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J. Biol. Chem., Vol. 276, Issue 42, 38602-38609, October 19, 2001

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Purification and Characterization of Membrane-associated CooC Protein and Its Functional Role in the Insertion of Nickel into Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum^*

Won Bae Jeon, Jiujun Cheng, and Paul W. Ludden

From the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, May 30, 2001, and in revised form, July 23, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (CODH). 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 mass of 61-63 kDa, contains less than 0.1 atom of Ni²⁺ or Fe²⁺ per dimer, and has a _max at 277.5 nm (_277.5 32.1 mM¹ cm¹) with no absorption peaks at the visible region. CooC catalyzes the hydrolysis of ATP and GTP with K_m values of 24.4 and 26.0 µM and V_max 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 apoCODH 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 apoCODH. 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Rhodospirillum rubrum, a purple nonsulfur photosynthetic bacterium, can use CO as the sole energy and carbon source during anaerobic growth in the dark (1, 2). The cooS-encoded carbon monoxide dehydrogenase (CODH)¹ from R. rubrum catalyzes the reversible oxidation of CO to CO₂ (3, 4). CODH contains a nickel-iron-sulfur cluster (C-center) and an iron-sulfur cluster (B-center). CO oxidation occurs at the C-center, and the B-center mediates the transfer of electrons from the C-center to external electron acceptors (5). A nickel-deficient apoCODH, which contains all of the iron clusters of holoCODH yet no CO-oxidation activity, is obtained by growing wild-type R. rubrum on nickel-depleted medium (6). The apoCODH can be activated both in vivo and in vitro by the addition of nickel (6, 7).

In contrast to the extensive knowledge about the catalytic (8) and spectroscopic properties (9, 10) of CODH, information about the biosynthesis of the nickel cluster is very limited. The basic studies on CO-dependent growth (11) and ⁶³Ni transport (12) demonstrate that three accessory proteins encoded by cooCTJ genes are involved in nickel incorporation into a nickel site. The cooT gene encodes a 7.1-kDa protein that shows marginal similarity to chaperone-type HypC protein required for the maturation of hydrogenase from Escherichia coli (13). The cooJ gene encodes a soluble, 12.6-kDa protein that has a histidine-rich nickel-binding domain. CooJ has been purified by IMAC from R. rubrum and has been shown to bind 4 Ni²⁺ atoms per apparent 19-kDa monomer with a K_d of 4.3 µM (14).

A mutant lacking a functional cooC gene requires a 1,000-fold higher nickel concentration than wild-type strain for CO-dependent anaerobic growth (11). The elevated nickel requirement of the cooC mutant suggests that CooC is required for the production of enzymatically active CODH in vivo at normal levels of nickel in the medium. Significantly, CooC has a P-loop (15) that is indicative of nucleotide binding in many proteins including HypB and UreG which play a role in nickel processing for hydrogenase (16) and urease (17), respectively. CooC also shows homology to NifH, which functions in the ATP-dependent insertion of FeMo-co into apodinitrogenase (18).

To gain information about the function of CooC in the insertion of nickel into CODH and insight into the nickel center maturation, we purified CooC and characterized its biochemical properties. A mutant form of CooC in which lysine 13 of the P-loop was replaced by glutamine was obtained by site-specific mutagenesis of the cooC gene. The K13Q CooC was unable to hydrolyze ATP in vitro, and a mutant strain that expresses K13Q CooC accumulated the catalytically inactive, nickel-deficient CODH. Nickel-dependent activation of apoCODH in the presence of nucleotides was tested to provide evidence that CooC couples ATP hydrolysis with nickel insertion into R. rubrum CODH.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Buffers and Anaerobic Conditions-- Metal-free MOPS (100 mM, pH 7.5), TE (100 mM Tris-HCl and 1 mM EDTA, pH 7.5), and phosphate (50 mM NaH₂PO₄, pH 7.5) buffers were prepared by passage through a column of Chelex-100 cation-exchange resin (Bio-Rad) and used in protein purification, enzyme assays, and nickel activation assays. All purification steps and assays were performed under anaerobic conditions using a buffer containing 1 mM sodium dithionite or in a Vacuum Atmosphere anaerobic glove box (Hawthorne, CA model HE-493).

Strains and Culture Conditions-- The genotypes of strains employed in this study were UR2 (wild type), UR445 ((cooFSCTJA)10::aacC1), UR469 (cooC16::aacC1linker), UR471 (cooJ18), UR479 (cooT19::linker), UR495 (cooC20::linker), and UR501 (cooJ23::aac1linker) (11). Strains were cultured, depending upon experiments, in nickel-depleted or nickel-supplemented medium according to published procedures (6, 7, 11).

Generation of K13Q CooC Variant-- The mutant producing K13Q CooC was constructed as follows. A 0.56-kilobase pair HindIII-PstI fragment was isolated from pCO39 in E. coli strain UQ1155 (11) and cloned into HindIII-PstI digested pBSKS. The resulting plasmid was termed pLJC3 and used as a template. Two mutagenic primers were designed to convert Lys-13 codon (AAG) to Gln (CAG) in the cooC gene by using a QuickChange (Stratagene) method. After mutagenesis, the mutation was verified by sequencing. The mutated cooC gene was excised by HindIII-PstI and inserted into pCO39 digested with the same enzymes, and the resulting plasmid was termed pLJC5. A 4.5-kilobase pair KpnI-SacI fragment carrying cooS'C(K13Q cooC)TJAnadC' from pLJC5 was excised and cloned into the mobilizable vector pUX19 (Km^r). This construct, in E. coli S17-1 (UQ324) (19), was conjugated into R. rubrum UR447 (11). The isolated R. rubrum Km^rGm^rNx^rSm^r recombinant was designated UR871.

