Structural and functional models of the active site of zinc phosphotriesterase

In an attempt to prepare structural and functional models for the active site of the hydrolytic enzyme zinc phosphotriesterase, five new zinc complexes of the ligands 2,6-bis[N-(N-(carboxylmethyl)-N-((1-methylimidazol)methyl)amine)methyl]-4-methylphenolate (BCIMP) and the corresponding asymmetric ligand 2-(N-isopropyl-N-((1-methylimidazolyl)methyl)aminomethyl)-6-(N-carboxylmethyl-N-((1-methylimidazolyl)methyl)aminomethyl)-4-methylphenol (ICIMP) have been synthesized, viz. Na[Zn(2)(BCIMP)Ac(2)] (1), [Zn(2)(BCIMP)(Ph(2)Ac)] (2), [Zn(2)(ICIMP)Ac(2)] (3), [Zn(4)(ICIMP)(2)(Me(3)Ac)(2)][ClO(4)](2) (4), and [Zn(4)(ICIMP)(2)(Ph(2)Ac)(2)][ClO(4)](2) (5). The X-ray structure of complex 5 has been determined and reveals that the complex is a dimer of dimers in the solid state, which in solution dissociates to potent structural models. Studies using NMR show that only one carboxylate coligand bridges the dizinc units in the case of diphenyl acetate and pivalate, while the steric bulk of acetate is sufficiently small to permit the coordination of two acetates/dizinc unit. Functional studies involving the hydrolysis/transesterification of 2-hydroxypropyl p-nitrophenyl phosphate (HPNP) show that the complex with ICIMP (compound 5) has a significantly higher rate of catalysis than the BCIMP complex (compound 2). This is attributed to the vacant/labile coordination site that is available in the ICIMP complex but not the BCIMP complex.     

Electronic state of the dimethyl sulfoxide reductase active site

Computations suggest that in contrast with small models the active site geometry of reduced dimethyl sulfoxide reductase might prefer a triplet over a singlet electronic state.     

Synthetic models of the reduced active site of superoxide reductase

We report the synthesis, structural and spectroscopic characterization, and magnetic and electrochemical studies of a series of iron(II) complexes of the pyridyl-appended diazacyclooctane ligand L(8)py(2), including several that model the square-pyramidal [Fe(II)(N(his))(4)(S(cys))] structure of the reduced active site of the non-heme iron enzyme superoxide reductase. Combination of L(8)py(2) with FeCl(2) provides [L(8)py(2)FeCl(2)] (1), which contains a trigonal-prismatic hexacoordinate iron(II) center, whereas a parallel reaction using [Fe(H(2)O)(6)](BF(4))(2) provides [L(8)py(2)Fe(FBF(3))]BF(4) (2), a novel BF(4)(-)-ligated square-pyramidal iron(II) complex. Substitution of the BF(4)(-) ligand in 2 with formate or acetate ions affords distorted pentacoordinate [L(8)py(2)Fe(O(2)CH)]BF(4) (3) and [L(8)py(2)Fe(O(2)CCH(3))]BF(4) (4), respectively. Models of the superoxide reductase active site are prepared upon reaction of 2 with sodium salts of aromatic and aliphatic thiolates. These model complexes include [L(8)py(2)Fe(SC(6)H(4)-p-CH(3))]BF(4) (5), [L(8)py(2)Fe(SC(6)H(4)-m-CH(3))]BF(4) (6), and [L(8)py(2)Fe(SC(6)H(11))]BF(4) (7). X-ray crystallographic studies confirm that the iron(II)-thiolate complexes model the square-pyramidal geometry and N(4)S donor set of the reduced active site of superoxide reductase. The iron(II)-thiolate complexes are high spin (S = 2), and their solutions are yellow in color because of multiple charge-transfer transitions that occur between 300 and 425 nm. The ambient temperature cyclic voltammograms of the iron(II)-thiolate complexes contain irreversible oxidation waves with anodic peak potentials that correlate with the relative electron donating abilities of the thiolate ligands. This electrochemical irreversibility is attributed to the bimolecular generation of disulfides from the electrochemically generated iron(III)-thiolate species.     

