Chemically distinct Ni sites in the A-cluster in subunit beta of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila: Ni L-edge absorption and X-ray magnetic circular dichroism analyses

The 5-subunit-containing acetyl-CoA decarbonylase/synthase (ACDS) complex plays an important role in methanogenic Archaea that convert acetate to methane, by catalyzing the central reaction of acetate C-C bond cleavage in which acetyl-CoA serves as the acetyl donor substrate reacting at the ACDS beta subunit active site. The properties of Ni in the active site A-cluster in the ACDS beta subunit from Methanosarcina thermophila were investigated. A recombinant, C-terminally truncated form of the beta subunit was employed, which mimics the native subunit previously isolated from the ACDS complex, and contains an A-cluster composed of an [Fe(4)S(4)] center bridged to a binuclear Ni-Ni site. The electronic structures of these two Ni were studied using L-edge absorption and X-ray magnetic circular dichroism (XMCD) spectroscopy. The L-edge absorption data provided evidence for two distinct Ni species in the as-isolated enzyme, one with low-spin Ni(II) and the other with high-spin Ni(II). XMCD spectroscopy confirmed that the species producing the high-spin signal was paramagnetic. Upon treatment with Ti(3+) citrate, an additional Ni species emerged, which was assigned to Ni(I). By contrast, CO treatment of the reduced enzyme converted nearly all of the Ni in the sample to low-spin Ni(II). The results implicate reaction of a high-spin tetrahedral Ni site with CO to form an enzyme-CO adduct transformed to a low-spin Ni(II) state. These findings are discussed in relation to the mechanism of C-C bond activation, in connection with the model of the beta subunit A-cluster developed from companion Ni and Fe K edge, XANES, and EXAFS studies.     

Genetic construction of truncated and chimeric metalloproteins derived from the alpha subunit of acetyl-CoA synthase from Clostridium thermoaceticum

In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha(2)beta(2) tetrameric 310 kDa bifunctional enzyme. ACS catalyzes the reversible reduction of CO(2) to CO and the synthesis of acetyl-CoA from CO (or CO(2) in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains seven metal-sulfur clusters of four different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kDa beta subunit, while the A-cluster, a Ni-X-Fe(4)S(4) cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kDa alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire beta subunit was removed, as long as CO, rather than CO(2) and a low-potential reductant, was used as a substrate. Truncating an approximately 30 kDa region from the N-terminus of the alpha subunit yielded a 49 kDa protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO-binding properties. Further truncation afforded a 23 kDa protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe(4)S(4)](2+) clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 and 82 kDa fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multicentered metalloenzymes and to generate new complex metalloenzymes with interesting properties.     

Structural models of the bimetallic subunit at the A-cluster of acetyl coenzyme a synthase/CO dehydrogenase: binuclear sulfur-bridged Ni-Cu and Ni-Ni complexes and their reactions with CO

The Ni(II)-dicarboxamido-dithiolato complexes (Et4N)2[Ni(NpPepS)] (1) and (Et4N)2[Ni(PhPepS)] (2) were used as Nid metallosynthons in the construction of higher nuclearity dinuclear Ni-Cu and Ni-Ni species to model the bimetallic Mp-Nid site of the A-cluster of acetyl coenzyme A synthase/CO dehydrogenase (ACS/CODH). Reaction of 1 with [Cu(neo)Cl] and [Ni(terpy)Cl2] in MeCN affords the dinuclear complexes (Et4N)[Cu(neo)Ni(NpPepS)] (3) and [Ni(terpy)Ni(NpPepS)] (4), respectively. Reaction of 2 with [Ni(dppe)Cl2] in MeCN yields [Ni(dppe)Ni(PhPepS)] (6). The Ni-Cu complex 3 exhibits no redox chemistry at the Nid site and no reaction with CO. In contrast, the Nip sites in 4 and 6 are readily reduced (characterized by their Ni(I) EPR spectra) and bind CO, exhibiting nuco bands at 2044 and 1997 cm-1, respectively, indicating terminal CO binding. The present Ni-Ni systems replicate the structural and chemical properties of the A-cluster site in ACS/CODH and support the presence of Ni at Mp in the catalytically active enzyme.     

