 1.0-ml IMAC column was poured and equilibrated with 0.1 M NaH2PO4, 0.01 M Tris, 8 M urea, pH 8.0. CooJ was loaded and washed with 0.1 M NaH2PO4, 0.01 M Tris, 8 M urea, pH 6.3. CooJ was eluted with 0.1 M NaH2PO4, 0.01 M Tris, 8 M urea, pH 4.5.

Interactions between CooJ and apoCODH-- Interactions between CooJ and apoCODH were tested using an IMAC column. CooJ was bound to the column and then apoCODH was loaded on the column. Controls for the individual proteins bound to the IMAC column showed that CooJ eluted in the elute buffer wash (500 mM imidazole) and purified apoCODH bound to the IMAC column and eluted in the wash buffer wash (60 mM imidazole). Purified apoCooJ was loaded onto the IMAC column, and the column was washed with binding buffer followed by loading purified apoCODH onto the column. The column was washed with wash buffer and elute buffer. The elution of the proteins was determined by SDS-PAGE.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Identification of the CooJ Protein-- The strategy used to identify the cooJ gene product took advantage of the proposed nickel-binding capability of the histidine-rich region. The cooJ gene encodes a polypeptide of 12.5 kDa with a nickel binding motif composed of 16 histidine residues in the final 34 amino acids (11). CO-induced R. rubrum cells were lysed and centrifuged, and the crude supernatant was passed over an IMAC column. Proteins that bound to the nickel affinity column were washed from the column with elute buffer and visualized on a 15% SDS gel. CooJ was identified as the major protein band observed in the gel at an apparent molecular mass of 19 kDa (Fig. 1, lane 3). 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). The addition of 10 µM nickel to the growing cells did not induce the expression of the 19-kDa protein in the absence of CO (Fig. 1, lane 2), and cells grown in the presence of CO and the absence of nickel still expressed the 19-kDa protein (Fig. 1, lane 4). N-terminal sequence analysis of the 19-kDa protein identified the first six amino acids as Thr-Glu-Ser-Pro-Glu-Arg, which is an exact match to the predicted amino acid sequence of residues 2-7 of CooJ. The N-terminal methionine was absent in the N-terminal sequencing, indicating that CooJ is posttranslationally processed.


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Fig. 1.   The identification of CooJ. Crude supernatants of R. rubrum grown under the stated conditions for each lane were passed over an IMAC column, and metal-binding proteins were eluted with elute buffer (see "Experimental Procedures"). The appropriate volume of each fraction was precipitated so that each lane contained 20 µg of protein. The protein was electrophoresed on 15% acrylamide gels. Lane 1 is from cells grown in the absence of Ni2+ and CO. Lane 2 is from cells grown with 10 µM Ni2+ but in the absence of CO. Lane 3 is from cells grown with CO and 10 µM nickel. Lane 4 is from cells grown in the presence of CO but in the absence of nickel. Lane 5 is from the mutant strain UR294 containing a kanamycin cassette insertion in cooC that is polar onto cooJ, grown in the absence of nickel. Molecular mass markers are as indicated under "Experimental Procedures."

Purification of CooJ-- To obtain sufficient amounts of CooJ for purification, the growth conditions of R. rubrum were scaled up from 0.5-liter cultures to 20-liter cultures. For efficient induction of the coo operon, the cells were sparged with 99% CO.

The first step of the purification was an IMAC column as described in the identification of CooJ (above). CooJ eluted from the IMAC column in the elute buffer wash (Fig. 2, lane 1). CooJ was observed at 19 kDa, with several additional protein bands at 38, 43, 62, and 72 kDa. This protein banding pattern is different from that shown in Fig. 1 (compare Fig. 1, lane 3, with Fig. 2, lane 1). The expression of the other proteins observed in the IMAC eluent appears to correlate with the vigorous sparging of the culture with CO.