Dihydrogen activation by a diruthenium analogue of the Fe-only hydrogenase active site

The photochemical reaction of Ru2(S2C3H6)(CO)4(PCy3)2 (1) and H2 gives the dihydride Ru2(S2C3H6)(mu-H)(H)(CO)3(PCy3)2 (2). NMR and crystallographic studies reveal mutually trans basal phosphine ligands and both bridging and terminal hydrides. Ru2(S2C2H4)(CO)4(PCy3)2 behaves similarly. Other HX substrates undergo photoaddition to 1, affording Ru2(S2C3H6)(mu-H)(X)(CO)3(PCy3)2 for X = OTs (3a), Cl (3b), and SPh (3c). Treatment of Ru2(S2C3H6)(mu-H)(H)(CO)3(PCy3)2 with [H(OEt2)]BArF4 (ArF = B(C6H3-3,5-(CF3)2) in CD2Cl2 gives [Ru2(S2C3H6)(mu-H)(CO)3(PCy3)2(H2)]+ (4), which catalyzes H2-D2 exchange. The reaction of 2 with [D(OEt2)]BArF4 gave [Ru2(S2C3H6)(mu-H)(CO)3(PCy3)2(HD)]+ (JH-D = 31 Hz). These studies provide the first models for the Fe-only hydrogenases that bear dihydrogen and terminal hydrido ligands.     

Interaction of product analogues with the active site of rhodobacter sphaeroides dimethyl sulfoxide reductase

We report a structural characterization using X-ray absorption spectroscopy of Rhodobacter sphaeroides dimethyl sulfoxide (DMSO) reductase reduced with trimethylarsine and show that this is structurally analogous to the physiologically relevant dimethyl sulfide reduced DMSO reductase. Our data unambiguously indicate that these species should be regarded as formal MoIV species and indicate a classical coordination complex of trimethylarsine oxide, with no special structural distortions. The similarity of the trimethylarsine and dimethyl sulfide complexes suggests, in turn, that the dimethyl sulfide reduced enzyme possesses a classical coordination of DMSO with no special elongation of the S-O bond, as previously suggested.     

Synthesis and incorporation into DNA of a chemically stable, functional abasic site analogue

[reaction: see text] The abasic site building block 7 for DNA synthesis, containing a methylenephosphinic acid group at C3', was prepared in six steps and was incorporated into DNA via a combination of H-phosphonate and phosphoramidite chemistry. Corresponding oligodeoxynucleotides were shown to be chemically stable under basic conditions and fully functional at the respective hemiacetal center.     

Identification of iron(III) peroxo species in the active site of the superoxide reductase SOR from Desulfoarculus baarsii

The active site of superoxide reductase SOR consists of an Fe2+ center in an unusual [His4 Cys1] square-pyramidal geometry. It specifically reduces superoxide to produce H2O2. Here, we have reacted the SOR from Desulfoarculus baarsii directly with H2O2. We have found that its active site can transiently stabilize an Fe3+-peroxo species that we have spectroscopically characterized by resonance Raman. The mutation of the strictly conserved Glu47 into alanine results in a stabilization of this Fe3+-peroxo species, when compared to the wild-type form. These data support the hypothesis that the reaction of SOR proceeds through such a Fe3+-peroxo intermediate. This also suggests that Glu47 might serve to help H2O2 release during the reaction with superoxide.     

High-energy intermediate or stable transition state analogue: theoretical perspective of the active site and mechanism of beta-phosphoglucomutase

A recent crystal structure of beta-phosphoglucomutase from Lactococcus lactis is reported to contain a five-coordinate phosphorus with five oxygen ligands that is a high-energy reaction intermediate during the phosphoryl transfer in the isomerization of beta-glucose 1-phosphate to beta-glucose 6-phosphate. Subsequently, it has been suggested that this structure is a transition state analogue with a five-coordinate magnesium with two oxygen and three fluorine ligands. Two layer ONIOM(B3LYP:PM3MM) calculations have been performed to address the nature of this intermediate and the mechanism of the phosphoryl transfer. These calculations provide evidence that (1) the observed crystal structure is consistent with a five-coordinate magnesium (a stable transition state analogue), not a five-coordinate phosphorus (a phosphorane) as a high-energy intermediate, (2) the active site is stabilized by the extensive hydrogen-bonding network, (3) the transfer of the phosphoryl group proceeds through a moderate barrier (14 kcal mol-1) five-coordinate phosphorus without a stable phosphorane or metaphosphate intermediate, (4) this concerted transition state is directly coupled to a proton transfer from the oxygen of glucose to the carboxylic group of aspartate 10, and (5) a stable glucose 1,6-bis-phosphoglucose intermediate is formed.     