Mossbauer evidence for an exchange-coupled {[Fe4S4]1+ Nip1+} A-cluster in isolated alpha subunits of acetyl-coenzyme A synthase/carbon monoxide dehydrogenase

The active site A-cluster in the alpha subunit of the title enzyme consists of an Fe4S4 cluster coordinated to a [Nip Nid] subcomponent. The cluster must be activated for catalysis using low-potential reductants such as Ti(III) citrate. Relative to the inactive {[Fe4S4]2+ Nip2+ Nid2+} state, the activated state appears to be 2-electrons more reduced, but the location of these electrons within the A-cluster is uncertain, with {[Fe4S4]2+ Nip0 Nid2+} and {[Fe4S4]1+ Nip1+ Nid2+} configurations proposed. Recombinant apo-alpha subunits oligomerize after activation with NiCl2. The dimer fraction, upon reduction with excess Ti(III)citrate, exhibited M?ssbauer spectra consisting of two quadrupole doublets representing 51% and 21% of the Fe, with parameters indicating [Fe4S4]1+ states. Spectra recorded in strong magnetic fields were typical of diamagnetic systems, indicating an exchange-coupled S = 0 {[Fe4S4]1+ Nip1+} state. Additional treatment with CO altered the doublet M?ssbauer parameters, suggesting an interaction with CO, but maintaining the cluster in the {[Fe4S4]1+ Nip1+} state. Reduction with substoichiometric equivalents of Ti(III) citrate afforded an EPR signal typical of Ni1+ ions, with g parallel = 2.10 and g perpendicular = 2.02. Addition of more Ti caused the signal intensity to decline, suggesting that it arises from the semireduced {[Fe4S4]2+ Nip1+} state.     

Probing the role of the bridging C509 between the [Fe4S4] cubane and the [Ni(p)Ni(d)] centre in the A-cluster of acetyl-coenzyme A synthase

The A-cluster of acetyl-coenzyme A synthase consists of an [Fe(4)S(4)] cubane bridged to a [Ni(p)Ni(d)] centre via C509 cysteinate. The bridging cysteinate, which could be substituted by histidine imidazole, mediates "communication" between the [Fe(4)S(4)] cubane and the [Ni(p)Ni(d)] centre during the synthesis of acetyl-coenzyme A.     

Helix-loop-helix peptides as scaffolds for the construction of bridged metal assemblies in proteins: the spectroscopic A-cluster structure in carbon monoxide dehydrogenase

Four helix-loop-helix 63mer peptides were designed and synthesized in order to assess the utility of peptides as scaffolds for the stabilization of complex metal sites in proteins. Bridged assembly [Ni(II)-(mu(2)-S.Cys)-Fe(4)S(4)], consistent with spectroscopic information on the A-cluster of carbon monoxide dehydrogenase, was chosen as the target assembly. The peptides consist of two helices with approximately 20 residues connected by a flexible loop containing the ferredoxin consensus sequence Cys-Ile-Ala-Cys-Gly-Ala-Cys to bind the Fe(4)S(4) cluster. A fourth cysteine was positioned to serve as the bridging ligand between the cluster and Ni(II). Three other binding residues were incorporated in appropriate positions to constitute a binding site for Ni(II). One of the peptides was designed with an N(3)S (His(3)Cys) site, and each of the other three with N(2)S(2) (His(2)Cys(2)) sites. A detailed account of the synthesis and characterization of the peptides and their metalloderivatives is presented. The four peptides were synthesized using an Fmoc/t-Bu-based solid-phase strategy, purified by reversed-phase HPLC, and characterized by ES-MS. On the basis of size-exclusion chromatography and circular dichroism spectropolarimetry, these peptides appear to dimerize in solution to form four-helix bundles of high helical contents. Reactions of the peptides with preformed cluster [Fe(4)S(4)(SCH(2)CH(2)OH)(4)](2)(-) and subsequent purification by column chromatography yield a product consistent with the incorporation of one [Fe(4)S(4)](2+) cluster per 63mer, as judged from absorption and M?ssbauer spectra. Addition of a Ni(II) salt to the [Fe(4)S(4)]-peptides results in an apparent equilibrium between free Ni(II) and a peptide-bound nickel form, as established by column chromatography studies. Nickel EXAFS data (Musgrave, K. B.; Laplaza, C. E.; Holm, R. H.; Hedman, B.; Hodgson, K. O. Results to be published.) provide strong evidence that the peptide-bound nickel binds in the desired site in two of the metallopeptides. This work represents the first exploration of peptides as scaffolds for the support of biologically relevant bridged assemblies containing iron-sulfur clusters.     