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Fig. 2.   Purification of CooJ. Lane 1, IMAC elution fraction containing nickel-binding proteins from the crude supernatant of CO-induced R. rubrum. Lane 2, CooJ eluted from the IMAC column that was washed with 1.0 M NaBr and 1.0% CHAPS to remove the other nickel-binding proteins observed in lane 1. Lane 3, pooled 200-500 mM NaCl eluent fraction from the Source Q column.

The CooJ-containing fraction from the IMAC column was chromatographed on ion exchange (DEAE and Mono Q), gel filtration (Superose 12 and G-75), and hydrophobic (phenyl-Sepharose) columns. The protein bands at 38, 43, 62, and 72 kDa co-migrated with CooJ on each of the columns tested. The co-migration of these proteins through each of these columns is indicative of a protein complex formed among these proteins. CooJ is proposed to deliver nickel to CODH; therefore, a protein complex containing CooJ might contain CODH and other proteins required for nickel insertion into CODH. An immunoblot using the anti-CODH antibody identified the protein band at 62 kDa as CODH. A CODH assay of this fraction showed a small amount of CODH activity. This fraction may represent a nickel-deficient form of CODH that requires interaction with nickel-processing proteins for full activation.

CooJ was purified by binding CooJ and the copurifying proteins to the IMAC column and washing the column with chaotropic buffer (see "Experimental Procedures") to disrupt protein-protein interactions. The chaotropic buffer wash was followed by a 50 mM Tris, 85 mM NaCl, pH 8.0, wash to remove the chaotropic buffer from the column. The proteins that remained bound on the column were eluted with elute buffer. The elute buffer fraction contained CooJ without the presence of the other contaminating protein bands (Fig. 2, lane 2). CooJ was purified from any remaining impurities by chromatography on a Source Q column using a 200-500 mM NaCl gradient (Fig. 2, lane 3).

Purification Table-- The amount of CooJ present in each fraction of the purification was estimated from immunoblots of each fraction. The results of a typical purification are shown in Table I. The largest loss of CooJ during the purification was the Source Q column, where the final impurities were removed from CooJ.

                              
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Table I
CooJ purification table

Metal Content of CooJ-- CooJ was analyzed for metal content after each chromatography step of the purification. CooJ contained 1-3 nickel atoms per monomer after the IMAC column, but treatment with EDTA prior to the Source Q column removed this nickel because CooJ contained less than 0.1 nickel atom per CooJ monomer after the Source Q column. Analysis for other divalent metals revealed that Cu2+, Co2+, Cd2+, Fe2+, and Zn2+ were present at less than 0.05 mol of M2+ per mol of CooJ monomer after the Source Q column.

Equilibrium Dialysis-- CooJ was analyzed for the ability to bind Ni2+ using equilibrium dialysis with the results shown in Fig. 3A. Analysis of the binding curve showed that CooJ binds 4.2 ± 0.1 Ni2+/CooJ monomer with a kD of 4.3 ± 0.3 µM. The optimal computer fit was found using one type of binding site, suggesting that all binding sites are similar.


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Fig. 3.   Nickel binding by purified CooJ. A, CooJ (5 µM) in 50 mM Tris, pH 8.0, was equilibrated under the indicated concentrations of NiCl2 with a constant 63NiCl2 (0.4 µCi) concentration. The curve represents a nonlinear fit of the data. B, effect of divalent metal ions on nickel binding to CooJ. Equilibrium dialysis of CooJ (4 µM) with 4 µM NiCl2 (0.4 µCi of 63Ni) was performed in the presence of the indicated concentrations of competitive metals. black-square, Ca2+; square , Mg2+; triangle , Mn2+; ×, Cu2+; open circle , Co2+; bullet , Cd2+; black-triangle, Zn2+.

