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Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/monomeric form that translocates into the nucleus, causing oncogene transcription. However, it is not known how these post-translational modifications (PTMs) disrupt the oligomeric state of PKM2. We explored this question via crystallographic and biophysical analyses of PKM2 mutants containing residues that mimic phosphorylation and acetylation. We find that the PTMs elicit major structural reorganization of the fructose 1,6-bisphosphate (FBP), an allosteric activator, binding site, impacting the interaction with FBP and causing a disruption in oligomerization. To gain insight into how these modifications might cause unique outcomes in cancer cells, we examined the impact of increasing the intracellular pH (pH(i)) from ∼7.1 (in normal cells) to ∼7.5 (in cancer cells). Biochemical studies of WT PKM2 (wtPKM2) and the two mimetic variants demonstrated that the activity decreases as the pH is increased from 7.0 to 8.0, and wtPKM2 is optimally active and amenable to FBP-mediated allosteric regulation at pH(i) 7.5. However, the PTM mimetics exist as a mixture of tetramer and dimer, indicating that physiologically dimeric fraction is important and might be necessary for the modified PKM2 to translocate into the nucleus. Thus, our findings provide insight into how PTMs and pH regulate PKM2 and offer a broader understanding of its intricate allosteric regulation mechanism by phosphorylation or acetylation.

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.

Cancer cells regulate key enzymes in the glycolytic pathway to control the glycolytic flux, which is necessary for their growth and proliferation. One of the enzymes is pyruvate kinase muscle isoform 2 (PKM2), which is allosterically regulated by various small molecules. Using detailed biochemical and kinetic studies, we demonstrate that cysteine inhibits wild-type (wt) PKM2 by shifting from an active tetramer to a mixture of a tetramer and a less active dimer/monomer equilibrium and that the inhibition is dependent on cysteine concentration. The cysteine-mediated PKM2 inhibition is reversed by fructose 1,6-bisphosphate, an allosteric activator of PKM2. Furthermore, kinetic studies using two dimeric PKM2 variants, S437Y PKM2 and G415R PKM2, show that the reversal is caused by the tetramerization of wtPKM2. The crystal structure of the wtPKM2-Cys complex was determined at 2.25 Å, which showed that cysteine is held to the amino acid binding site via its main chain groups, similar to that observed for phenylalanine, alanine, serine, and tryptophan. Notably, ligand binding studies using fluorescence and isothermal titration calorimetry show that the presence of phosphoenolpyruvate alters the binding affinities of amino acids for wtPKM2 and vice versa, thereby unravelling the existence of a functionally bidirectional coupling between the amino acid binding site and the active site of wtPKM2.

The prolyl 4-hydroxylases (P4Hs) are mononuclear nonheme iron enzymes that catalyze the formation of 4R-hydroxyproline from many different substrates, with various biological implications. P4H is a key player in collagen accumulation, which has implications in fibrotic disorders. The stabilization of collagen triple-helical structureviaprolyl hydroxylation is the rate-limiting step in collagen biosynthesis, and therefore P4H has been extensively investigated as a potential therapeutic target of fibrotic disease. Understanding how these enzymes recognize cofactors and substrates is important and will aid in the future design of inhibitors of P4H. In this article, X-ray crystal structures of a metallocofactor- and α-ketoglutarate (αKG)-bound form of P4H fromBacillus anthracis(BaP4H) are reported. Structures of BaP4H were solved at 1.63 and 2.35 Å resolution and contained a cadmium ion and αKG bound in the active site. The αKG–Cd–BaP4H ternary complex reveals conformational changes of conserved residues upon the binding of metal ion and αKG, resulting in a closed active-site configuration required for dioxygen, substrate binding and catalysis.