Structural characterization of metallopeptides designed as scaffolds for the stabilization of nickel(II)-Fe(4)S(4) bridged assemblies by X-ray absorption spectroscopy

In earlier work, de novo designed peptides with a helix-loop-helix motif and 63 residues have been synthesized as potential scaffolds for stabilization of the [Ni(II)-X-Fe(4)S(4)] bridged assembly that is the spectroscopically deduced structure of the A-Cluster in clostridial carbon monoxide dehydrogenase. The 63mers contain a consensus tricysteinyl ferredoxin domain in the loop for binding an Fe(4)S(4) cluster and Cys and His residues proximate to the loop for binding Ni(II), with one Cys residue designed as the bridge X. The metallopeptides HC(4)H(2)-[Fe(4)S(4)]-Ni and HC(5)H-[Fe(4)S(4)]-M, containing three His and one Cys residue for Ni(II) coordination and two His and two Cys residues for binding M = Ni(II) and Co(II), have been examined by Fe-, Ni-, and Co-K edge spectroscopy and EXAFS. All peptides bind an [Fe(4)S(4)](2+) cubane-type cluster. Interpretation of the Ni and Co data is complicated by the presence of a minority population of six-coordinate species with low Z ligands, designated for simplicity as [M(OH(2))(6)](2+). Best fits of the data were obtained with ca. 20% [M(OH(2))(6)](2+) and ca. 80% M(II) with mixed N/S coordination. The collective XAS results for HC(4)H(2)-[Fe(4)S(4)]-Ni and HC(5)H-[Fe(4)S(4)]-M demonstrate the presence of an Fe(4)S(4) cluster and support the existence of the distorted square-planar coordination units [Ni(II)(S.Cys)(N.His)(3)] and [Ni(II)(S.Cys)(2)(N.His)(2)] in the HC(4)H(2) and HC(5)H metallopeptides, respectively. In the HC(5)H metallopeptide, tetrahedral [Co(II)(S.Cys)(2)(N.His)(2)] is present. We conclude that the designed scaffolded binding sites, including Ni-(mu(2)-S.Cys)-Fe bridges, have been achieved. This is the first XAS study of a de novo designed metallopeptide intended to stabilize a bridged biological assembly, and one of a few XAS analyses of metal derivatives of designed peptides. The scaffolding concept should be extendable to other bridged metal assemblies.     

Synthetic analogues of the active site of the A-cluster of acetyl coenzyme A synthase/CO dehydrogenase: syntheses, structures, and reactions with CO

Two metallosynthons, namely (Et4N)2[Ni(NpPepS)] (1) and (Et4N)2[Ni(PhPepS)] (2) containing carboxamido-N and thiolato-S as donors have been used to model the bimetallic M(p)-Ni(d) subsite of the A-cluster of the enzyme acetyl coenzyme A synthase/CO dehydrogenase. A series of sulfur-bridged Ni/Cu dinuclear and trinuclear complexes (3-10) have been synthesized to explore their redox properties and affinity of the metal centers toward CO. The structures of (Et4N)2[Ni(PhPepS)] (2), (Et4N)[Cu(neo)Ni(NpPepS)] x 0.5 Et2O x 0.5 H2O (3 x 0.5 Et2O x 0.5 H2O), (Et4N)[Cu(neo)Ni(PhPepS)] x H2O (4 x H2O), (Et4N)2[Ni{Ni(NpPepS)}2] x DMF (5 x DMF), (Et4N)2[Ni(DMF)2{Ni(NpPepS)}2] x 3 DMF (6 x 3 DMF), (Et4N)2[Ni(DMF)2{Ni(PhPepS)}2] (8), and [Ni(dppe)Ni(PhPepS)] x CH2Cl2 (10 x CH2Cl2) have been determined by crystallography. The Ni(d) mimics 1 and 2 resist reduction and exhibit no affinity toward CO. In contrast, the sulfur-bridged Ni center (designated Ni(C)) in the trinuclear models 5-8 are amenable to reduction and binds CO in the Ni(I) state. Also, the sulfur-bridged Ni(C) center can be removed from the trimers (5-8) by treatment with 1,10-phenanthroline much like the "labile Ni" from the enzyme. The dinuclear Ni-Ni models 9 and 10 resemble the Ni(p)-Ni(d) subsite of the A-cluster more closely, and only the modeled Ni(p) site of the dimers can be reduced. The Ni(I)-Ni(II) species display EPR spectra typical of a Ni(I) center in distorted trigonal bipyramidal and distorted tetrahedral geometries for 9(red) and 10(red), respectively. Both species bind CO, and the CO-adducts 9(red)-CO and 10(red)-CO display strong nu(co) at 2044 and 1997 cm(-1), respectively. The reduction of 10 is reversible. The CO-affinity of 10 in the reduced state and the nu(co) value of 10(red)-CO closely resemble the CO-bound reduced A-cluster (nu(co) = 1996 cm(-1)).     