DFT investigation of the interaction between gold(I) complexes and the active site of thioredoxin reductase

The binding of the [Au(PMe₃)]⁺ fragment to cysteine, selenocysteine and the tetrapeptides H₂NGlyCysAGlyCOOH (A = Cys, Sec) has been investigated by DFT methods as a model for the binding of gold(I) to the selenium-containing active site of thioredoxin reductase. The calculations demonstrate both a higher acidity of Se-H compared to S-H and a stronger binding of gold at the selenium site compared to sulphur. Se-H dissociation at the selenium site increases the reducing power of the tetrapeptide H₂NGlyCysSecGlyCOOH whilst gold coordination at selenium has the opposite effect.     

Variable coordination geometries at the diiron(II) active site of ribonucleotide reductase R2

The R2 subunit of Escherichia coli ribonucleotide reductase contains a dinuclear iron center that generates a catalytically essential stable tyrosyl radical by one electron oxidation of a nearby tyrosine residue. After acquisition of Fe(II) ions by the apo protein, the resulting diiron(II) center reacts with O(2) to initiate formation of the radical. Knowledge of the structure of the reactant diiron(II) form of R2 is a prerequisite for a detailed understanding of the O(2) activation mechanism. Whereas kinetic and spectroscopic studies of the reaction have generally been conducted at pH 7.6 with reactant produced by the addition of Fe(II) ions to the apo protein, the available crystal structures of diferrous R2 have been obtained by chemical or photoreduction of the oxidized diiron(III) protein at pH 5-6. To address this discrepancy, we have generated the diiron(II) states of wildtype R2 (R2-wt), R2-D84E, and R2-D84E/W48F by infusion of Fe(II) ions into crystals of the apo proteins at neutral pH. The structures of diferrous R2-wt and R2-D48E determined from these crystals reveal diiron(II) centers with active site geometries that differ significantly from those observed in either chemically or photoreduced crystals. Structures of R2-wt and R2-D48E/W48F determined at both neutral and low pH are very similar, suggesting that the differences are not due solely to pH effects. The structures of these "ferrous soaked" forms are more consistent with circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopic data and provide alternate starting points for consideration of possible O(2) activation mechanisms.     

Stabilization of a transition-state analogue at the active site of yeast cytosine deaminase: importance of proton transfers

It is believed that the binding of pyrimidin-2-one to cytosine deaminase (CD) leads to the formation of 4-[R]-hydroxyl-3,4-dihydropyrimidine (DHP). Here the formation of transition-state analogue (TSA) at the active site of yeast cytosine deaminase (yCD) is investigated by quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations. It is shown that DHP may in fact be unstable in the active site and a proton transfer from the Zn hydroxide group to Glu-64 may occur during the nucleophilic attack, leading to an alkoxide-like TSA complex instead. The free energy simulations for the nucleophilic attack process show that the proton transfer from the Zn hydroxide to Glu-64 may play an important role in stabilizing the TSA complex.     