The specificity of CooJ for nickel was tested using competition dialysis against other divalent metals (Fig. 3B). Ca2+, Mg2+, and Mn2+ competed very poorly, whereas Cu2+, Co2+, Cd2+, and Zn2+ competed well and are listed with increasing effectiveness as inhibitors of Ni2+ binding. The calculated Ki values for the competing metals (32) are as follows: Zn2+, 5 µM; Cd2+, 19 µM; Co2+, 23 µM; Cu2+, 122 µM.

Molecular Size-- CooJ migrated as an apparent 19-kDa band in SDS-PAGE gels (Fig. 1, lanes 3 and 4, and Fig. 2), which is larger than the predicted molecular mass of 12.5 kDa for CooJ. Table II shows a summary of other histidine-rich proteins that were observed to migrate anomalously large in SDS-PAGE. Rey et al. (22) reported that several cysteine residues in HypB from R. leguminosarum were modified and proposed that a posttranslational modification of these cysteine residues could be responsible for the anomalous migration of HypB. The possibility that the migration of CooJ at 19 kDa was caused by a posttranslational modification prompted us to look for a smaller species of CooJ that existed in an unmodified form at the predicted molecular mass of 12.5 kDa. The major contaminating protein from the IMAC column migrated at ~12 kDa (Fig. 2, lane 2), and this 12-kDa band was absent in extracts of mutants lacking a functional cooJ gene (data not shown). N-terminal sequence analysis of the 12-kDa protein also identified it as a cooJ product. Two possibilities explain the presence of two species of CooJ migrating at 19 and 12 kDa. The first is that the 19-kDa CooJ band is a covalently modified form of the 12-kDa CooJ. The second is that CooJ migrates anomalously large (19 kDa), and the 12 kDa band is a cleavage fragment at the C terminus.

                              
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Table II
Comparison of histidine-rich proteins
All of the reported CooJ analogs (UreE and HypB proteins) have migrated at molecular masses larger than the predicted value. The WHP protein from E. coli is a nickel-binding protein that has been purified and characterized, but its function in nickel metabolism is unknown.

Two experiments were performed to test the hypothesis that the C terminus of the 12-kDa CooJ was cleaved. The C-terminal region of CooJ contains the histidine-rich region, and removal of this portion of the protein may not permit the 12-kDa CooJ to bind to an IMAC column. The purified 12-kDa CooJ was chromatographed on an IMAC column and it flowed through; this result is consistent with a C-terminal cleavage of the histidine-rich region of the protein. However, the 12-kDa CooJ was initially identified in the eluent fraction from IMAC chromatography. The binding of the 12-kDa CooJ to the IMAC column was proposed to occur due to affinity of the 12-kDa CooJ for the 19 kDa CooJ that contained the histidine-rich region, and not due to affinity for the nickel on the column. To verify that the 12-kDa CooJ bound to the IMAC column only in the presence of the native 19-kDa CooJ, a fraction containing both the 19-kDa CooJ and the 12-kDa CooJ was chromatographed under denaturing conditions using 8 M urea. This CooJ fraction was loaded onto an IMAC column equilibrated for denaturing conditions. The 12-kDa CooJ flowed through and the 19-kDa CooJ remained bound (data not shown), indicating that the 12-kDa CooJ lacked the histidine-rich region and under the denaturing conditions could no longer interact with the 19-kDa CooJ. Mass spectrometry of fractions containing both the 12- and 19-kDa CooJ proteins showed peaks at 9 and 12.5 kDa, indicating that both species migrated anomalously large in the gel (see "Mass Spectrometry," below).

The native molecular size of CooJ was estimated by gel filtration using a Superose 12 column. Purified CooJ was loaded onto the Superose 12 column and eluted as a broad shoulder leading into peaks that correspond to sizes of 54.3 and 32.8 kDa, respectively (Fig. 4A). When 5 mM DTT was added to the CooJ sample prior to chromatography on Superose 12, the protein eluted as one major peak at 39 kDa with a slight shoulder (~50 kDa), as seen in Fig. 4B.