The organosulfur metabolite dimethylsulfoniopropionate (DMSP) and its enzymatic breakdown product dimethyl sulfide (DMS) have important implications in the global sulfur cycle and in marine microbial food webs. Enormous amounts of DMSP are produced in marine environments where microbial communities import and catabolize it via either the demethylation or the cleavage pathways. The enzymes that cleave DMSP are termed “DMSP lyases” and generate acrylate or hydroxypropionate, and ~ 107 tons of DMS annually. An important environmental factor affecting DMS generation by the DMSP lyases is the availability of metal ions as these enzymes use various cofactors for catalysis. This chapter summarizes advances on bacterial DMSP catabolism, with an emphasis on various biochemical methods employed for the isolation and characterization of bacterial DMSP lyases. Strategies are presented for the purification of DMSP lyases expressed in bacterial cells. Specific conditions for the efficient isolation of apoproteins in Escherichia coli are detailed. DMSP cleavage is effectively inferred, utilizing the described HPLC-based acrylate detection assay. Finally, substrate and metal binding interactions are examined using fluorescence and UV–visible assays. Together, these methods are rapid and well suited for the biochemical and structural characterization of DMSP lyases and in the assessment of uncharacterized DMSP catabolic enzymes, and new metalloenzymes in general.

Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant, and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC-SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC-SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.

Enormous amounts of the organic osmolyte dimethylsulfoniopropionate (DMSP) are produced in marine environments where bacterial DMSP lyases cleave it, yielding acrylate and the climate-active gas dimethyl sulfide (DMS). SAR11 bacteria are the most abundant clade of heterotrophic bacteria in the oceans and play a key role in DMSP catabolism. An important environmental factor affecting DMS generation via DMSP lyases is the availability of metal ions because they are essential cofactors for many of these enzymes. Here we examine the structure and activity of DddK in the presence of various metal ions. We have established that DddK containing a double-stranded β-helical motif utilizes various divalent metal ions as cofactors for catalytic activity. However, nickel, an abundant metal ion in marine environments, adopts a distorted octahedral coordination environment and conferred the highest DMSP lyase activity. Crystal structures of cofactor-bound DddK reveal key metal ion binding and catalytic residues and provide the first rationalization for varying activities with different metal ions. The structures of DddK along with site-directed mutagenesis and ultraviolet-visible studies are consistent with Tyr 64 acting as a base to initiate the β-elimination reaction of DMSP. Our biochemical and structural studies provide a detailed understanding of DMS generation by one of the ocean's most prolific bacteria.

The biochemistry of dimethylsulfoniopropionate (DMSP) catabolism is reviewed. The microbes that catalyze the reactions central to DMSP catabolic pathways are described, and the focus is on the enzymology of the process. Approximately 109tons of DMSP is released annually by marine eukaryotes as an osmolyte. A vast majority of DMSP is assimilated by bacteria through either a demethylation or lyase pathways, producing either the methane thiol or the volatile dimethylsulfide (DMS), respectively. Enzymatic breakdown of DMSP generates ~107tons of DMS annually, which may have impact on global climate. DMS also acts as a chemoattractant for zooplanktons and seabirds. Both DMSP and DMS play a key role in the global sulfur cycle and are key nutrients for marine microbial growth. Important enzymes in the biochemical pathways of DMSP catabolism are covered in this review, with a focus on the latest developments in their mechanism.

The marine microbial catabolism of dimethylsulfoniopropionate (DMSP) by the lyase pathway liberates ∼300 million tons of dimethyl sulfide (DMS) per year, which plays a major role in the biogeochemical cycling of sulfur. Recent biochemical and structural studies of some DMSP lyases, including DddQ, reveal the importance of divalent transition metal ions in assisting DMSP cleavage. While DddQ is believed to be zinc-dependent primarily on the basis of structural studies, excess zinc inhibits the enzyme. We examine the importance of iron in regulating the DMSP β-elimination reaction catalyzed by DddQ as our as-isolated purple-colored enzyme possesses ∼0.5 Fe/subunit. The UV-visible spectrum exhibited a feature at 550 nm, consistent with a tyrosinate-Fe(III) ligand-to-metal charge transfer transition. Incubation of as-isolated DddQ with added iron increases the intensity of the 550 nm peak, whereas addition of dithionite causes a bleaching as Fe(III) is reduced. Both the Fe(III) oxidized and Fe(II) reduced species are active, with similar k

Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that uses dioxygen to catalyze the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid (S-HPP) in the biosynthesis of the antibiotic fosfomycin. Additionally, the enzyme converts the R-enantiomer of the substrate (R-HPP) to 2-oxo-propylphosphonic acid. To probe the mechanism of HppE regiospecificity, we determined three X-ray structures: R-HPP with inert cobalt-containing enzyme (Co(II)–HppE) at 2.1 Å resolution; R-HPP with active iron-containing enzyme (Fe(II)–HppE) at 3.0 Å resolution; and S-HPP–Fe(II)–HppE in complex with dioxygen mimic NO at 2.9 Å resolution. These structures, along with previously determined structures of S-HPP–HppE, identify the dioxygen binding site on iron and elegantly illustrate how HppE is able to recognize both substrate enantiomers to catalyze two completely distinct reactions.

Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in methanogenesis by using N-7-mercaptoheptanolyl-threonine phosphate (CoBSH) as the two-electron donor to reduce 2-(methylthiol)-ethane sulfonate (methyl-SCoM) to methane, and producing the heterodisulfide, CoBS-SCoM. The active site of MCR includes a noncovalently bound Ni tetrapyrrolic cofactor called coenzyme F 430 , which is in the Ni(I) state in the active enzyme (McR red1 ). Bromopropanesulfonate (BPS) is a potent inhibitor and reversible redox inactivator that reacts with MCR red1 to form an EPR-active state called MCRps, which is an alkyl-nickel species. When MCRps is treated with free thiol containing compounds, the enzyme is reconverted to the active McR red1 state. In this paper, we demonstrate that the reactivation of MCRps to MCR red1 by thiols involves formation of a thioether product. MCRps also can be converted to active MCR red1 by treatment with sodium borohydride. Reactivation is highest with the smallest free thiol HS - . Interestingly, MCRps can also be reductively activated with analogues of CoBSH such as COB 8 SH and COB 9 SH, but not CoBSH itself. Unambiguous demonstration of the formation of a methylthioether product from thiolysis of an alkyl-Ni species provides support for a methyl-Ni intermediate in the MCR-catalyzed last step in methanogenesis and the first proposed step in anaerobic methane oxidation.

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the final step in the biological synthesis of methane. Using coenzyme B (CoBSH) as the two-electron donor, MCR reduces methyl-coenzyme M (methyl-SCoM) to methane and the mixed disulfide, CoB-S-S-CoM. MCR contains coenzyme F 430 , an essential redox-active nickel tetrahydrocorphin, at its active site. The active form of MCR (MCR red1 ) contains Ni(I)-F 430 . When 3-bromopropane sulfonate (BPS) is incubated with MCR red1 , an alkyl-Ni(III) species is formed that elicits the MCR PS EPR signal. Here we used EPR and UV-visible spectroscopy and transient kinetics to study the reaction between MCR from Methanothermobacter marburgensis and a series of brominated carboxylic acids, with carbon chain lengths of 4-16. All of these compounds give rise to an alkyl-Ni intermediate with an EPR signal similar to that of the MCRps species. Reaction of the alkyl-Ni(III) adduct, formed from brominated acids with eight or fewer total carbons, with HSCoM as nucleophile at pH 10.0 results in the formation of a thioether coupled to regeneration of the active MCR red1 state. When reacted with 4-bromobutyrate, MCR red1 forms the alkyl-Ni(III) MCR XA state and then, surprisingly, undergoes "self-reactivation" to regenerate the Ni(I) MCR red1 state and a bromocarboxy ester. The results demonstrate an unexpected reactivity and flexibility of the MCR active site in accommodating a broad range of substrates, which act as molecular rulers for the substrate channel in MCR.

