Portal de Periódicos da CAPES

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês

10.1074/jbc.m210484200

1083-351X

Simonida Gencic, David A. Grahame,

Microbial Metabolic Engineering and Bioproduction

The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C–C bond cleavage in methanogens growing on acetate and is also responsible for synthesis of acetyl units during growth on C-1 substrates. The ACDS β subunit contains nickel and an Fe/S center and reacts with acetyl-CoA forming an acetyl-enzyme intermediate presumably directly involved in acetyl C–C bond activation. To investigate the role of nickel in this process two forms of the Methanosarcina thermophila β subunit were overexpressed in anaerobically grown Escherichia coli. Both contained an Fe/S center but lacked nickel and were inactive in acetyl-enzyme formation in redox-dependent acetyltransferase assays. However, high activity developed during incubation with NiCl2. The native and nickel-reconstituted proteins both contained iron and nickel in a 2:1 ratio, with insignificant levels of other metals, including copper. Binding of nickel elicited marked changes in the UV-visible spectrum, with intense charge transfer bands indicating multiple thiolate ligation to nickel. The kinetics of nickel incorporation matched the time course for enzyme activation. Other divalent metal ions could not substitute for nickel in yielding catalytic activity. Acetyl-CoA was formed in reactions with CoA, CO, and methylcobalamin, directly demonstrating C–C bond activation by the β subunit in the absence of other ACDS subunits. Nickel was indispensable in this process too and was needed to form a characteristic EPR-detectable enzyme-carbonyl adduct in reactions with CO. In contrast to enzyme activation, EPR signal formation did not require addition of reducing agent, indicating indirect catalytic involvement of the paramagnetic species. Site-directed mutagenesis indicated that Cys-278 and Cys-280 coordinate nickel, with Cys-189 essential for Fe/S cluster formation. The results are consistent with an Ni2[Fe4S4] arrangement at the active site. A mechanism for C–C bond activation is proposed that includes a specific role for the Fe4S4 center and accounts for the absolute requirement for nickel. The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C–C bond cleavage in methanogens growing on acetate and is also responsible for synthesis of acetyl units during growth on C-1 substrates. The ACDS β subunit contains nickel and an Fe/S center and reacts with acetyl-CoA forming an acetyl-enzyme intermediate presumably directly involved in acetyl C–C bond activation. To investigate the role of nickel in this process two forms of the Methanosarcina thermophila β subunit were overexpressed in anaerobically grown Escherichia coli. Both contained an Fe/S center but lacked nickel and were inactive in acetyl-enzyme formation in redox-dependent acetyltransferase assays. However, high activity developed during incubation with NiCl2. The native and nickel-reconstituted proteins both contained iron and nickel in a 2:1 ratio, with insignificant levels of other metals, including copper. Binding of nickel elicited marked changes in the UV-visible spectrum, with intense charge transfer bands indicating multiple thiolate ligation to nickel. The kinetics of nickel incorporation matched the time course for enzyme activation. Other divalent metal ions could not substitute for nickel in yielding catalytic activity. Acetyl-CoA was formed in reactions with CoA, CO, and methylcobalamin, directly demonstrating C–C bond activation by the β subunit in the absence of other ACDS subunits. Nickel was indispensable in this process too and was needed to form a characteristic EPR-detectable enzyme-carbonyl adduct in reactions with CO. In contrast to enzyme activation, EPR signal formation did not require addition of reducing agent, indicating indirect catalytic involvement of the paramagnetic species. Site-directed mutagenesis indicated that Cys-278 and Cys-280 coordinate nickel, with Cys-189 essential for Fe/S cluster formation. The results are consistent with an Ni2[Fe4S4] arrangement at the active site. A mechanism for C–C bond activation is proposed that includes a specific role for the Fe4S4 center and accounts