Anti-CooC Antibodies and Western Blots-- A 1.5-kilobase pair EcoRV fragment from pCO5 (E. coli UQ1193) (11), bearing coo'CTJA' genes, was ligated into XmnI-cut pMAL-c2 expression vector (New England Biolabs Inc.) and transformed into E. coli DH5 to generate strain UQ1430. This ligation created an in-frame fusion between malE and coo'C, starting with codon 89 of cooC, and the orientation and plasmid insert junctions were verified by DNA sequencing. Expression and isolation of the fusion protein were performed by the manufacturer's protocol. Purified MalE-CooC fusion protein (32 mg) was digested with trypsin (48 µg) at 4 °C for 40 min. The CooC was separated by preparative SDS-PAGE (15% acrylamide gel). After electrophoresis, the CooC band was excised, and the acrylamide fragment was washed three times with shaking for 10 min in 50 ml of distilled water and then minced through a syringe. The gel pieces were incubated 12 h in PBS buffer (20 mM sodium phosphate and 150 mM NaCl, pH 7.4) containing 1 mM dithiothreitol. After centrifugation, supernatant was collected and reanalyzed by SDS-PAGE to confirm that pure CooC was obtained. Antibodies to CooC were generated in rabbits at the University of Wisconsin-Madison Medical School Animal Care Unit Polyclonal Antibody Service. Western blots of protein with anti-CooC antibodies were performed as described previously (20-22) and used to follow the purification of CooC.

Solubilization of CooC from Membrane-- Chromatophore membranes isolated according to published methods (23) were suspended in TE buffer (4.72 mg of protein/ml). Salt extraction (24), alkaline treatment (25), (NH₄)₂SO₄ fractionation (26), and detergent solubilization (4) were performed according to published methods. The yield and purity of solubilized CooC were estimated by Western blots (14).

Purification of Wild-type CooC and K13Q CooC-- Chromatophore membranes were treated with 50% (NH₄)₂SO₄ with gentle stirring for 1 h in an anaerobic glove box. After centrifugation at 15,000 × g for 30 min, the pellet fraction was resuspended in TE buffer and subjected to 40% (NH₄)₂SO₄ precipitation. Precipitated proteins were collected by centrifugation at 15,000 × g for 30 min, resuspended in TE buffer, and dialyzed twice against 4 liters of TE buffer containing 10% glycerol at 4 °C. After dialysis, membrane debris and dark red pigments were removed by centrifugation at 270,000 × g for 1 h. At this point, the CooC protein was solubilized in TE buffer.

The dialyzed protein solution was applied to a Whatman DE52 column (2.5 × 18.5 cm) that had been packed and equilibrated with TE buffer. The column was washed with 2 column volumes of TE buffer and then proteins were eluted with a linear gradient of NaCl (0-350 mM; 300 ml total volume) in TE buffer with a flow rate of 5.0 ml/min; fractions of 15 ml each were collected. CooC eluted from the DE52 column between 170 and 240 mM NaCl. Active fractions were combined, and (NH₄)₂SO₄ was added to a final concentration of 250 mM. The protein solution was loaded onto the first phenyl-Sepharose column (2.5 × 12.5 cm) equilibrated with TE buffer containing 250 mM of (NH₄)₂SO₄. The column was washed with 120 ml of 250 mM (NH₄)₂SO₄, and proteins were eluted with a 300-ml linear gradient of (NH₄)₂SO₄ (from 250 to 0 mM) in TE buffer at a flow rate of 5.0 ml/min; fractions of 10 ml each were collected. A second phenyl-Sepharose column (16 × 10 cm) was connected to an FPLC system and equilibrated with TE buffer containing 250 mM (NH₄)₂SO₄. CooC-containing fractions from the first phenyl-Sepharose column were combined. After estimating the concentration of (NH₄)₂SO₄ in the pooled fractions by measuring the conductivity, (NH₄)₂SO₄ was added to a final concentration of 250 mM, and the solution was loaded onto the second phenyl-Sepharose column. The column was washed with 60 ml of TE buffer containing 250 mM (NH₄)₂SO₄, and proteins were eluted with a linear decreasing gradient of (NH₄)₂SO₄ (250 to 0 mM) in TE buffer over 50 min with a flow rate of 2.5 ml/min; fractions of 10 ml were collected. CooC eluted from the phenyl-Sepharose column in buffer with 80 to 30 mM (NH₄)₂SO₄.

Fractions containing CooC were transferred anaerobically to a nitrogen-filled ultrafiltration kit (Amicon) equipped with XM50 membrane filter (Millipore). After desalting, the protein solution was recovered anaerobically. A hydroxylapatite column (10 × 10 cm) was connected to an FPLC system and equilibrated with TE buffer. Desalted protein solution was loaded onto the column at a flow rate of 2 ml/min. The column was washed with 20 ml of TE buffer, after which a linear gradient from TE buffer to phosphate buffer was applied at a flow rate of 2.0 ml/min over 50 min; fractions of 15 ml each were collected. CooC eluted between 10 and 15 mM NaH₂PO₄ from the hydroxylapatite column. Finally, a Mono Q column (Amersham Pharmacia Biotech HR 5/5, 0.8 ml) was equilibrated with TE buffer. The active fractions from the hydroxylapatite column were applied to the Mono Q column. The column was washed with 20 ml of TE buffer, and proteins were eluted with a linear gradient of NaCl (0-300 mM) in TE buffer over 30 min. The flow rate was 1 ml/min, and fractions of 1 ml were collected. CooC eluted from the Mono Q column between 170 and 190 mM NaCl. NaCl was removed by gel filtration on Sephadex G-25 using TE buffer, and purified CooC was kept in liquid nitrogen for further studies. The same procedures were applied to purify K13Q CooC.