Methyl-coenzyme M reductase (MCR) catalyzes the final step in methane biosynthesis by methanogenic archaea and contains a redox-active nickel tetrahydrocorphin, coenzyme F430, at its active site. Spectroscopic and computational methods have been used to study a novel form of the coenzyme, called F330, which is obtained by reducing F430 with sodium borohydride (NaBH4). F330 exhibits a prominent absorption peak at 330 nm, which is blue shifted by 100 nm relative to F430. Mass spectrometric studies demonstrate that the tetrapyrrole ring in F330 has undergone reduction, on the basis of the incorporation of protium (or deuterium), upon treatment of F430 with NaBH4 (or NaBD4). One- and two-dimensional NMR studies show that the site of reduction is the exocyclic ketone group of the tetrahydrocorphin. Resonance Raman studies indicate that elimination of this pi-bond increases the overall pi-bond order in the conjugative framework. X-ray absorption, magnetic circular dichroism, and computational results show that F330 contains low-spin Ni(II). Thus, conversion of F430 to F330 reduces the hydrocorphin ring but not the metal. Conversely, reduction of F430 with Ti(III) citrate to generate F380 (corresponding to the active MCR(red1) state) reduces the Ni(II) to Ni(I) but does not reduce the tetrapyrrole ring system, which is consistent with other studies [Piskorski, R., and Jaun, B. (2003) J. Am. Chem. Soc. 125, 13120-13125; Craft, J. L., et al. (2004) J. Biol. Inorg. Chem. 9, 77-89]. The distinct origins of the absorption band shifts associated with the formation of F330 and F380 are discussed within the framework of our computational results. These studies on the nature of the product(s) of reduction of F430 are of interest in the context of the mechanism of methane formation by MCR and in relation to the chemistry of hydroporphinoid systems in general. The spectroscopic and time-dependent DFT calculations add important insight into the electronic structure of the nickel hydrocorphinate in its Ni(II) and Ni(I) valence states.

Thioredoxins are small soluble proteins that contain a redox-active disulfide (CXXC). These disulfides are tuned to oxidizing or reducing potentials depending on the function of the thioredoxin within the cell. The mechanism by which the potential is tuned has been controversial, with two main hypotheses: first, that redox potential (E[subscript m]) is specifically governed by a molecular ‘rheostat’—the XX amino acids, which influence the Cys pK[subscript a] values, and thereby, E[subscript m]; and second, the overall thermodynamics of protein folding stability regulates the potential. Here, we use protein film voltammetry (PFV) to measure the pH dependence of the redox potentials of a series of wild-type and mutant archaeal Trxs, PFV and glutathionine-equilibrium to corroborate the measured potentials, the fluorescence probe BADAN to measure pK[subscript a] values, guanidinium-based denaturation to measure protein unfolding, and X-ray crystallography to provide a structural basis for our functional analyses. We find that when these archaeal thioredoxins are probed directly using PFV, both the high and low potential thioredoxins display consistent 2H+:2e- coupling over a physiological pH range, in conflict with the conventional ‘rheostat’ model. Instead, folding measurements reveals an excellent correlation to reduction potentials, supporting the second hypothesis and revealing the molecular mechanism of reduction potential control in the ubiquitous Trx family., National Science Foundation (U.S.) (MCB 1122977)

A series of mono-, di- and tri-nuclear Ni(II) complexes of N, O-donating molecules possessing ---H2C---NH--- and ---HC=N--- moieties have been synthesized and characterized and the structures have been determined by single crystal X-ray diffraction. All these exhibited interesting molecular packing in their crystal lattices. Di-nuclear complexes were found to be cleaved in pyridine to result in mononuclear ones with additional coordinations being provided by pyridine. Di-nuclear complexes were found to form urea adducts as demonstrated based on absorption and vibrational studies., © Elsevier

This paper deals with the development of a receptor with selectively functionalized at the lower rim of p-tert-butyl calix[4]arene linked through amide bond, resulting in the diamide derivatives (L 1 , L 2 and L 3 ) suitable for sensing ions and molecules. All the derivatives were thoroughly characterized by analytical and spectral methods. These derivatives were structurally characterized by single crystal X-ray diffraction and the conformational features of the calix[4]arene as well as the pendants are discussed. The L 1 and L 2 exhibited intramolecular hydrogen bond interaction between the N–H and the lower rim phenolic oxygen in addition to the circular hydrogen bonding between the phenolic O–H and the ether oxygen of the adjacent strand. The corresponding H-bond is absent when no ‘H’ is present on amide-N as in case of L 3 . Such intramolecular N–H···O interactions observed with the pendant results in a conformational bend responsible for the orientation of the arms and there by the nature of the binding core formed. Thus the binding cores formed differ largely between L 3 and the other two.