for the absolute requirement for nickel. The methanogenic Archaea utilize a unique metabolic pathway for degradation of acetate under anaerobic conditions, and cleavage of acetate thereby accounts for a major proportion of the methane formed in the environment. The central reaction in this pathway is carried out by an unusual multienzyme complex, designated acetyl-CoA decarbonylase/synthase (ACDS), 1The abbreviations used are: ACDS, acetyl-CoA decarbonylase/synthase; H4SPt, tetrahydrosarcinapterin; MOPS, 3-(N-morpholino)propanesulfonic acid; HPLC, high pressure liquid chromatography; CODH/ACS, CO dehydrogenase/acetyl-CoA synthase which contains five different polypeptide subunits and accounts for as much as 25% of the soluble protein in species such as Methanosarcina thermophila and Methanosarcina barkeri growing on acetate. The ACDS complex catalyzes cleavage of the acetyl C–C bond using the substrates acetyl-CoA and tetrahydrosarcinapterin (H4SPt), a tetrahydrofolate analog which serves as methyl acceptor, and yields the products CoA,N 5-methyltetrahydrosarcinapterin, CO2, and two reducing equivalents, as given in Reaction 1 (1Grahame D.A. J. Biol. Chem. 1991; 266: 22227-22233Google Scholar). acetyl­CoA+H4SPt+H2O⇄CoASH+CH3­H4SPt+CO2+2H++2e−REACTION 1 This overall reaction is made up of a series of partial reactions catalyzed by different protein subcomponents of the ACDS complex as shown in Scheme FS1 (2Grahame D.A. DeMoll E. J. Biol. Chem. 1996; 271: 8352-8358Google Scholar). Acetyl-CoA binds to the β subunit, and under low redox potential conditions, as required for activity, transfers the acetyl group to a nucleophilic center on the enzyme forming an acetyl-enzyme species and releasing CoA (Scheme FS1, acetyl transfer) (2Grahame D.A. DeMoll E. J. Biol. Chem. 1996; 271: 8352-8358Google Scholar, 3Bhaskar B. DeMoll E. Grahame D.A. Biochemistry. 1998; 37: 14491-14499Google Scholar). The acetyl intermediate then undergoes C–C bond cleavage by a reaction that is presumed to involve metal-based decarbonylation and/or methyl group migration (Scheme FS1, cleavage). The nascent methyl group is then transferred to a corrinoid cofactor present on the γδ subcomponent, which catalyzes subsequent methyl transfer to the substrate H4SPt (Scheme FS1, methyl transfer) (2Grahame D.A. DeMoll E. J. Biol. Chem. 1996; 271: 8352-8358Google Scholar). The carbonyl group is oxidized to CO2 by a process involving the αε CO dehydrogenase subcomponent, with regeneration of the reduced form of the β subunit. Previous studies on the β subunit have focused on a C-terminally truncated form of the protein purified from the native ACDS complex following partial proteolytic digestion (2Grahame D.A. DeMoll E. J. Biol. Chem. 1996; 271: 8352-8358Google Scholar, 3Bhaskar B. DeMoll E. Grahame D.A. Biochemistry. 1998; 37: 14491-14499Google Scholar, 4Murakami E. Ragsdale S.W. J. Biol. Chem. 2000; 275: 4699-4707Google Scholar). The genes encoding the five ACDS subunits are arranged together in an operon along with one additional open reading frame in all species ofMethanosarcina and in certain other methanogens as well (5Maupin-Furlow J.A. Ferry J.G. J. Bacteriol. 1996; 178: 6849-6856Google Scholar, 6Galagan J.E. Nusbaum C. Roy A. Endrizzi M.G. Macdonald P. FitzHugh W. Calvo S. Engels R. Smirnov S. Atnoor D. Brown A. Allen N. Naylor J. Stange-Thomann N. DeArellano K. Johnson R. Linton L. McEwan P. McKernan K. Talamas J. Tirrell A. Ye W. Zimmer A. Barber R.D. Cann I. Graham D.E. Grahame D.A. Guss A.M. Hedderich R. Ingram-Smith C. Kuettner H.C. Krzycki J.A. Leigh J.A. Li W. Liu J. Mukhopadhyay B. Reeve J.N. Smith K. Springer T.A. Umayam L.A. White O. White R.H. Conway de Macario E. Ferry J.G. Jarrell K.F. Jing H. Macario A.J.L. Paulsen I. Pritchett M. Sowers K.R. Swanson R.V. Zinder S.H. Lander E. Metcalf W.W. Birren B. Genome Res. 2002; 12: 532-542Google Scholar, 7Deppenmeier U. Johann A. Hartsch T. Merkl R. Schmitz R.A. Martinez-Arias R. Henne A. Wiezer A. Bäumer S. Jacobi C. Brüggemann H. Lienard T. Christmann A. Bömeke M. Steckel S. Bhattacharyya A. Lykidis A. Overbeek R. Klenk H.P. Gunsalus R.P. Fritz H.J. Gottschalk G. J. Mol. Microbiol. Biotechnol. 