Purification of CODH and CooJ-- Purification of CODH (3, 4) and CooJ (14) were carried out according to the established methods.

Electrophoresis-- SDS-PAGE was performed as described previously (27). All SDS-polyacrylamide gels were 12% acrylamide. Nondenaturing polyacrylamide gel electrophoresis was performed by the modified method of Hedrick and Smith (28).

Molecular Weight Determination-- Purified CooC sample was desalted with ZipTip_c18 (Millipore) according to manufacturer's protocol and was analyzed by MALDI-MS using a Shimadzu Kratos Kompact MALDI 4, version 5.0.1 mass spectrometer at the University of Wisconsin-Madison Biotechnology Center. FPLC gel filtration was also performed anaerobically using a Superose 12 HR 10/30 column. CooC was eluted from the column with a TE buffer containing 150 mM NaCl, and fractions were precipitated with trichloroacetic acid to concentrate CooC for SDS-PAGE analysis (29). SDS-PAGE followed by Western blot with anti-CooC antibodies was performed on all fractions to detect CooC.

UV-Visible Spectra-- UV-visible spectra of purified CooC anaerobically sealed in quartz cuvettes were recorded using a Shimadzu UV-1601PC spectrometer. Dithionite was separated from CooC by gel filtration on Sephadex G-25 prior to recording the spectra. The concentration of CooC was 0.1 mg/ml in TE buffer.

Metal Analysis-- Metal contents of CooC and CODH were determined by inductively coupled plasma mass spectrometry at the University of Georgia Chemical Analysis Laboratory.

ATPase and GTPase Activity Assay-- Nucleotide triphosphatase activities of wild type and K13Q CooC were analyzed by determining the release of -³²P from [-³²P]ATP (3,000 Ci/mmol) and [-³²P]GTP (5,000 Ci/mmol, Amersham Pharmacia Biotech). The assay solution consisted of 100 mM Tris-HCl (pH 7.5) with 50 mM KCl, 5 mM MgCl₂, 1 mM EDTA, and 1 mM dithionite in a total volume of 50 µl, unless noted otherwise. To determine the K_m and V_max values, [-³²P]ATP and [-³²P]GTP were diluted to 2,000-3,000 cpm/pmol in 500 µM of ATP and GTP, respectively. The concentration of purified CooC in each reaction was 0.2 µM (0.62 µg). Reactions were initiated by the addition of substrate and carried out at 37 °C. After 10 min, triplicate 15-µl aliquots were analyzed using a charcoal precipitation method (30).

Nickel Activation Assay-- All the vials used for nickel incorporation experiments were soaked in 4 N HCl for a day, and then rinsed with metal-free distilled water and MOPS buffer before use. In vivo nickel incorporation was analyzed by a CO-dependent, methyl viologen reduction assay (3). In vitro nickel activation was performed with the modification of the published methods (6, 7). R. rubrum cells were grown on nickel-depleted malate-ammonium (MN) medium (70 ml) in stoppered glass vials (100 ml). Cultures were grown in the light to an A₆₀₀ of 2.0 and then induced with bubbling of CO to the medium. When A₆₀₀ reached 4.5, cells were collected by centrifugation of the culture (50 ml), disrupted by osmotic shock (31), and resuspended in MOPS buffer (5 ml). Nickel activation was initiated by mixing the cell extract (0.5 ml, 1.82 mg of total protein) with CO-saturated MOPS buffer (4.5 ml) containing 0.5 mM NiCl₂, 0.2 mM methyl viologen, and 5.0 mM MgCl₂ in the presence or absence of 0.5 mM nucleotides. Reactions were performed at 25 °C. Every 10 min, a 10-µl aliquot of the activation mixture was taken and assayed for CO-dependent methyl viologen reduction. The specific activity of CODH was expressed as micromoles of CO oxidized/min/mg protein. Assay data represent the average of three independent determinations.