A nickel hydride complex in the active site of methyl-coenzyme m reductase: implications for the catalytic cycle

Methanogenic archaea utilize a specific pathway in their metabolism, converting C1 substrates (i.e., CO2) or acetate to methane and thereby providing energy for the cell. Methyl-coenzyme M reductase (MCR) catalyzes the key step in the process, namely methyl-coenzyme M (CH3-S-CoM) plus coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. The active site of MCR contains the nickel porphinoid F430. We report here on the coordinated ligands of the two paramagnetic MCR red2 states, induced when HS-CoM (a reversible competitive inhibitor) and the second substrate HS-CoB or its analogue CH3-S-CoB are added to the enzyme in the active MCR red1 state (Ni(I)F430). Continuous wave and pulse EPR spectroscopy are used to show that the MCR red2a state exhibits a very large proton hyperfine interaction with principal values A((1)H) = [-43,-42,-5] MHz and thus represents formally a Ni(III)F430 hydride complex formed by oxidative addition to Ni(I). In view of the known ability of nickel hydrides to activate methane, and the growing body of evidence for the involvement of MCR in "reverse" methanogenesis (anaerobic oxidation of methane), we believe that the nickel hydride complex reported here could play a key role in helping to understand both the mechanism of "reverse" and "forward" methanogenesis.     

Harnessing the mechanism of glutathione reductase for synthesis of active site bound metallic nanoparticles and electrical connection to electrodes

It is demonstrated herein that the FAD-dependent enzyme glutathione reductase (GR) catalyzes the NADPH-dependent reduction of AuCl4-, forming gold nanoparticles at the active site that are tightly bound through the catalytic cysteines. The nanoparticles can be removed from the GR active site with thiol reagents such as 2-mercaptoethanol. The deep enzyme active site cavity stabilizes very small metallic clusters and prevents them from aggregating in the absence of capping ligands. The behavior of the GR-nanoparticle complexes in solution, and their electrochemical properties when immobilized on graphite paper electrodes are presented. It is shown that the borohydride ion, a known reducing agent for GR, is catalytically oxidized by larger GR-nanoparticle (>or=150 gold atoms) complexes generating catalytic currents, whereas NADPH (the natural reducing agent for GR) is not. It is proposed that the surface of the Toray graphite paper electrode employed here interferes with NADPH binding to the GR-nanoparticle complex. The catalytic currents with borohydride begin at the potential of GR-bound FAD, showing that there is essentially zero resistance to electron transfer (i.e., zero overpotential) from GR-bound FAD through the gold nanoparticle to the electrode.     

Synthesis of nitric oxide reductase active site models bearing key components at both distal and proximal sites

Porphyrins 1ab and 2ab were successfully synthesized from cis-alpha2-bisimidazole-beta-imidazole-tail porphyrins and two newly synthesized imidazole pickets containing an aliphatic ester chain following a [2+1] approach. The four compounds possess a distal trisimidazole set, a distal carboxylic acid, and a proximal imidazole, which constitute all the key features of the coordination environment of the active site in Bacterial Nitric Oxide Reductase (NOR) and make them the closest synthetic NOR model ligands to date.     

Structure of the active site of sulfite dehydrogenase from Starkeya novella

In this paper, we report the results of molybdenum K-edge X-ray absorption studies performed on the oxidized and reduced active sites of the sulfite dehydrogenase from Starkeya novella. Our results provide the first direct structural information on the active site of the oxidized form of this enzyme and confirm the conclusions derived from protein crystallography that the molybdenum coordination is analogous to that of the sulfite oxidases. The molybdenum atom of the oxidized enzyme is bound by two Mo=O ligands at 1.73 A and three thiolate Mo-S ligands at 2.42 A, whereas the reduced enzyme has one oxo at 1.74 A, one long oxygen at 2.19 A (characteristic of Mo-OH2), and three Mo-S ligands at 2.40 A.     

Spectroscopic and density functional studies of the red copper site in nitrosocyanin: role of the protein in determining active site geometric and electronic structure