2002; 4: 453-461Google Scholar, 9Smith D.R. Doucette-Stamm L.A. Deloughery C. Lee H. Dubois J. Aldredge T. Bashirzadeh R. Blakely D. Cook R. Gilbert K. Harrison D. Hoang L. Keagle P. Lumm W. Pothier B. Qiu D. Spadafora R. Vicaire R. Wang Y. Wierzbowski J. Gibson R. Jiwani N. Caruso A. Bush D. Safer H. Patwell D. Prabhakar S. McDougall S. Shimer G. Goyal A. Pietrokovski S. Church G.M. Daniels C.J. Mao J.-I. Rice P. Nölling J. Reeve J.N. J. Bacteriol. 1997; 179: 7135-7155Google Scholar). 2M. barkeri Fusaro Genome Project, the United States Department of Energy Joint Genome Institute (www.jgi.doe.gov). The operon structure is shown in Scheme FS2, with the designated genes and corresponding subunit molecular masses indicated for M. thermophila TM-1. The additional open reading frame encodes an accessory protein thought to be involved in nickel insertion, and nickel is present in both the large CO dehydrogenase subunit α (CdhA) and in the β subunit (CdhC) containing the active site for acetyl-enzyme formation. In addition to nickel, CdhA and CdhC also contain iron in the form of Fe/S clusters. Early indications that the β subunit contains a unique Ni-Fe/S center, the spectroscopically designated A cluster, came from studies on detergent dissociation of the clostridial CO dehydrogenase/acetyl-CoA synthase enzyme (CODH/ACS), an α2β2 heterodimer, with α subunit homologous to the β subunit from methanogens (10Xia J. Lindahl P.A. Biochemistry. 1995; 34: 6037-6042Google Scholar, 11Xia J. Lindahl P.A. J. Am. Chem. Soc. 1996; 118: 483-484Google Scholar). When reacted with CO in the presence of a reducing agent, the isolated clostridial α subunit exhibited an EPR spectrum similar to the A cluster NiFeC signal found in earlier studies on the intact ACDS complex and the native clostridial CODH/ACS enzyme (11Xia J. Lindahl P.A. J. Am. Chem. Soc. 1996; 118: 483-484Google Scholar). Characterization of the ability of the ACDS β subunit to bind acetyl-CoA and CoA, and to catalyze acetyl group transfer reactions by way of a high energy acetyl-enzyme intermediate formed on the enzyme at low redox potentials (3Bhaskar B. DeMoll E. Grahame D.A. Biochemistry. 1998; 37: 14491-14499Google Scholar), and other studies (4Murakami E. Ragsdale S.W. J. Biol. Chem. 2000; 275: 4699-4707Google Scholar), further implicated the β subunit A center as the site where acetyl C–C bond activation takes place. Functional preparations of the clostridial α subunit have now been obtained that catalyze acetyl-CoA formation from CoA and CO in the presence of methylated clostridial corrinoid iron-sulfur protein (12Loke H.-K. Tan X. Lindahl P.A. J. Am. Chem. Soc. 2002; 124: 8667-8672Google Scholar). Recently, a crystallographic structure of the clostridial CODH/ACS enzyme was published in which the A center was shown to contain an Fe4S4 cluster in close proximity to a binuclear metal site containing nickel and another metal ion suggested to be copper (13Doukov T.I. Iverson T.M. Seravalli J. Ragsdale S.W. Drennan C.L. Science. 2002; 298: 567-572Google Scholar). The purpose of the present study was to characterize the role of the β subunit in cleavage of the acetyl C–C bond and to better understand the role of nickel at the active site. Therefore, we developed methods to generate large quantities of the protein using recombinant techniques to overexpress the ACDS β subunit inEscherichia coli under anaerobic growth conditions. UV-visible spectroscopic analyses were employed to investigate the kinetics and stoichiometry of nickel binding to the β subunit apoprotein. The requirements for nickel were established for activity in acetyltransferase, and for the ability to form the C–C bond of acetyl-CoA in the absence of all other protein components. EPR analyses were used to examine the role of nickel in forming a paramagnetic A-center enzyme-CO adduct, with the non-requirement for reducing agent indicative of the oxidation state of nickel in the NiFeC species, relevant to indirect involvement of this species in the mechanism of C–C bond activation. In efforts to define the nickel and Fe/S coordination environments site-directed mutagenic analysis was employed to identify amino acid residues providing ligands to nickel and iron. Additional insight into the structure and catalytic function of the Ni-Fe/S active center was obtained from spectroscopic and enzymological characterization of the