Protein Assay-- Protein concentrations were determined with the BCA assay using bovine serum albumin as a standard (32).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of cooCTJ Mutations on CODH Activity-- Fig. 1 shows the effects of Ni²⁺ concentration in the growth medium on extractable CODH activity in various mutants. The R. rubrum strains were grown in non-metal-chelexed SMN medium to prevent Ni²⁺ toxicity that inhibits cell growth at high Ni²⁺ concentrations (11). Even without addition of Ni²⁺, wild-type (UR2) strain showed considerable CODH activity (38 µmol/min/mg). This activity level indicates that the Ni²⁺ insertion system is so efficient that wild-type cells can utilize the contaminating Ni²⁺ present in the SMN medium. Addition of an increasing concentration of Ni²⁺ to a wild-type culture resulted in an increase in CODH activity, and activity was saturated at 50 µM of Ni²⁺ with a maximum activity of 110 µmol/min/mg protein. For cooT nonpolar (UR479) and cooJ polar (UR501) mutants, the CODH activity was increased as the medium Ni²⁺ concentration increased, but specific activities were only 40-50% of wild-type activity at all Ni²⁺ concentrations. The activity of UR471, which has a deletion in the portion of the cooJ gene that encodes the polyhistidine tail, was also about 41% of wild-type activity but was not significantly affected by Ni²⁺ concentrations. This result confirms the previous observation that the polyhistidine domain of CooJ is dispensable (11).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effects of nickel concentration on the CODH activities of cooCTJ mutants. The indicated concentration of Ni2+ was added to the non-metal-chelexed SMN medium, which is an MN medium enriched with 0.3% casein enzyme hydrolysate plus 0.3% yeast extract (11). After growth with CO, the cells were collected by centrifugation and ruptured by osmotic shock (31). Extractable CODH activity was determined using the CO-dependent methyl viologen reduction assay (3). All cultures except the UR445 strain accumulated similar amounts of CODH protein when analyzed by SDS-PAGE followed by Western blots of the proteins with anti-CODH antibodies. , UR2 (wild type); , UR445 (cooFSCTJA deletion); , UR469 (cooC polar insertion); , UR471 (cooJ polyhistidine deletion); , UR479 (cooT nonpolar insertion); , UR495 (cooC nonpolar insertion); , UR501 (cooJ polar insertion). Genotypes are given under "Experimental Procedures" and in Ref. 11.

A drastic reduction in CODH activity was observed in all strains containing mutations in the cooC gene. As the Ni²⁺ concentration increased from 25 to 700 µM, the activity of cooC polar mutant (UR469) was increased from 6.3 to 36% of wild-type activity. The CO oxidation activity of cooC nonpolar mutant (UR495) lacking functional CooC was very low regardless of the medium Ni²⁺ level. Even at high concentrations of Ni²⁺, none of the strains lacking cooCTJ exhibited CODH activity at the level of wild-type strain. Because the coo operon proteins do not function in nickel transport (11, 12), these results clearly implicate the CooC, CooT, and CooJ proteins in the nickel-dependent maturation of CODH. To further study CooC and its role in nickel insertion, the CooC protein was purified.

Solubilization of CooC from Membrane-- CooC was identified and localized by SDS-PAGE and Western blots. CooC was observed in the chromatophore membrane fraction of UR2 (wild type) cells grown under CO-inducing conditions as an immunoreactive band migrating at a molecular mass of 28 kDa (Fig. 2A). But it was detected neither in the membrane or soluble fraction of UR2 grown under non-CO-inducing conditions nor in extracts from UR445 lacking cooCTJ genes (data not shown). The mutant form of CooC (K13Q) was also observed in the membrane fraction of UR871 grown in the presence of CO (Fig. 2A). These results demonstrate that CooC is a CO-inducible membrane protein. Prior to CooC purification, extraction efficiencies of reagents were tested to characterize the nature of association with membrane. Membranes were treated with solutions containing the following salts or detergents, and solubilized CooC was then separated from treated membranes by ultracentrifugation. The yield and purity of solubilized CooC are given in parentheses: 0.5 M NaCl (27.7 and 0.53%); 2.0 M NaCl (65.3 and 0.44%); alkaline treatment with 100 mM Na₂CO₃ buffer at pH 11.3 (71.2 and 0.40%); 30% (NH₄)₂SO₄ (63.8 and 0.47%); 50% (NH₄)₂SO₄ (80.4 and 0.38%); 70% (NH₄)₂SO₄ (86.5 and 0.25%); Triton X-100 (62.6 and 0.33%); and CHAPS (82.1 and 0.30%). Treatment of the membrane fraction with 50% (NH₄)₂SO₄ saturation was selected based upon a compromise between yield and purity. Since CooC can be released by mild extraction methods such as high salt concentration or alkaline treatment, it seems likely that CooC is not an integral protein. This is consistent with the hydropathy analysis that CooC is a moderately hydrophobic protein with no transmembrane segments (11).

View larger version (63K): [in this window] [in a new window]   Fig. 2.   Identification, localization, and purification of wild-type CooC and K13Q CooC. A, identification and localization of CooC and K13Q CooC in the membrane of UR2 (wild type) and UR871 (K13Q CooC) cells. Cells were grown in MN medium in the presence of CO, and intracellular localization was analyzed by SDS-PAGE and Western blots of membrane (M) and soluble fraction (S) with anti-CooC antibodies. B, SDS-PAGE and Western blots of the cell extracts from UR2 (0.92 mg of protein) and UR871 (1.93 mg of protein) with anti-CODH antibodies. C, Coomassie Blue-stained SDS-polyacrylamide gel of purification fractions from membrane (lane 2), 50% (NH4)2SO4 solubilization (lane 3), 40% (NH4)2SO4 fractionation (lane 4), DE52 (lane 5), first phenyl-Sepharose (lane 6), second phenyl-Sepharose (lane 7), hydroxylapatite (lane 8), and Mono Q (3.5 µg of wild-type CooC) (lane 9). Lane 10 shows 0.26 µg of K13Q CooC. Molecular mass standards (lane 1) are phosphorylase b (97.4 kDa), bovine serum albumin (67.0 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa).