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Fig. 4.   The elution of CooJ from a Superose 12 filtration column. A, CooJ as isolated from the Source Q column (Fig. 2) and chromatographed on Superose 12. B, CooJ from Source Q purification step treated with DTT prior to loading onto the Superose 12 column.

Mass Spectrometry-- To test the hypothesis that CooJ was posttranslationally modified and that the modification was the source of the anomalous migration of CooJ on SDS-PAGE, mass spectrometry was performed on CooJ. Matrix-assisted laser desorption was used to analyze CooJ. CooJ was observed at a mass of 12,430 kDa. The predicted molecular mass of CooJ lacking the N-terminal methionine is 12,448 kDa, indicating that a posttranslational modification is not the source of the anomalous migration of CooJ in SDS-PAGE.

UV Visible Spectrophotometry-- The UV visible spectrum of CooJ was recorded on a CooJ sample containing less than 0.1 Ni2+ atom/CooJ monomer, with all other measured divalent metals below the limits of detection. CooJ begins to precipitate at concentrations greater than 0.2 mg/ml; therefore, the spectrum of CooJ was performed on a dilute sample of CooJ. The spectrum of CooJ (Fig. 5) shows several interesting features. The absorbance peak at 275 nm is due to the single tyrosine residue in CooJ. Other interesting features that were observed are a small peak at 410 nm and a shoulder at 310 nm. A peak at 420 nm has been observed in nickel-containing human serum albumin, and the intensity of this peak increased with increasing pH and was attributed to Ni2+ coordinated in a square planar or square pyramidal geometry (36). The nickel content of this CooJ sample is less than 0.1 Ni2+ atom/CooJ monomer, and the feature at 410 nm did not change with increasing pH, ruling out a nickel species similar to that found in human serum albumin. No changes in the spectrum of CooJ were observed when CooJ was incubated with divalent metals (Ni2+, Zn2+, Cu2+, Co2+, or Fe2+) or when CooJ was incubated with EDTA, DTT, or dithionite (data not shown). Treatment with 2.5% SDS did not change the spectrum of the CooJ sample (data not shown). Treatment of the protein with hydrochloric acid changed the spectrum of CooJ (Fig. 5). The 275-nm peak was present with a new peak at 325 nm, but the peaks at 310 and 410 nm were no longer observed (Fig. 5). When the protein was precipitated with trichloroacetic acid and resuspended in buffer, the only peak observed was the 275-nm peak assigned to the tyrosine of the protein (data not shown). These data suggest that the 310- and 410-nm peaks are from a tightly bound molecule that is released by acid, but with the current data, no assignments have been made to the peaks at 310 and 410 nm.


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Fig. 5.   UV visible spectra of CooJ. CooJ (0.2 mg/ml) as prepared by incubation with EDTA prior to the Source Q column. Metal analysis showed that nickel was present at 0.1 Ni2+/CooJ monomer, and all other divalent metals were not detected.

Interactions between CooJ and apoCODH-- Gel filtration chromatography or native gel electrophoresis may be used to determine protein-protein interactions. Unfortunately, CooJ migrates very poorly in native gels and on gel filtration columns, and attempts to use these tools to show interactions between CooJ and CODH did not give interpretable results. An alternate method was devised using an IMAC column as an affinity column by binding CooJ to the IMAC column and testing for apoCODH binding (37). Purified apoCODH elutes from IMAC in the wash buffer (60 mM imidazole), whereas purified CooJ elutes in the elute buffer (500 mM imidazole). The elution of apoCODH at higher imidazole concentrations would indicate interaction with CooJ that stabilized the ability of apoCODH to bind to the IMAC column. Purified apoCODH was loaded onto the column containing CooJ, and the column was washed with wash buffer and elute buffer. The apoCODH eluted in the wash buffer, and CooJ eluted in the elute buffer, indicating that CooJ did not stabilize the binding of apoCODH on the IMAC column and that the two purified proteins do not interact under these conditions.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The products of the cooCTJ genes are proposed to facilitate the insertion of nickel into CODH, forming the nickel-iron-sulfur C cluster required for the oxidation of CO to CO2. The histidine-rich, nickel binding motif of the CooJ protein led to the proposal that CooJ is a nickel-binding and delivery protein. Mutational analysis of the cooJ gene has provided evidence to support this hypothesis. A mutant strain lacking a functional cooJ gene was able to grow on CO, but it required a 10-fold increase of nickel in the growth medium (11). The phenotype of the cooJ mutant suggests that the product of the cooJ gene is not absolutely required for the production of an active nickel-containing CODH in vivo. The requirement for more nickel to overcome the effect of the cooJ mutation is consistent with less efficient nickel delivery to CODH, supporting the proposed role of CooJ as a nickel delivery protein. To gain further information about the role of the cooJ gene product, the protein was identified, purified, and characterized.