mutants. The absolute requirement for nickel and a defined role for the Fe/S center are incorporated in a mechanism proposed for C–C bond activation that is consistent with the properties of the Ni-Fe/S center described here and in accordance with findings from previous investigations. Restriction enzymes were obtained from New England Biolabs. Unless otherwise indicated, all other chemicals were from Sigma and of the highest purity grade offered. All solutions were prepared using Milli-Q deionized water (Millipore Corp.). Anaerobic procedures were performed under an atmosphere of nitrogen containing 1–3% H2 using a Coy-type anaerobic chamber. Oxygen levels were monitored by a Teledyne model 3190 trace oxygen analyzer and were typically in the range of 0.5–2 ppm O2. Previously we determined the complete sequence of the ACDS operon from M. thermophila strain TM-1 (GenBankTM accession number AF173830). The gene for the full-length ACDS β subunit (cdhC) and a truncated form of the gene coding for a protein lacking 75 amino acids at the C terminus (cdhC*) were overexpressed using a modified version of the pQE60 (Qiagen) vector from which the His6 tag was deleted, designated pQE60ΔHis. Modifications to remove the His6tag were as described previously (14Gencic S. LeClerc G.M. Gorlatova N. Peariso K. Penner-Hahn J.E. Grahame D.A. Biochemistry. 2001; 40: 13068-13078Google Scholar). PCR amplifications ofcdhC and cdhC* were performed using genomic DNA as template isolated from M. thermophila strain TM-1 with forward primers incorporating an NcoI site and reverse primers containing a BamHI site. PCR products were initially subcloned into the plasmid pCRII-TOPO (Invitrogen). After digestion with NcoI and BamHI and purification by agarose gel electrophoresis, the eluted fragments were cloned into the pQE60ΔHis vector, previously cut with NcoI andBamHI. E. coli strain M15 [pREP4] was then transformed with the pQE60ΔHis expression constructs and used for overexpression as described below. Site-directed mutagenesis ofcdhC* was performed using the QuikChange Multisite-directed Mutagenesis kit, according to instructions provided by the manufacturer (Stratagene). The sequences of all genetic constructs and bordering regions were verified by sequencing of isolated plasmid DNAs using the method of dideoxynucleotide termination with the ABI PRISM Big-Dye Terminator cycle sequencing kit version 3.0 with AmpliTaq DNA polymerase FS (Applied Biosystems). Overexpression of the wild type and all mutant forms of the ACDS β subunit was carried out by growth at 31 °C under strictly anaerobic conditions in Luria-Bertani medium containing 1% glucose and 40 mm sodium fumarate as electron acceptor. Hydrogen gas evolved during growth was allowed to escape through a 22-gauge syringe needle fitted to one of two ports constructed in the stopper used to seal the culture vessels. A second port provided the means to remove aliquots for monitoring the growth and for required additions. Cultures were inoculated at 1:100 using an anaerobic starter culture grown overnight. About 3 h later, after reaching an OD600 nm of 0.7–0.9, the cells were induced by addition of 0.4 mm isopropyl-β-d-thiogalactopyranoside (Research Organics) and immediately supplemented with iron, sulfide, and nickel, 100 μmFe(NH4)2(SO4)2, 200 μm Na2S, and 5 μmNiCl2 added from sterile, anaerobic stock solutions. The cultures were further supplemented after 2 and 4 h following induction. These subsequent additions were needed to provide sufficient iron, as was evident from small amounts of black iron sulfide that remained in the final cell pellets under these conditions, but not when either lower levels or fewer additions of iron and sulfide were employed. Cells were harvested by centrifugation under anaerobic conditions 5 h after induction (at an OD600 nm of around 2–2.5), and the cell paste was frozen in liquid nitrogen. Crude buffer-soluble extracts were prepared by French press cell lysis at 20,000 pounds/square inch at 4 °C of 6.5 g of cell paste resuspended in 30 ml of 50 mm Tris·SO4, 25 mm Na2SO4, 10% glycerol, pH 7.5, and the lysate was centrifuged at 34,000 × g for 20 min at 4 °C. These steps as well as all others subsequently used for purification of various forms of the ACDS β subunit were