Purification of CooC-- Fig. 2C shows Coomassie-stained SDS-polyacrylamide gel of the proteins from various steps of purification. About 65% of total membrane proteins were extracted by 50% (NH₄)₂SO₄ with a wide range of molecular weights. CooC was enriched by 40% (NH₄)₂SO₄ fractionation. Western blot with anti-CODH antibodies and CODH activity assays of DE52 fractions showed that CooC co-eluted with CODH and CooF from the DE52 column. Two consecutive hydrophobic phenyl-Sepharose columns successfully separated the major membrane proteins such as CODH and CooF from CooC. CooF eluted when washing the column with 250 mM (NH₄)₂SO₄ and CooC eluted between 80 and 30 mM (NH₄)₂SO₄; CODH remained bound tightly on the hydrophobic column. After the second hydrophobic column, CooC appeared as a major protein (Fig. 2C, lane 7). Further purification with hydroxylapatite (lane 8) and Mono Q columns (lane 9) provided homogeneous CooC as shown in lane 9 of Fig. 2C. The purification resulted in a 3,464-fold overall purification with a 0.8% recovery. The mutant form of K13Q CooC was purified with reduced yield (0.6%) and purity (87%) from the UR871 cells grown under CO-inducing conditions. The wild-type CooC and K13Q CooC migrated to the same position in a SDS-polyacrylamide gel. The results of a typical purification are summarized in Table I.

Characterization of CooC-- The Ni²⁺ content of CooC was analyzed to be less than 0.1 atom per dimer. Fe²⁺, Cd²⁺, Co²⁺, Cu²⁺, Mn²⁺, and Zn²⁺ were not detected. In order to test the nickel binding ability of CooC, Ni²⁺ was added into a CooC solution, and unbound Ni²⁺ was removed by a Sephadex G-25 gel column. The content of Ni²⁺ in the Ni²⁺-treated CooC was less than 0.1 atom per dimer, demonstrating that CooC does not bind Ni²⁺ tightly under the conditions tested.

In SDS-polyacrylamide gels, purified CooC migrated at 29 kDa (Fig. 2C, lane 9). MALDI-MS spectrum recorded the molecular mass of CooC as 28.9 kDa. These values are ~1 kDa larger than the DNA sequence-deduced molecular mass of 27.8 kDa. The data can be explained by either of two possibilities: 1) membrane components or nucleotides are tightly bound and not removed from CooC during a ZipTip_c18 cleaning step, or 2) CooC is modified posttranslationally. To test these hypotheses, CooC was precipitated with trichloroacetic acid, resuspended in desalted water, and then cleaned again with a ZipTip_c18. In this case MALDI-MS recorded the molecular mass of 29.1 kDa, indicating posttranslational modification. The results prompted us to search the possible modification residues in the amino acid sequence of CooC. Interestingly, CooC shared the conserved Gly-Arg-Gly segment centered in residues 140-142 with NifH from Azotobacter vinelandii (11). In addition, this arginine residue is conserved in all CooC homologous proteins found in the CODH/ACS systems of a number of Archaea (33-35). In NifH, the arginine residue at the conserved position (Arg-101) can be modified by ADP-ribosylation (36). However, we could not assign the identities of modifying molecules or the identities of modified residues with the MALDI-MS data.

The native molecular mass of CooC was estimated to be 63 kDa by analytical gel filtration. To confirm the gel filtration result, a native gel electrophoresis was carried out under a variety of acrylamide concentrations, ranging from 6 to 14% arylamide (28). The graph of migration rate versus gel percentage gave an estimated molecular mass of 61 kDa. Thus, it appears that native CooC is a homodimer.

The UV absorption maximum (_max) assigned to a single tryptophan residue in CooC was recorded at 277.5 nm with a molar extinction coefficient (_277.5) of 32.1 mM¹ cm¹. The spectrum was not appreciably changed upon oxidation by air or upon addition of nickel. Upon treatment with hydrochloric acid, absorption intensities below 265 nm were increased, but the _max and the _277.5 values remained unchanged, suggesting that chromophores are not acid-labile. CooC contains six cysteine residues, and thus we examined the visible region of the spectrum in detail to see whether CooC has an absorbance at 420 nm that might suggest the presence of an Fe₄S₄ cluster (31, 37) or of nickel d  d transition of nickel-binding protein (38), but A₄₂₀ was not observed. Analyses of iron content and of the UV spectrum confirmed that CooC is not an iron-sulfur protein.

ATPase and GTPase Activities of Wild-type CooC and K13Q CooC-- CooC was found to hydrolyze ATP and GTP at rates of 35.2 and 6.1 pmol/min, respectively. The specific activities for ATP and GTP hydrolysis were calculated to be 0.81 and 0.09 mol of P_i released/mol of CooC/min, at 37 °C (Fig. 3, A and B). Under the same assay conditions, the rate constants for ATP and GTP hydrolysis by K13Q CooC were 7.5 and 2.7 pmol/min (Fig. 3, A and B). These values were the same as the rate constants for spontaneous hydrolysis, indicating K13Q CooC has no NTPase activities for either nucleotide. NTPase activities were not detected from the Mono Q column fractions that were enriched in the minor contaminant found in our CooC preparations. These results ruled out a possibility that NTPase activities arose from contaminants. The effect of nickel ion on the ATPase activity was examined by adding 2 eq of Ni²⁺ to the assay mixture. The ATPase activity was decreased to 94% of the reaction without Ni²⁺.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   ATPase and GTPase activities of CooC. A and B, time course assays for nucleotide hydrolysis. Purified CooC (2 µg) was incubated with 20 µM [32Pi]ATP (A) or [32Pi]GTP (B) at 37 °C in 500-µl reaction volume. Aliquots (25 µl) were withdrawn at times indicated from CooC-catalyzed (), K13Q CooC-catalyzed (), and control reaction (). The release of [32Pi] was assayed as described under "Experimental Procedures." C and D, effect of [32Pi]ATP (C) or [32Pi]GTP (D) concentration on the hydrolysis rate. At each substrate concentration, the rate of substrate hydrolysis in the absence of CooC was determined as a control and subtracted from the CooC-catalyzed hydrolytic rate.