Stopped-Flow Kinetics of Methyl Group Transfer between the Corrinoid-Iron-Sulfur Protein and Acetyl-Coenzyme A Synthase from Clostridium thermoaceticum

Kinetics of methyl group transfer between the Ni-Fe-S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the stopped-flow method at 390 nm. Rates of the reaction CH(3)-Co(3+)FeSP + ACS(red) <==> Co(1+)FeSP + CH(3)-ACS(ox) in both forward and reverse directions were determined using various protein and reductant concentrations. Ti(3+)citrate, dithionite, and CO were used to reductively activate ACS (forming ACS(red)). The simplest mechanism that adequately fit the data involved formation of a [CH(3)-Co(3+)FeSP]:[ACS(red)] complex, methyl group transfer (forming [Co(1+)FeSP]:[CH(3)-ACS(ox)]), product dissociation (forming Co(1+)FeSP + CH(3)-ACS(ox)), and CO binding yielding a nonproductive enzyme state (ACS(red) + CO <==> ACS(red)-CO). Best-fit rate constants were obtained. CO inhibited methyl group transfer by binding ACS(red) in accordance with K(D) = 180 +/- 90 microM. Fits were unimproved when >1 CO was assumed to bind. Ti(3+)citrate and dithionite inhibited the reverse methyl group transfer reaction, probably by reducing the D-site of CH(3)-ACS(ox). This redox site is oxidized by 2e(-) when the methyl cation is transferred from CH(3)-Co(3+)FeSP to ACS(red), and is reduced during the reverse reaction. Best-fit K(D) values for pre- and post-methyl-transfer complexes were 0.12 +/- 0.06 and 0.3 +/- 0.2 microM, respectively. Intracomplex methyl group transfer was reversible with K(eq) = 2.3 +/- 0.9 (k(f)/k(r) = 6.9 s(-1)/3.0 s(-1)). The nucleophilicity of the [Ni(2+)D(red)] unit appears comparable to that of Co(1+) cobalamins. Reduction of the D-site may cause the Ni(2+) of the A-cluster to behave like the Ni of an organometallic Ni(0) complex.     

High-nuclearity metal-cyanide clusters: synthesis,magnetic properties,and inclusion behavior of open-cage species incorporating [(tach)M(CN)3] (M = Cr,Fe, Co) complexes

The use of 1,3,5-triaminocyclohexane (tach) as a capping ligand in generating metal-cyanide cage clusters with accessible cavities is demonstrated. The precursor complexes [(tach)M(CN)(3)] (M = Cr, Fe, Co) are synthesized by methods similar to those employed in preparing the analogous 1,4,7-triazacyclononane (tacn) complexes. Along with [(tach)Fe(CN)(3)](1)(-), the latter two species are found to adopt low-spin electron configurations. Assembly reactions between [(tach)M(CN)(3)] (M = Fe, Co) and [M'(H(2)O)(6)](2+) (M' = Ni, Co) in aqueous solution afford the clusters [(tach)(4)(H(2)O)(12)Ni(4)Co(4)(CN)(12)](8+), [(tach)(4)(H(2)O)(12)Co(8)(CN)(12)](8+), and [(tach)(4)(H(2)O)(12)Ni(4)Fe(4)(CN)(12)](8+), each possessing a cubic arrangement of eight metal ions linked through edge-spanning cyanide bridges. This geometry is stabilized by hydrogen-bonding interactions between tach and water ligands through an intervening solvate water molecule or bromide counteranion. The magnetic behavior of the Ni(4)Fe(4) cluster indicates weak ferromagnetic coupling (J = 5.5 cm(-)(1)) between the Ni(II) and Fe(III) centers, leading to an S = 6 ground state. Solutions containing [(tach)Fe(CN)(3)] and a large excess of [Ni(H(2)O)(6)](2+) instead yield a trigonal pyramidal [(tach)(H(2)O)(15)Ni(3)Fe(CN)(3)](6+) cluster, in which even weaker ferromagnetic coupling (J = 1.2 cm(-)(1)) gives rise to an S = (7)/(2) ground state. Paralleling reactions previously performed with [(Me(3)tacn)Cr(CN)(3)], [(tach)Cr(CN)(3)] reacts with [Ni(H(2)O)(6)](2+) in aqueous solution to produce [(tach)(8)Cr(8)Ni(6)(CN)(24)](12+), featuring a structure based on a cube of Cr(III) ions with each face centered by a square planar [Ni(CN)(4)](2)(-) unit. The metal-cyanide cage differs somewhat from that of the analogous Me(3)tacn-ligated cluster, however, in that it is distorted via compression along a body diagonal of the cube. Additionally, the compact tach capping ligands do not hinder access to the sizable interior cavity of the molecule, permitting host-guest chemistry. Mass spectrometry experiments indicate a 1:1 association of the intact cluster with tetrahydrofuran (THF) in aqueous solution, and a crystal structure shows the THF molecule to be suspended in the middle of the cluster cavity. Addition of THF to an aqueous solution containing [(tach)Co(CN)(3)] and [Cu(H(2)O)(6)](2+) templates the formation of a closely related cluster, [(tach)(8)(H(2)O)(6)Cu(6)Co(8)(CN)(24) superset THF](12+), in which paramagnetic Cu(II) ions with square pyramidal coordination are situated on the face-centering sites. Reactions intended to produce the cubic [(tach)(4)(H(2)O)(12)Co(8)(CN)(12)](8+) cluster frequently led to an isomeric two-dimensional framework, [(tach)(H(2)O)(3)Co(2)(CN)(3)](2+), exhibiting mer rather than fac stereochemistry at the [Co(H(2)O)(3)](2+) subunits. Attempts to assemble larger edge-bridged cubic clusters by reacting [(tach)Cr(CN)(3)] with [Ni(cyclam)](2+) (cyclam = 1,4,8,11-tetraazacyclotetradecane) complexes instead generated extended one- or two-dimensional solids. The magnetic properties of one of these solids, two-dimensional [(tach)(2)(cyclam)(3)Ni(3)Cr(2)(CN)(6)]I(2), suggest metamagnetic behavior, with ferromagnetic intralayer coupling and weak antiferromagnetic interactions between layers.     