performed under anaerobic conditions. The supernatant obtained following centrifugation was applied at ∼1.6 ml/min to a 12 × 2.5 cm diameter column of Q-Sepharose Fast Flow (Amersham Biosciences) equilibrated in buffer A (50 mm Tris·SO4, 25 mm Na2SO4, pH 7.5) at room temperature. The column was then washed with ∼270 ml of buffer A and eluted with 400 ml of linear gradient of 0–0.4 mNa2SO4 in buffer A. Fractions (8 ml) were collected and analyzed by SDS-PAGE and for UV-visible absorbance. Fractions with the maximum absorbance ratioA 400 nm/A 280 nm and displaying the highest purity on SDS gels were pooled, concentrated by ultrafiltration using an Amicon stirred cell, and diafiltered to remove salt and low molecular mass contaminants. Samples were adjusted to contain 10% glycerol prior to storage by freezing in liquid nitrogen. Protein was determined on the basis of absorbance at 280 nm using an absorptivity coefficient for CdhC of 72,300m−1 cm−1 (66,600m−1 cm−1 for CdhC*) obtained from the expression 1.03 × (5550 ΣTrp + 1340 ΣTyr + 150 ΣCysnon-cluster) + 4500 ΣCysFe/S-cluster, modified from the original method of Perkins (15Perkins S.J. Eur. J. Biochem. 1986; 157: 169-180Google Scholar) to include the number of cysteine residues involved in Fe/S clusters in order to account for absorbance contributed by Fe/S clusters. Comparable results were obtained from protein measurements performed by the method of Bradford (16Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) using bovine serum albumin as a standard. Metal analyses were carried out by ICP-atomic emission spectroscopy using a Thermo Jarrell-Ash 965 AtomComp plasma emission spectrometer on samples submitted to the University of Georgia, Research Services, Chemical Analysis Laboratory. Purified recombinant CdhC at a concentration of 7–35 μm that contained iron but lacked nickel was reconstituted with Ni2+ by incubation under anaerobic conditions in the presence of 100 μmNiCl2 in 30 mm HEPES, pH 7.2, at room temperature. Aliquots were removed at different times, diluted with sufficient water to give 1.4 μm CdhC, and immediately assayed by transferring 10 μl of the diluted sample to 110 μl of acetyltransferase assay solution, and the reactions were completed as described below. Large scale reconstitution of the enzyme employed 60–80 μm CdhC* and 180 μmNiCl2 in reaction volumes of up to 12 ml. Sufficient time was allowed for metal incorporation (as determined in separate trials), and the reaction mixtures were thereafter concentrated to 1.3 ml by ultrafiltration on an Amicon YM30 ultrafiltration membrane, 44.5 mm diameter. Excess Ni2+ was removed by applying the concentrated enzyme to a 1.5-cm diameter, 10-ml bed volume column of Sephadex G-25 equilibrated in 40 mm HEPES, 10 mm Na2SO4, pH 7.2, and 1.0-ml fractions were collected. The protein was recovered in three brown fractions, which were combined and reconcentrated to yield a preparation of holoenzyme at a final concentration equal to that of the starting apoenzyme. The ability of recombinant CdhC to catalyze redox-dependent transfer of the acetyl group from acetyl-CoA (acetyltransferase) was measured by a modified method similar to that described previously (3Bhaskar B. DeMoll E. Grahame D.A. Biochemistry. 1998; 37: 14491-14499Google Scholar). The reaction mixtures contained 100 μm acetyl-CoA, 100 μm3′-dephospho-CoA, 50 μm aquocobalamin, 4.0 mmTi3+-EDTA, 50 mm MOPS buffer, pH 7.2. Reactions were carried out in 12 × 75-mm glass tubes at 25 °C and were initiated by addition of a 10-μl aliquot of sufficiently diluted protein to 110 μl of an assay solution that contained all the above components at concentrations needed to give the final values indicated in a total reaction volume of 120 μl. A series of reactions were carried out for each enzymatic rate determination and stopped at different time points (from 0 to 8 min) by addition of 120 μl of 2 mm TiCl3 in 0.5 m sodium citrate, pH 4.0, and the mixtures were frozen in liquid nitrogen prior to analysis by HPLC for the products CoA andS-acetyl-3′-dephospho-CoA. HPLC analyses and calculation of reaction rates were performed as described previously (3Bhaskar B. DeMoll E. Grahame D.A. Biochemistry. 