Kinetic parameters of NTPase activities were determined by measuring the specific activity of the CooC with a varying concentration of nucleotide. The plot of hydrolysis rate versus substrate concentration showed saturation kinetics (Fig. 3, C and D). The K_m and V_max values for ATP hydrolysis determined by Lineweaver-Burk plot were 24.4 µM and 58.7 nmol/min/mg protein, respectively. Wild-type CooC showed a low level of GTPase activity with a K_m of 26.0 µM and a V_max of 3.7 nmol/min/mg protein.

Requirement of Functional CooC with Intact P-loop for in Vivo Nickel Insertion into CODH-- The K13Q mutant form of CooC was tested in CODH maturation. Based on the previous report that the increased accumulation of ⁶³Ni in CO-induced cells results from the insertion of ⁶³Ni into CODH (12), kinetic measurements of in vivo nickel insertion were conducted with the simultaneous addition of CO and Ni²⁺ to UR2 (wild-type) and UR871 (K13Q CooC) strains. UR2 and UR871 cells grown on nickel-depleted medium under non-CO-induced conditions showed very low basal CODH activity (0.9 µmol/min/mg). After 30 min of exposure to CO and Ni²⁺, the linear increase in CODH activity was observed from the wild-type culture with an apparent rate of 1.7 units/mg/h (Fig. 4). However, the UR871 cells showed only the basal activity of non-CO-induced wild-type cells, indicating that the K13Q CooC variant is not functional in production of a catalytically active CODH.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of P-loop mutation (K13Q) on the in vivo nickel activation of apoCODH. UR2 (wild-type) () and UR871 (K13Q CooC) () cells were grown on nickel-depleted MN medium. When A600 reached about 1.5, CO (20% gas phase) and Ni2+ (25 µM final concentration) were added simultaneously to the cultures. The control (non-CO-induced) contains the same concentration of Ni2+ added to UR2 (). The cells were collected by centrifugation, ruptured by osmotic shock (31), and assayed for CODH activity by CO-dependent, methyl viologen assay (6).

For further confirmation, CODH was purified from the UR871 cells grown with 25 µM of Ni²⁺, a normal concentration for wild-type culture (3, 4), and the metal content and specific activity were compared with those of apo- and holoCODH purified from wild-type cells (Table II). The CODH from UR871 cells contained 8.4 iron atoms but had a low level of Ni²⁺ compared with holoCODH. Approximately 0.18 atom of Ni²⁺ was detected per mol of CODH. The CODH had a specific activity of 26 µmol/min/mg protein. Upon Ni²⁺ treatment, the specific activity was increased to 580 µmol/min/mg protein, about 15% of the activity of Ni²⁺-activated apoCODH isolated from wild-type cells. The increased Ni²⁺ content compared with apoCODH (from nickel-starved cells) may result from the nonspecific association of Ni²⁺ during the cell breakage for CODH purification (39). The in vivo nickel insertion data suggest that K13Q CooC lacks the ability to promote incorporation of nickel into CODH, and the in vitro analyses confirm the absence of stoichiometric amounts of Ni²⁺ in CODH purified from the K13Q cooC mutant strain (UR871).

Nickel Activation of ApoCODH with Purified CooC and CooJ-- Purified CooC and CooJ were assayed for their ability to facilitate the nickel activation of apoCODH that is isolated from the wild-type strain. With a low level of Ni²⁺ (2 µM), apoCODH was activated to a specific activity of 67 µmol/min/mg CODH (control reaction) (Fig. 5). The addition of CooC to the control reaction did not lead to enhancement in activation. Further addition of CooJ resulted in a decrease in specific activity to 42 µmol/min/mg CODH, indicating that CooJ competes for nickel with apoCODH. Preincubation of apoCODH with CooC, CooJ, and ATP or GTP prior to the addition of Ni²⁺ did not cure the inhibitory effect of CooJ and not show the nucleotide-stimulated activation.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Nickel activation of apoCODH in the presence of purified CooC and CooJ. Activation mixture (250 µl total volume) contained apoCODH (10.5 µg), methyl viologen (0.2 mM), and MgCl2 (2 mM). Purified CooC (9.7 µg), CooJ (3.0 µg), and ATP (0.2 mM) were preincubated with apoCODH, and nickel activation was initiated by adding 2 or 25 µM of Ni2+ at 25 °C according to the published method (7). Aliquots (5 µl) were removed at the indicated time points and were assayed for CODH activity by CO-dependent, methyl viologen assay (6). , apoCODH without nickel (negative control); , apoCODH + 2 µM Ni2+; , apoCODH + CooC + CooJ + 2 µM Ni2+; , apoCODH + CooC + CooJ + ATP + 2 µM Ni2+; , apoCODH + 25 µM Ni2+; , apoCODH + CooC + CooJ + 25 µM Ni2+; , apoCODH + CooC + CooJ + ATP + 25 µM Ni2+.

Similar experiments were performed with a normal (25 µM) (Fig. 5) and a high (0.5 mM) level of Ni²⁺. The apoCODH was activated by 25 µM and 0.5 mM Ni²⁺ to specific activities of 159 and 1,200 µmol/min/mg CODH, respectively. When apoCODH incubated with CooC, CooJ, and ATP or GTP was treated with Ni²⁺, CODH activity increased to the level of control reaction with the same rate, but enhanced activation was not observed.