Reduction and methyl transfer kinetics of the alpha subunit from acetyl coenzyme a synthase

Stopped-flow was used to evaluate the methylation and reduction kinetics of the isolated alpha subunit of acetyl-Coenzyme A synthase from Moorella thermoacetica. This catalytically active subunit contains a novel Ni-X-Fe4S4 cluster and a putative unidentified n = 2 redox site called D. The D-site must be reduced for a methyl group to transfer from a corrinoid-iron-sulfur protein, a key step in the catalytic synthesis of acetyl-CoA. The Fe4S4 component of this cluster is also redox active, raising the possibility that it is the D-site or a portion thereof. Results presented demonstrate that the D-site reduces far faster than the Fe4S4 component, effectively eliminating this possibility. Rather, this component may alter catalytically important properties of the Ni center. The D-site is reduced through a pathway that probably does not involve the Fe4S4 component of this active-site cluster.     

On [Fe4S4]2+ -(mu2-SR)-M II bridge formation in the synthesis of an A-cluster analogue of carbon monoxide dehydrogenase/acetylcoenzyme A synthase

The construction of a synthetic analogue of the A-cluster of carbon monoxide dehydrogenase/acetylcoenzyme synthase, the site of acetylcoenzyme A formation, requires as a final step the formation of an unsupported [Fe(4)S(4)]-(mu(2)-SR)-Ni(II) bridge to a preformed cluster. Our previous results (Rao, P. V.; Bhaduri, S.; Jiang, J.; Holm, R. H. Inorg. Chem. 2004, 43, 5833) and the work of others have addressed synthesis of dinuclear complexes relevant to the A-cluster. This investigation concentrates on reactions pertinent to bridge formation by examining systems containing dinuclear and mononuclear Ni(II) complexes and the 3:1 site-differentiated clusters [Fe(4)S(4)(LS(3))L'](2-) (L' = TfO(-) (14), SEt (15)). The system 14/[{Ni(L(O)-S(2)N(2))}M(SCH(2)CH(2)PPh(2))](+) results in cleavage of the dinuclear complex and formation of [{Ni(L(O)-S(2)N(2))}Fe(4)S(4)(LS(3))]- (18), in which the Ni(II) complex binds at the unique cluster site with formation of a Ni(mu(2)-SR)(2)Fe bridge rhomb. Cluster 18 and the related species [{Ni(phma)}Fe(4)S(4)(LS(3))](3)- (19) are obtainable by direct reaction of the corresponding cis-planar Ni(II)-S(2)N(2) complexes with 14. The mononuclear complexes [M(pdmt)(SEt)]- (M = Ni(II), Pd(II)) with 14 in acetonitrile or Me(2)SO solution react by thiolate transfer to give 15 and [M(2)(pdmt)(2)]. However, in dichloromethane the Ni(II) reaction product is interpreted as [{Ni(pdmt)(mu(2)-SEt)}Fe(4)S(4)(LS(3))](2-) (20). Reaction of Et(3)NH(+) and 15 affords the double cubane [{Fe(4)S(4)(LS(3))}(2)(mu(2)-SEt)](3-) (21). Cluster 18 contains two mutually supportive Fe-(mu(2)-SR)-Ni(II) bridges, 19 exhibits one strong and one weaker bridge, 20 has one unsupported bridge (inferred from the (1)H NMR spectrum), and 21 has one unsupported Fe-(mu(2)-SR)-Fe bridge. Bridges in 18, 19, and 21 were established by X-ray structures. This work demonstrates that a bridge of the type found in the enzyme A-clusters is achievable by synthesis and implies that more stable, unsupported single thiolate bridges may require reinforcement by an additional covalent linkage between the Fe(4)S(4) and nickel-containing components. (LS(3) = 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-); L(O)-S(2)N(2) = N,N'-diethyl-3,7-diazanonane-1,9-dithiolate(2-); pdmt = pyridine-2,6-methanedithiolate(2-); phma = N,N'-1,2-phenylenebis(2-acetylthio)acetamidate(4-); TfO = triflate.).     