CooJ was identified by analyzing the proteins from the crude supernatant of CO induced cells that bound to an IMAC column. CooJ was distinguished from other proteins that bound to the IMAC column by comparison to proteins from cultures grown in the absence of CO and by comparison to proteins from a mutant strain lacking CooJ. The identification of CooJ as a 19-kDa protein band in an SDS-PAGE gel was confirmed by N-terminal amino acid sequencing.

The purification of CooJ was complicated by several proteins that co-purified with CooJ on the IMAC column, perhaps suggesting the presence of a protein complex. CooJ was separated from the proteins that co-purified on the IMAC column by washing the IMAC column with the chaotropic agent NaBr and the nondenaturing, zwitterionic detergent CHAPS prior to elution of CooJ from the IMAC column with elute buffer. The removal of the copurifying proteins under these conditions suggests that binding to the IMAC column was due to interaction with CooJ or to a metal binding site that was denatured in the chaotropic/detergent washing of the column.

Purified CooJ was shown by equilibrium dialysis to bind 4 Ni2+ atoms/CooJ monomer, with a Kd of 4.3 µM. These values are similar to those reported for other histidine-rich proteins (Table II). The ability of other divalent metals to compete for the binding sites of nickel on CooJ was analyzed by equilibrium dialysis. The competition by divalent metals paralleled the reported data for the analogous proteins discussed above, with Ca2+, Mg2+, and Mn2+ exhibiting very little competition; Cu2+, Co2+, and Cd2+ competing for the binding sites at elevated concentrations; and Zn2+ competing significantly, with affinity similar to that of nickel for the binding sites on CooJ.

Brayman and Hausinger (19) reported that the binding of Cu2+ altered the spectrum of the truncated H144* UreE, providing a spectroscopic probe of the binding site of this protein. The addition of divalent metals to CooJ did not alter the spectrum from the spectrum recorded for metal-deficient CooJ. The spectrum of metal-deficient CooJ had several interesting peaks at 310 and 410 nm. The addition of reducing agents (DTT or dithionite) and chelators (EDTA) had no effect on the spectrum. Acid denaturation of the protein appears to release or destroy the entity responsible for the absorbance observed at 310 and 410 nm. At present, no assignment have been made for these peaks, which do not correspond to any of the peaks observed by Brayman and Hausinger (19).

CooJ migrated in SDS gels at 19 kDa, which is larger than its predicted molecular mass of 12.5 kDa. Slow migration in SDS-PAGE gels has been reported for other histidine-rich proteins. Table II provides a summary of some histidine-rich proteins, their predicted molecular masses, and their apparent molecular masses in SDS gels. Mass spectrometry data confirmed that CooJ, observed at 19 kDa in SDS-PAGE, has a molecular mass of 12,430 Da. This value is consistent with the size predicted by the gene and the removal of the N-terminal methionine; therefore, a posttranslational modification is not the cause of the slower migration of CooJ in SDS-PAGE.