The electronic structure of the red copper site in nitrosocyanin is defined relative to that of the well understood blue copper site of plastocyanin by using low-temperature absorption, circular dichroism, magnetic circular dichroism, resonance Raman, EPR and X-ray absorption spectroscopies, combined with DFT calculations. These studies indicate that the principal electronic structure change in the red copper site is the sigma rather than the pi donor interaction of the cysteine sulfur with the Cu 3d(x2-y2) redox active molecular orbital (RAMO). Further, MCD data show that there is an increase in ligand field strength due to an increase in coordination number, whereas resonance Raman spectra indicate a weaker Cu-S bond. The latter is supported by the S K-edge data, which demonstrate a less covalent thiolate interaction with the RAMO of nitrosocyanin at 20% relative to plastocyanin at 38%. EXAFS results give a longer Cu-S(Cys) bond distance in nitrosocyanin (2.28 A) compared to plastocyanin (2.08 A) and also show a large change in structure with reduction of the red copper site. The red copper site is the only presently known blue copper-related site with an exogenous water coordinated to the copper. Density functional calculations reproduce the experimental properties and are used to determine the specific protein structure contributions to exogenous ligand binding in red copper. The relative orientation of the CuNNS and the CuSC(beta) planes (determined by the protein sequence) is found to be key in generating an exchangeable coordination position at the red copper active site. The exogenous water ligation at the red copper active site greatly increases the reorganization energy (by approximately 1.0 eV) relative to that of the blue copper protein site, making the red site unfavorable for fast outer-sphere electron transfer, while providing an exchangeable coordination position for inner-sphere electron transfer.     

The active site of arsenite oxidase from Alcaligenes faecalis

Arsenite oxidase, a member of the DMSO reductase family of molybdenum enzymes, has two molecules of guanosine dinucleotide molybdenum cofactor coordinating the molybdenum at the active site. X-ray absorption spectroscopy indicates that the Mo-S bonds shorten from 2.47 to 2.37 A upon reduction with the physiological substrate. It also indicates the presence of an oxo ligand at 1.70 A in both oxidized and reduced forms of the enzyme, together with a short, 1.83 A, Mo-O bond in the oxidized form that is lost upon reduction. Resonance Raman spectroscopy indicates that the two pterin dithiolene moieties have different aromaticities, with one, the Q-pterin, having a more discrete dithiolate structure while the other, the P-pterin, has considerable pi-delocalization. Our results indicate that the structure of arsenite oxidase is intermediate between that seen in other molybdenum enzymes, in which one ligand to the metal is provided by the polypeptide (serine, cysteine, or selenocysteine), and tungsten enzymes that lack a peptide ligand.     

Photonics of the hemoglobin active site

The results of a pico- and nanosecond laser absorption spectroscopy study of the rebinding of the oxygen molecule by the triliganded hemoglobin tetramer in the R-state are presented.     

Structural insights into the ferroxidase site of ferritins from higher eukaryotes

The first step of iron biomineralization mediated by ferritin is the oxidation at the ferroxidase active site of two ferrous ions to a diferric oxo/hydroxo species. Metal-loaded ferritin crystals obtained by soaking crystals of frog ferritin in FeSO(4) and CuSO(4) solutions followed by flash freezing provided X-ray crystal structures of the tripositive iron and bipositive copper adducts at 2.7 and 2.8 ? resolution, respectively. At variance with the already available structures, the crystal form used in this study contains 24 independent subunits in the asymmetric unit permitting comparison between them. For the first time, the diferric species at the ferroxidase site is identified in ferritins from higher eukaryotes. Anomalous difference Fourier maps for crystals (iron crystal 1) obtained after long soaking times in FeSO(4) solution invariantly showed diferric species with a Fe-Fe average distance of 3.1 ± 0.1 ?, strongly indicative of the presence of a μ-oxo/hydroxo bridge between the irons; protein ligands for each iron ion (Fe1 and Fe2) were also unequivocally identified and found to be the same in all subunits. For copper bound ferritin, dicopper(II) centers are also observed. While copper at site 1 is essentially in the same position and has the same coordination environment as Fe1, copper at site 2 is displaced toward His54, now acting as a ligand; this results in an increased intermetal distance (4.3 ± 0.4 ?). His54 coordination and longer metal-metal distances might represent peculiar features of divalent cations at the ferroxidase site. This oxidation-dependent structural information may provide key features for the mechanistic pathway in ferritins from higher eukaryotes that drive uptake of bivalent cation and release of ferric products at the catalytic site. This mechanism is supported by the X-ray picture obtained after only 1 min of soaking in FeSO(4) solutions (iron crystal 2) which reasonably contain the metal at different oxidation states. Here two different di-iron species are trapped in the active site, with intermetal distances corresponding to those of the ferric dimer in crystal 1 and of the dicopper centers and corresponding rearrangement of the His54 side chain.