ATP Hydrolysis Enhances the Nickel Activation of apoCODH in Whole Cell Extract-- To test if ATP and/or GTP could enhance the nickel insertion, whole extract from wild-type cells grown on nickel-depleted MN medium was used as a source of apoCODH and accessory proteins. The quantitative analyses of the activation rates and specific activities are summarized in Table III. The CODH activity was increased in the presence of 0.5 mM Ni²⁺ from 2.3 (row A) to 5.9 µmol/min/mg protein with an initial rate of 0.17 unit/mg/min (row B). The presence of both Ni²⁺ and ATP resulted in a 1.8- and 2.4-fold increase in activation rate and specific activity compared with the control reaction (row C). ATP-regenerating mixture also increased the activation rate and specific activity (row D). In contrast to ATP, GTP did not enhance activation rate or specific activity (row E). When ATP and GTP were incubated together in the activation mixture, both activation rate and specific activity were decreased to the 60 and 80% levels of the reaction without GTP (row F).

In order to identify whether the ATP-enhanced nickel activation resulted from hydrolysis or from binding, nickel activation was performed with ADP or ,-AMP-PCP, a nonhydrolyzable ATP analogue. Neither ADP (row G) nor ,-AMP-PCP (row H) enhanced or decreased the nickel activation of apoCODH. The presence of ,-AMP-PCP in the ATP reaction mixture reduced the specific activity to 58% of ATP-enhanced activation (row I). The results suggest that ATP hydrolysis is required for the stimulation of nickel activation.

Nickel activation was performed with the extract from UR871 (K13Q CooC) grown on nickel-depleted MN medium to provide evidence that ATP-enhanced activation is associated with the ATPase activity of CooC. Specific activity of Ni²⁺-treated extracts from UR871 mutant was only 10% of the wild-type level (row J), and ATP-stimulated activation was not observed (row K). The same results were obtained with the extract from UR495 (cooC nonpolar) grown with 25 µM of Ni²⁺ (rows L and M).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The effects of cooCTJ mutations on the CODH activities of mutants show that all accessory gene products are required for optimal biosynthesis of the nickel center of R. rubrum CODH. Mutations in cooT and cooJ genes impair nickel insertion but do not block it completely, suggesting that CooT and CooJ are important although not essential for nickel insertion. Strains containing mutations in the cooC gene exhibit minimal CODH activity when grown at the normal concentration of nickel (25 µM), confirming the previous proposal that CooC is an essential protein for nickel processing. The results are also consistent with the report of ⁶³Ni accumulation study (12) that CooC, CooT, and CooJ are involved in nickel insertion into CODH.

Biochemical properties of CooC are compared with the traits of homologous proteins involved in metallocenter assembly and maturation (Table IV). Purified CooC is a homodimer and has NTPase activities. These properties are similar to those of A. vinelandii NifH (nitrogen fixation) (37, 40) or E. coli HypB (nickel-processing for hydrogenase) (41) which are also homodimers with NTPase activities; CooC has sequence similarity to both of these proteins. However, CooC and NifH reveal a higher ATPase/GTPase ratio compared with E. coli HypB. Conversely, HypB from Bradyrhizobium japonicum, a homodimer of molecular mass of 78 kDa with a polyhistidine nickel-binding domain, hydrolyzes GTP but not ATP (42). The similarities may reflect the common functions of these homologous proteins in nickel or FeMo-co insertion processes, whereas differences might indicate the special protein-protein interactions or the unique mechanisms of metal center formation.

In an effort to identify the features of CooC required for its role in the nickel insertion process, the P-loop mutated form of the K13Q CooC variant was generated by site-directed substitution of lysine with glutamine, and the effects of amino acid change on the NTPase activities and on the resultant CODH activity of the mutant strain were investigated. The K13Q substitution abolished the NTPase activities of CooC, and the mutant strain UR871 that produces K13Q CooC was found to accumulate enzymatically inactive, nickel-deficient CODH as evidenced by CO oxidation assay and metal analysis. Therefore, the important findings obtained with the P-loop altered form of K13Q CooC are that the ability of CooC to function in nickel insertion is dependent upon its NTP-binding or -hydrolyzing property, and that nickel center maturation is an energy-requiring process. Our observations are consistent with the study reported by Rangaraji et al. (43) with the altered form of K15R NifH that was inactive both in ATP hydrolysis and maturation of apodinitrogenase from A. vinelandii and are also similar to the report of Maier et al. (16) for HypB of E. coli. The K117N HypB substitution in GTP-binding motif 1 (P-loop) motif decreased GTPase activity to 2.9% of wild-type HypB, and a mutant strain that expresses K117N HypB was unable to incorporate nickel into apohydrogenase (16). It has also been reported that intact P-loop sequence in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease (17) and that UreG catalyzes the GTP-dependent activation of UreDFG-apourease in vitro (44).