The non-haem-iron-dependent enzyme HppE catalyses the final step in the biosynthesis of fosfomycin, a broad-spectrum, clinically useful antibiotic; here it is shown that HppE can also catalyse a 1,2-phosphono-migration reaction, previously undocumented for any enzyme. The last step in the biosynthesisof fosfomycin, an antibiotic used to treat diabetic foot infections and lower urinary tract infections, is catalysed by the non-haem-iron-dependent enzyme (S)-2-hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE). The reaction involves an unusual dehydrogenation reaction in which the secondary alcohol of (S)-2-HPP is converted to the epoxide ring of fosfomycin. In this manuscript, the authors show that the enzyme can also catalyse a 1,2-phosphono-migration reaction, which is a biologically unprecedented chemical transformation. This finding could pave the way for the production of novel phosphonate-containing natural products and new phosphonate derivatives. (S)-2-hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE) is a mononuclear non-haem-iron-dependent enzyme1,2,3 responsible for the final step in the biosynthesis of the clinically useful antibiotic fosfomycin4. Enzymes of this class typically catalyse oxygenation reactions that proceed via the formation of substrate radical intermediates. By contrast, HppE catalyses an unusual dehydrogenation reaction while converting the secondary alcohol of (S)-2-HPP to the epoxide ring of fosfomycin1,5. Here we show that HppE also catalyses a biologically unprecedented 1,2-phosphono migration with the alternative substrate (R)-1-HPP. This transformation probably involves an intermediary carbocation, based on observations with additional substrate analogues, such as (1R)-1-hydroxyl-2-aminopropylphosphonate, and model reactions for both radical- and carbocation-mediated migration. The ability of HppE to catalyse distinct reactions depending on the regio- and stereochemical properties of the substrate is given a structural basis using X-ray crystallography. These results provide compelling evidence for the formation of a substrate-derived cation intermediate in the catalytic cycle of a mononuclear non-haem-iron-dependent enzyme. The underlying chemistry of this unusual phosphono migration may represent a new paradigm for the in vivo construction of phosphonate-containing natural products that can be exploited for the preparation of new phosphonate derivatives.

We present the 1.2 Å resolution X-ray crystal structure of a Ni-methyl species that is a proposed catalytic intermediate in methyl-coenzyme M reductase (MCR), the enzyme that catalyzes the biological formation of methane. The methyl group is situated 2.1 Å proximal of the Ni atom of the MCR coenzyme F(430). A rearrangement of the substrate channel has been posited to bring together substrate species, but Ni(III)-methyl formation alone does not lead to any observable structural changes in the channel.

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the terminal step in methanogenesis using coenzyme B (CoBSH) as the two-electron donor to reduce methyl-coenzyme M (methyl-SCoM) to form methane and the heterodisulfide, CoBS-SCoM. The active site of MCR contains an essential redox-active nickel tetrapyrrole cofactor, coenzyme F(430), which is active in the Ni(I) state (MCR(red1)). Several catalytic mechanisms have been proposed for methane synthesis that mainly differ in whether an organometallic methyl-Ni(III) or a methyl radical is the first catalytic intermediate. A mechanism was recently proposed in which methyl-Ni(III) undergoes homolysis to generate a methyl radical (Li, X., Telser, J., Kunz, R. C., Hoffman, B. M., Gerfen, G., and Ragsdale, S. W. (2010) Biochemistry 49, 6866-6876). Discrimination among these mechanisms requires identification of the proposed intermediates, none of which have been observed with native substrates. Apparently, intermediates form and decay too rapidly to accumulate to detectible amounts during the reaction between methyl-SCoM and CoBSH. Here, we describe the reaction of methyl-SCoM with a substrate analogue (CoB(6)SH) in which the seven-carbon heptanoyl moiety of CoBSH has been replaced with a hexanoyl group. When MCR(red1) is reacted with methyl-SCoM and CoB(6)SH, methanogenesis occurs 1000-fold more slowly than with CoBSH. By transient kinetic methods, we observe decay of the active Ni(I) state coupled to formation and subsequent decay of alkyl-Ni(III) and organic radical intermediates at catalytically competent rates. The kinetic data also revealed substrate-triggered conformational changes in active Ni(I)-MCR(red1). Electron paramagnetic resonance (EPR) studies coupled with isotope labeling experiments demonstrate that the radical intermediate is not tyrosine-based. These observations provide support for a mechanism for MCR that involves methyl-Ni(III) and an organic radical as catalytic intermediates. Thus, the present study provides important mechanistic insights into the mechanism of this key enzyme that is central to biological methane formation.