1998; 37: 14491-14499Google Scholar). Net synthesis of acetyl-CoA by the recombinant ACDS β subunit from carbon monoxide, CoA, and methylcobalamin was measured in reaction mixtures containing 0.65–3.9 μm nickel-reconstituted CdhC, 500 μm methylcobalamin, 100 μm CoA, 1 mm Ti3+-citrate in 50 mm MOPS buffer, pH 7.2, under an atmosphere of 100% CO at room temperature. Reactions were initiated by addition of CdhC and were followed over time by removal of aliquots that were mixed with an equal volume of 2 mm TiCl3 in 0.5 m sodium citrate, pH 4.0, frozen in liquid nitrogen, and subsequently analyzed by HPLC for the formation of acetyl-CoA. Metal ion incorporation into nickel-deficient CdhC* in titration experiments was monitored spectrophotometrically on the basis of increase in absorbance around 336–340 nm observed due to ligand-to-metal charge transfer band formation when the nickel-deficient protein was exposed to different divalent metal ions in the late 3d transition series. Titration of 16.4 nmol of CdhC* contained in 590 μl of 50 mm HEPES, pH 8.0, was carried out in a semi-microspectrophotometer cuvette by addition of 5.0-μl aliquots of 1.20 mm stock solutions of divalent metal ions (e.g. MnCl2, Fe(NH4)2(SO4)2, CoCl2, NiCl2, CuSO4). UV-visible scans were recorded on a Hewlett-Packard 8452A spectrophotometer set up inside the anaerobic chamber. After addition of each aliquot of divalent metal solution, scans were recorded over time until no further changes were observed in the spectrum. The gene encoding the β subunit of the ACDS complex from M. thermophila was overexpressed in two separate forms in E. coli. One of these was the full-length 472-amino acid protein, CdhC, and the other was a 397-amino acid form truncated at the C terminus, CdhC*. CdhC* includes all major regions of conservation among β subunit homologs and is about 30 amino acids smaller than the estimated size of the native protein isolated in truncated form following disruption of the ACDS complex by partial proteolysis (2Grahame D.A. DeMoll E. J. Biol. Chem. 1996; 271: 8352-8358Google Scholar, 3Bhaskar B. DeMoll E. Grahame D.A. Biochemistry. 1998; 37: 14491-14499Google Scholar). SDS-gel electrophoresis showed that similar amounts of CdhC and CdhC* were produced over time after induction of E. coli cultures grown under anaerobic conditions, as described under "Materials and Methods." However, CdhC was considerably less soluble than CdhC* as found by analysis of cell extracts and fractions from subsequent purification procedures. Yields of the purified proteins were around 3 mg per g of cell paste for CdhC versus 20–22 mg per g of cells for CdhC*. As shown in Fig. 1, Q-Sepharose anion exchange chromatography of the supernatant obtained from an extract of E. coli expressing CdhC* resulted in elution of a major peak of protein with absorbance at 280 and at 400 nm (due to Fe/S clusters), with fractions containing highly purified CdhC*. The final preparation consisting of the pooled and concentrated peak fractions was ∼90–92% pure as judged by densitometric analysis of SDS gels (Fig. 1, inset). Samples analyzed for metal content by plasma emission spectroscopy contained 2.7 to 3.0 g atom of iron per mol of CdhC*, but nickel was not detected, even though 5 μm NiCl2 had been added to the growth medium. Samples of purified CdhC and CdhC* were assayed for acetyltransferase activity to determine their ability to react with acetyl-CoA under low redox potential conditions generating an acetyl-enzyme intermediate. As isolated, both recombinant proteins showed very low levels of acetyltransferase activity, less than 1% of the specific activity of the native β subunit. However, high activity was found for both proteins, CdhC and CdhC*, after incubation with Ni2+. A progressive increase in activity was observed over time, reaching a maximum after several hours of incubation of CdhC, 7.1 μm, with NiCl2, 100 μm, as shown in Fig. 2. Activation of the enzyme under these conditions in which [Ni2+] ≫ [apoCdhC] followed pseudo-first order kinetics with an apparent half-time for activation of about 32 min. Activity in the assay showed absolute dependence on low redox potential, as expected, because it is a characteristic property of the subunit isolated from the ACDS complex (3Bhaskar B. DeMoll E. Grahame D.A. Biochemistry. 