Incubation of purified CooC and CooJ with apoCODH did not affect the nucleotide-stimulated nickel activation of apoCODH. Since in vivo and in vitro nickel incorporation suggests a requirement of all CooCTJ accessory proteins and ATP-enhanced activation was observed from the wild-type cell extract (Table III), we propose that unknown factor(s) perhaps including the CooT protein are required for optimal activation to occur. At present, we have no information about the role(s) of CooT in nickel center formation. The N-terminal sequence (Cys-X-Ala) of CooT is identical to that of chaperone-type HypC from E. coli (11). Cysteine is essential for the formation of a HypC-HycE complex to keep the hydrogenase subunit HycE in a nickel-accessible conformation, and a mutant containing C2A, C2R, or C2S HypC variant accumulates the nickel-deficient precursor form of hydrogenase (13). By analogy and N-terminal similarity to HypC, the lack of significant, ATP-stimulated activation of apoCODH may be attributable to the absence of CooT in the purified activation systems.

With the information that CooC functions in nickel center maturation by binding or hydrolyzing NTP, we tried to identify the physiologically relevant substrate for CooC. Nickel activation, which is the reductant-dependent in vitro insertion of nickel into apoCODH (7), was performed in the presence of ATP or GTP to test if either could enhance activation rate and extent of specific activity. When a cell extract from wild-type strain grown on nickel-depleted MN medium was employed as a source of apoCODH and CooCTJ accessory proteins, ATP was able to enhance both activation rate and specific activity. However, GTP showed no stimulatory effect on nickel activation and behaved as an inhibitor of ATP-enhanced nickel insertion (Table III). The results are explained based on the nucleotide hydrolysis kinetics. As shown earlier, the apparent affinities of CooC for ATP and GTP are nearly identical, but maximum rate for ATP hydrolysis is 16 times faster than GTP hydrolysis. Thus, in the reaction containing both ATP and GTP, GTP may compete for the P-loop with ATP, showing an inhibition of nickel activation compared with the reaction without GTP. In contrast to ATP, both ADP and ,-AMP-PCP failed to stimulate the nickel activation of apoCODH, and ,-AMP-PCP reduced the ATP-enhanced nickel activation. The integrated interpretations of the in vitro nickel insertion experiments suggest that ATP might be a physiological substrate for nickel center maturation and that hydrolysis of ATP drives nickel insertion into R. rubrum CODH.

The nickel center assembly systems of hydrogenase, urease, and CODH from R. rubrum all require two common accessory proteins, a nickel-binding and a nucleotide-binding protein. HypB from Rhizobium leguminosarum (45) and B. japonicum (46) contain nickel- and nucleotide-binding domains in a single protein. These may represent a gene fusion of proteins analogous to nucleotide-binding CooC and nickel-binding CooJ. Based on our results and proposals for the roles of HypB (41) and UreG (44) for apoenzyme maturation, two possible functions of CooC during nickel processing at the active site of CODH are envisioned. First, CooC functions as a nickel insertase that mobilizes nickel from CooJ to apoCODH using the energy released from ATP hydrolysis. Nickel enters the cells and CooJ binds nickel, but nickel bound in CooJ may be a form that apoCODH cannot access efficiently without the aid of CooC. Second, CooC might act as a chaperone and use the ATP hydrolysis to construct or fold the proper nickel center, and CooJ might deliver the nickel to the vacant nickel site. This model is based upon the observation that the nickel-activated CODH purified from UR871 (K13Q CooC) (Table II) and the nickel-treated extract from UR495 (cooC nonpolar) show only 10-20% of the wild-type level of CODH activity (Table III, rows J-M). The above results indicate that apoCODH from the mutants lacking functional CooC is less competent for nickel activation than that from the wild-type strain because similar amounts of CODH were expressed in both cooC mutant and wild-type strains, and no degradation fragments were detected by Western blots (Fig. 2B). A similar phenomenon had been published in the FeMo-co insertion system of dinitrogenase from K. pneumoniae by Homer et al. (47). Recently, Heo et al. (8) suggest that the vacant nickel site of apoCODH is not in the proper conformation for nickel insertion, probably due to the absence of coo-encoded accessory proteins. The role of CooC in this hypothesis is reminiscent of the ATP-dependent chaperoning function of GroEL or GroES (48). Actually, several reports have shown that these chaperones are involved in the activation and stabilization of urease from Helicobacter pylori (49, 50) and in nickel incorporation into hydrogenase from E. coli (51).

In conclusion, the data presented here show that CooC couples ATP hydrolysis with nickel insertion into the active site of R. rubrum CODH, although the exact mechanism remains to be clarified. Cell extract complementation assays in the presence of ATP will be performed to identify unknown factor(s) and to establish optimum conditions for ATP-dependent activation. We will also focus on the relations of accessory proteins with the nickel-activable conformation of apoCODH. The detailed mechanism of nickel center maturation will be resolved once all components are available to reconstitute the nickel insertion system in vitro.

	ACKNOWLEDGEMENTS

We are grateful to Michael A. Halbisen and Dr. Robert L. Kerby for the generation of anti-CooC antibodies.

	FOOTNOTES

^* This work was supported by Department of Energy Science Grant DE-FG02-87ER13691 (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.

To whom correspondence should be addressed: Dept. of Biochemistry, 433 Babcock Dr., Madison, WI 53706. Tel.: 608-262-6859; Fax: 608-262-3453; E-mail: pludden@cals.wisc.edu.

Published, JBC Papers in Press, August 15, 2001, DOI 10.1074/jbc. M104945200

	ABBREVIATIONS

The abbreviations used are: CODH, carbon monoxide dehydrogenase; MOPS, 3-(N-morpholino)propanesulfonate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; NTPase, nucleotide triphosphatase; , -AMP-PCP, adenylylmethylenediphosphate; PAGE, polyacrylamide gel electrophoresis; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; FPLC, fast protein liquid chromatography; FeMo-co, iron-molybdenum cofactor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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