Geometric and electrostatic study of the [4Fe-4S] cluster of adenosine-5'-phosphosulfate reductase from broken symmetry density functional calculations and extended X-ray absorption fine structure spectroscopy

Adenosine-5'-phosphosulfate reductase (APSR) is an iron-sulfur protein that catalyzes the reduction of adenosine-5'-phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X(~80)-CXXC motif, and the cluster is essential for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the iron-sulfur cluster in APS reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe-4S](2+) state in both native and substrate-bound APSR while EXAFS data recorded at ~0.1 ? resolution indicates that there is no significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On the other hand, DFT calculations provide an insight into the subtle differences between the geometry of the cluster in the native and APS-bound forms of APSR. A comparison between models with and without the tandem cysteine pair coordination of the cluster suggests a role for the unique coordination in facilitating a compact geometric structure and "fine-tuning" the electronic structure to prevent reduction of the cluster. Further, calculations using models in which residue Lys144 is mutated to Ala confirm the finding that Lys144 serves as a crucial link in the interactions involving the [4Fe-4S] cluster and APS.     

Carbon monoxide induced decomposition of the active site [Ni-4Fe-5S] cluster of CO dehydrogenase

During the past two years, crystal structures of Cu- and Mo-containing carbon monoxide dehydrogenases (CODHs) and Ni- and Fe-containing CODHs have been reported. The active site of CODHs from anaerobic bacteria (cluster C) is composed of Ni, Fe, and S for which crystallographic studies of the enzymes from Carboxydothermus hydrogenoformans, Rhodospirillum rubrum, and Moorella thermoaceticarevealed structural similarities in the overall protein fold but showed substantial differences in the essential Ni coordination environment. The [Ni-4Fe-5S] cluster C in the fully catalytically competent dithionite-reduced CODH II from C. hydrogenoformans (CODHII(Ch)) at 1.6 A resolution contains a characteristic mu(2)-sulfido ligand between Ni and Fe1, resulting in a square-planar ligand arrangement with four S-ligands at the Ni ion. In contrast, the [Ni-4Fe-4S] clusters C in CO-treated CODH from R. rubrum resolved at 2.8 A and in CO-treated acetyl-CoA synthase/CODH complex from M. thermoacetica at 2.2 and 1.9 A resolution, respectively, do not contain the mu(2)-sulfido ligand between Ni and Fe1 and display dissimilar geometries at the Ni ion. The [Ni-4Fe-4S] cluster is composed of a cubane [Ni-3Fe-4S] cluster linked to a mononuclear Fe site. The described coordination geometries of the Ni ion in the [Ni-4Fe-4S] cluster of R. rubrum and M. thermoacetica deviate from the square-planar ligand geometry in the [Ni-4Fe-5S] cluster C of CODHII(Ch). In addition, the latter was converted into a [Ni-4Fe-4S] cluster under specific conditions. The objective of this study was to elucidate the relationship between the structure of cluster C in CODHII(Ch) and the functionality of the protein. We have determined the CO oxidation activity of CODHII(Ch) under different conditions of crystallization, prepared crystals of the enzyme in the presence of dithiothreitol or dithionite as reducing agents under an atmosphere of N(2) or CO, and solved the corresponding structures at 1.1 to 1.6 A resolutions. Fully active CODHII(Ch) obtained after incubation of the enzyme with dithionite under N(2) revealed the [Ni-4Fe-5S] cluster. Short treatment of the enzyme with CO in the presence of dithiothreitol resulted in a catalytically competent CODHII(Ch) with a CO-reduced [Ni-4Fe-5S] cluster, but a prolonged treatment with CO caused the loss of CO-oxidizing activity and revealed a [Ni-4Fe-4S] cluster, which did not contain a mu(2)-S. These data suggest that the [Ni-4Fe-4S] cluster of CODHII(Ch) is an inactivated decomposition product originating from the [Ni-4Fe-5S] cluster.     