1998; 37: 14491-14499Google Scholar). Turnover rates for acetyl transfer were up to 1250 min−1 for CdhC and 4500 min−1 for CdhC*, as compared with the value of 3100 min−1 (3Bhaskar B. DeMoll E. Grahame D.A. Biochemistry. 1998; 37: 14491-14499Google Scholar) for the native β subunit isolated from the ACDS complex. Plasma emission spectroscopic analyses on samples of the β subunit isolated from the ACDS complex and on samples from large scale nickel reconstitution of CdhC* showed significant levels of iron and nickel but only trace amounts of other metals. In particular, levels of copper were extremely low, as shown in Fig. 3. Notably, the measured iron/nickel ratio was 1.9 for nickel-reconstituted CdhC*, and a similar value of 2.2 was observed for the isolated β subunit. These results indicate that binding of nickel is required for activation of the enzyme and that the enzyme contains iron and nickel in a ratio of ∼2:1. Therefore, direct spectrophotometric methods were developed to examine further the interaction of nickel with the β subunit. Preparations of the brown, iron-containing, nickel-deficient β subunit (CdhC and CdhC*) exhibited absorbance at around 400 nm typical of a simple Fe/S protein with minimum values for the ratioA 280 nm/A 400 nm of around 5.1. Upon addition of NiCl2 a marked, time-dependent change was observed in the UV-visible spectrum of the recombinant protein resulting in a final spectrum closely resembling that of the subunit isolated from the ACDS complex. Difference spectra obtained by subtracting the spectrum of the apoenzyme from that of the nickel-reconstituted protein showed a sharp peak of absorbance increase centered around 332–336 nm and a broader peak at around 550 nm, as shown in Fig.4 A (middle panel). A third peak with the highest intensity was found at 262 nm (not shown). These features are attributed to ligand-to-metal charge transfer absorption formed when nickel binds to the enzyme, with multiple S ligation indicated by the high values of molar absorptivity. In addition, similar features were observed for the enzyme after incubation with other divalent first row transition metals including Co2+ and Cu2+, Fig. 4 A(top and bottom panels), with lower intensityd-d transitions at 680 and 720 nm found in the spectrum of the Co2+-substituted protein. Titration of the β subunit apoprotein (CdhC*) was carried out with various different metal ions using the changes in absorbance to monitor the extent of binding. The results, shown in Fig. 4 B, indicated that Co2+, Ni2+, and Cu2+bind to the β subunit with end points all significantly greater than 1 eq. The values for Ni2+ and Cu2+ were close to 2, indicating a stoichiometry of 2 g atom of nickel per mol of CdhC*. Evidence for binding of Mn2+ and Fe2+was also obtained; however, much higher levels were required to saturate the enzyme, suggesting a weaker affinity for these metals. Acetyltransferase assays were performed on samples removed after the titrations and showed expected high levels of activity in the presence of nickel; however, no activity could be detected with any of the other metals (including manganese, iron, cobalt, and copper). These results indicate that the enzyme is capable of binding other divalent metal ions at the nickel sites, involving multiple thiolate ligation, but that only nickel is able to generate the active enzyme. The kinetics of nickel binding to the β subunit were monitored spectrophotometrically by following the increase in absorbance over time after a single addition of excess Ni2+ to CdhC*. Under conditions in which the concentration of CdhC* was significant relative to [Ni2+], the reaction followed second order kinetics in which the rate was dependent upon the concentration of both apoenzyme and [Ni2+], with an apparent second order rate constant of ∼3 × 10−4 μm−1min−1, as shown in Fig. 5. These results agree with the time course for activation of the enzyme in Fig. 2 in which [Ni2+] ≫ [apoenzyme], corresponding to pseudo-first order conditions, indicating that activation is limited by the rate of nickel binding. Nickel reconstitution at pH 7.2 (Fig. 5) was much faster than at pH 6.5 but markedly slower than the reaction at pH 8.0. The magnitude of these differences was consistent with deprotonation of more than one cysteine thiol grou