This title is not available to purchase from Royal Society of Chemistry. Please visit www.bioinorganic-chemistry.org/mils for title information. This volume, closely related to MILS-6, deals mainly with metal(loid)-alkyl derivatives but also with the rarer aryl compounds. Most of these (commonly toxic) compounds are formed in the environment by microorganisms, but some anthropogenic input occurs as well. MILS-7, providing a most up-to-date view, is of special relevance for researchers in analytical and bioinorganic chemistry, enzymology, environmental chemistry, physiology, toxicology, and related medical fields.

Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in the formation of biological methane from methyl-coenzyme M (Me-SCoM) and coenzyme B (CoBSH). The active site in MCR contains a Ni-F(430) cofactor, which can exist in different oxidation states. The catalytic mechanism of methane formation has remained elusive despite intense spectroscopic and theoretical investigations. On the basis of spectroscopic and crystallographic data, the first step of the mechanism is proposed to involve a nucleophilic attack of the Ni(I) active state (MCR(red1)) on Me-SCoM to form a Ni(III)-methyl intermediate, while computational studies indicate that the first step involves the attack of Ni(I) on the sulfur of Me-SCoM, forming a CH(3)(*) radical and a Ni(II)-thiolate species. In this study, a combination of Ni K-edge X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been performed on the Ni(I) (MCR(red1)), Ni(II) (MCR(red1-silent)), and Ni(III)-methyl (MCR(Me)) states of MCR to elucidate the geometric and electronic structures of the different redox states. Ni K-edge EXAFS data are used to reveal a five-coordinate active site with an open upper axial coordination site in MCR(red1). Ni K-pre-edge and EXAFS data and time-dependent DFT calculations unambiguously demonstrate the presence of a long Ni-C bond ( approximately 2.04 A) in the Ni(III)-methyl state of MCR. The formation and stability of this species support mechanism I, and the Ni-C bond length suggests a homolytic cleavage of the Ni(III)-methyl bond in the subsequent catalytic step. The XAS data provide insight into the role of the unique F(430) cofactor in tuning the stability of the different redox states of MCR.

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.

Cu(II) complex of the double-armed p-tert-butylcalix[4]arene conjugate possessing imine functionality at the lower rim was synthesized and was characterized by analytical, spectral and crystallographic methods and its catecholase activity was evaluated., © Elsevier

Mn(IV) and Co(III) complexes of tridentate –OH–rich ligands possessing O2N, O3N and O4N donor sets were synthesized, characterized and their structures were established by single crystal X-ray diffraction, where the binding core is O4N2. In the structurally characterized complexes, the coordination geometry about the metal ion was found to be distorted octahedral., © Elsevier

Four-, five- and six-coordinated complexes of Zn-II with OH-rich molecules possessing an ONO binding core were synthesized, characterized and their structures were established by single-crystal X-ray diffraction, The corresponding metal ion geometries were found to be distorted tetrahedral, square pyramidal and octahedral, respectively. The complexes exhibit interesting lattice structures such as layered and corrugated sheets owing to the presence of a number of weak intermolecular interactions. The five-coordinated, water-bound Zn-II complex was studied because of its putative hydrolysis property towards p-nitrophenyl acetate. (C) Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002.

Tetranuclear Cu(II) complexes having linear, cubane and pseudodouble-cubane cores were synthesized using hydroxy-rich molecules possessing amine and imine groups. The products were structurally characterized and were studied for their ability to oxidize catechol as well as for their inter-conversion between mono- and tetra-nuclear complexes., © Elsevier