Synthesis, crystal structures and magnetic properties of cyanide- and phenolate-bridged [M(III)NiII]2 tetranuclear complexes (M=Fe and Cr)

The binuclear complex NiII2L(H2O)2(ClO4)2(1) and the neutral tetranuclear bimetallic compounds [{M(III)(phen)(CN)4}2{NiII2L(H2O)2}].2CH3CN with M=Fe (2) and Cr (3)[H2L=11,23-dimethyl-3,7,15,19-tetraazatricyclo[19.3.1.1(9,13)]hexacosa-2,7,9,11,13(26),14,19,21(25),22,24-decaene-25,26-diol] have been synthesized and the structures of and determined by single crystal X-ray diffraction. and are isostructural compounds whose structure is made up of centrosymmetric binuclear cations [Ni2(L)(H2O)2]2+ and two peripheral [M(phen)(CN)4]- anions [M=Fe (2) and Cr (3)] acting as monodentate ligands towards the nickel atoms through one of their four cyanide nitrogen atoms. The environment of the metal atoms in 2 and 3 is six-coordinated: two phen-nitrogen and four cyanide-carbon atoms at the iron and chromium atoms and a water molecule, one cyanide-nitrogen and two phenolate-oxygens and two imine-nitrogens from the binucleating ligand L2- at the nickel atom build distorted octahedral surroundings. The values of the FeNi and CrNi separations through the single cyanide bridge are 5.058(1) and 5.174(2)A respectively, whereas the Ni-Ni distances across the double phenolate bridge are 3.098(2)(2) and 3.101(1) A (3). The magnetic properties of have been investigated in the temperature range 1.9-290 K. The magnetic behaviour of corresponds to that of an antiferromagnetically coupled nickel(II) dimer with J=-61.0(1) cm-1, the Hamiltonian being defined as H=-J S(A).S(B). An overall antiferromagnetic behaviour is observed for and with a low-lying singlet spin state. The values of the intramolecular magnetic couplings are J(Fe-Ni)=+17.4(1) cm-1 and J(Ni-Ni(a))=-44.4(1) cm-1 for and J(Cr-Ni)=+11.8(1) cm-1 and J(Ni-Ni(a))=-44.6(1) cm-1 for [H=-J(M-Ni)(S(M).S(Ni)+S(Ma).S(Nia))-J(Ni-Nia)S(Ni)S(Nia)]. Theoretical calculations using methods based on density functional theory (DFT) have been employed on in order to analyze the efficiency of the exchange pathways involved and also to substantiate the exchange coupling parameters.     

Structures and energetics of models for the active site of acetyl-coenzyme a synthase: role of distal and proximal metals in catalysis

Acetyl-coenzyme A (CoA) synthase/carbon monoxide dehydrogenase (ACS/CODH) is a bifunctional enzyme that generates CO from carbon dioxide in the C-cluster of the beta subunit and synthesizes acetyl-CoA from carbon monoxide (CO), CoA, and CH3+ at the active site of the A-cluster in the alpha subunit. On the basis of density functional calculations, we predict that methylation of Nip occurs first, and CO then adds to the NipII-CH3 species to form the intermediate, NipII(CO)(CH3), in which Nip deligates one of its SNid bonds. The CO-insertion/CH3-migration occurs on one metal, the proximal Ni, forming the trigonal planar NipII-acetyl intermediate. The thiolate can bind to NipII and reductively eliminate the thioester. Our calculations disfavor the unprecedented bimetallic CO-insertion/CH3-migration. Ni in the proximal site produces a better catalyst than does Cu.     

A Synthetic Pathway to Cys-Xxx-Cys (N2S2) Analogue Ligands: An Improved Synthesis of HSCH2CH2C(O)NHCH2C(O)NHCH2CH2SH

Herein, two improved synthetic pathways to biologically relevant Cys-Xxx-Cys analogue ligands used in conjunction with metals as metalloenzyme models (Ni for carbon monoxide dehydrogenase/acetyl-CoA synthase A-cluster; Co and Fe for nitrile hydratase) are reported.     

Synthesis and Characterization of Four Consecutive Members of the Five-Member [Fe(6)S(8)(PEt(3))(6)](n)()(+) (n = 0-4) Cluster Electron Transfer Series