The subunit composition of native CooJ was studied by both nondenaturing gel electrophoresis and gel filtration chromatography. Purified CooJ migrated as multiple bands on a nondenaturing gel (data not shown) and on a Superose 12 gel filtration column (Fig. 4A). When CooJ was treated with DTT, CooJ migrated as one band both in native gel electrophoresis (data not shown) and on the gel filtration column (Fig. 4B). CooJ, reduced with DTT, eluted from the Superose 12 column at a volume consistent with a molecular size of 39 kDa. A molecular size of 39 kDa could be achieved by three 12.5-kDa CooJ subunits forming a timer; however, all characterized UreE and HypB proteins, except for the R. leguminosarum HypB, are reported to be dimeric. The apparent trimeric form of CooJ may be a result of anomalous migration of CooJ on gel filtration columns. The ability of the histidine-rich region to bind to the IMAC column under native conditions suggests that the histidine-rich region is solvent-exposed. A protein containing a hydrophilic, solvent-exposed region of 34 amino acids would not migrate the same as a globular protein with a rigid structure on a gel filtration column. Therefore, the elution of CooJ from a gel filtration column at a volume consistent with a 39-kDa globular protein is most likely an overestimate of the size of CooJ. Incidentally, the elution at 39 kDa could be interpreted as a dimer of two 19-kDa subunits, which is the size observed in SDS-PAGE. If the property that causes CooJ to migrate anomalously large in SDS-PAGE (i.e. at 19 kDa) also affects the elution from the gel filtration column, then the elution observed at a position suggesting a molecular mass of 39 kDa could be interpreted as evidence for a dimer of CooJ.

The observation that CooJ exists in several multimeric forms is similar to the observations of Rey et al. (22) for the HypB of R. leguminosarum. HypB was observed to elute in the void volume of S200 and S300 gel filtration columns, consistent with a molecular mass of >100 kDa, and it was observed to interact significantly with Superose 6 and Superose 12 columns. HypB ran as multiple bands in O'Farrell 2-dimensional gels and was reported to be found at multiple sizes both larger and smaller than the major protein band in Western blots of SDS gels.

Purified apoCODH did not bind to an IMAC column to which CooJ had been loaded. This result appears to conflict with the data observed during the purification when CODH appeared to co-purify with CooJ (Fig. 2, lane 1). One explanation for this apparent discrepancy is that the other proteins present (Fig. 2, lane 1) are required for CooJ and apoCODH to interact. CooCT may also be required for the interaction of CooJ with apoCODH. No direct evidence has been shown for UreE or HypB binding to the apo-urease or apo-hydrogenase in vitro. Several reports indicate that chaperonins, such as GroEL and GroES, may play roles in nickel insertion into nickel-containing enzymes (38, 39). Perhaps the interaction between CooJ and apoCODH requires chaperonin-like proteins to stabilize this interaction.

In summary, we have identified, purified, and characterized CooJ from wild type extracts of R. rubrum and have demonstrated the ability of CooJ to bind nickel. CooJ has been shown to bind other divalent metals, but with less affinity than nickel. These properties are consistent with its proposed role as an accessory protein for CODH nickel cluster assembly. Finally, other CODH protein complexes have been observed that may play a role in metallocluster assembly.

    ACKNOWLEDGEMENTS

We are grateful to Myron Crawford for the total amino acid analysis and Gerald Porter for N-terminal sequence analysis. We thank Bob Kerby, Gary Roberts, and Judith Burstyn for helpful suggestions.

    FOOTNOTES

* This work was supported by DOE grant DE-FGO2-87ER13891 (to P. W. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, 420 Henry Mall, Madison, WI 53706. Tel.: 608-262-6859; Fax: 608-262-3453; E-mail: ludden{at}biochem.wisc.edu.

1 The abbreviations used are: CODH, CO dehydrogenase; IMAC, immobilized metal affinity chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; PBS, 0.15 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 6.4 mM Na2HPO4 (pH 7.4); PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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