The clusters [Fe(6)S(8)(PEt(3))(6)](+,2+) have been shown by other investigators to be formed by the reaction of [Fe(OH(2))(6)](2+) and H(2)S, to contain face-capped octahedral Fe(6)S(8) cores, and to be components of the five-membered electron transfer series [Fe(6)S(8)(PEt(3))(6)](n)()(+) (n = 0-4) estalished electrochemically. We have prepared two additional series members. Reaction of [Fe(6)S(8)(PEt(3))(6)](2+) with iodine in dichloromethane affords [Fe(6)S(8)(PEt(3))(6)](3+), isolated as the perchlorate salt (48%). Reduction of [Fe(6)S(8)(PEt(3))(6)](2+) with Na(Ph(2)CO) in acetonitrile/THF produces the neutral cluster [Fe(6)S(8)(PEt(3))(6)] (65%). The structures of the four clusters with n = 0, 1+, 2+, 3+ were determined at 223 K. The compounds [Fe(6)S(8)(PEt(3))(6)](ClO(4))(3), [Fe(6)S(8)(PEt(3))(6)] crystallize in trigonal space group R&thremacr;c with a = 21.691(4), 16.951(4) ?, c = 23.235(6), 19.369(4) ?, and Z = 6, 3. The compounds [Fe(6)S(8)(PEt(3))(6)](BF(4))(2), [Fe(6)S(8)(PEt(3))(6)](BF(4)).2MeCN were obtained in monoclinic space groups P2(1)/c, C2/c with a = 11.673(3), 16.371(4) ?, b = 20.810(5), 16.796(4) ?, c = 12.438(4), 23.617(7) ?, beta = 96.10(2), 97.98(2) degrees, and Z = 2, 4. [Fe(6)S(8)(PEt(3))(6)](BPh(4))(2) occurred in trigonal space group P&onemacr; with a = 11.792(4) ?, b = 14.350(5) ?, c = 15.536(6) ?, alpha = 115.33(3) degrees, beta = 90.34(3) degrees, gamma = 104.49(3) degrees, and Z = 1. Changes in metric features across the series are slight but indicate increasing population of antibonding Fe(6)S(8) core orbitals upon reduction. Zero-field M?ssbauer spectra are consistent with this result, isomer shifts increasing by ca. 0.05 mm/s for each electron added, and indicate a delocalized electronic structure. Magnetic susceptibility measurements together with previously reported results established the ground states S = (3)/(2) (3+), 3 (2+), (7)/(2) (1+), 3 (0). The clusters [Fe(6)S(8)(PEt(3))(6)](n)()(+) possess the structural and electronic features requisite to multisequential electron transfer reactions. This work provides the first example of a cluster type isolated over four consecutive oxidation states. Note is also made of the significance of the [Fe(6)S(8)(PEt(3))(6)](n)()(+) cluster type in the development of iron-sulfur-phosphine cluster chemistry.     

Structural studies of Na-montmorillonite exchanged with Fe2+, Cr3+, and Ti4+ by N2 adsorption and EXAFS

The structures of Fe(2+)-, Cr(3+)-, and Ti(4+)-modified montmorillonite prepared from ion exchange of the Na-clay with Fe(2+), Cr(3+), and Ti(4+) were investigated. Conventional BET surface area and spectroscopic analysis by extended adsorption fine structure (EXAFS) were applied. It was shown that the BET surface area of Na-clay was similar to that of Fe-clay, but somewhat different from those of Cr- and Ti-clay; it decreased in the order Na- > Fe- > Ti- > Cr-montmorillonite. This sequence appeared to be consistent with the ion size Na(+) (0.95 nm)>Fe(2+) (0.65 nm)>Cr(3+) (0.62 nm), except for Ti(4+) (0.69 nm). EXAFS data showed that some Si atoms within montmorillonite were replaced by Ti atoms and that a neostructure of titanium oxide was formed.     

Cyano-bridged homometallic 1-D and bimetallic 2-D assemblies: synthesis,structures, and magnetic properties of [Ni(baepn)(CN)]n(ClO4)n and [Ni(baepn)]2n[Fe(CN)(6)]n(H2O)8n

Cyano-bridged homometallic complex [Ni(baepn)(CN)](n)(ClO(4))(n)(1) and bimetallic complex [Ni(baepn)](2)(n)[Fe(CN)(6)](n)(H(2)O)(8)(n)(2) [baepn = N,N'-bis(2-aminoethyl)-1,3-propanediamine] were synthesized and characterized. 1 crystallizes in the monoclinic space group P2(1)/n with a = 9.560(3) A, b = 10.700(3) A, c = 14.138(9) A, beta = 90.18(6) degrees, and Z = 4; 2 crystallizes in the monoclinic space group P2(1)/c with a = 8.951(2) A, b = 13.672(3) A, c = 14.392(3) A, beta = 98.906(4) degrees, and Z = 4. The complex 1 has one-dimensional structure whose chain vector runs along the b axis with baepn ligands and perchlorate anions alternately arranged up and down in the c direction. The antiferromagnetic nature of 1 was explained in terms of the infinite chain model and Haldane gap, giving g = 2.33, J = -29.4 cm(-1), and the magnitude of Haldane gap E(g) = 5.22 K. The complex 2 that constitutes the first example of 2-D bimetallic assembly of Ni(II) ion and ferrocyanide anion is composed of the neutral layers based on the [Ni(4)Fe(4)] square grid spanning in the bc plane. For 2, the analysis with the Curie-Weiss law in 2-300 K range results in THETA = 0.200 K and the magnetism was explained in terms of the ability of ferrocyanide in the -Ni-NC-Fe-CN-Ni unit to promote ferromagnetic Ni-Ni interaction.