FAD binding,cobinamide binding and active site communication in the corrin reductase (CobR)

Adenosylcobalamin, the coenzyme form of vitamin B₁₂, is one Nature''s most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I).     

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica serovar typhimurium LT2

Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in Salmonella enterica serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC 1.6.8.1) as the enzyme responsible for this activity. The fre gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His-tagged Fre reduced flavins (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) and cob(III)alamin to cob(II)alamin very efficiently. Photochemically reduced FMN substituted for Fre in the reduction of cob(III)alamin to cob(II)alamin, indicating that the observed cobalamin reduction activity was not Fre dependent but FMNH(2) dependent. Enzyme-independent reduction of cob(III)alamin to cob(II)alamin by FMNH(2) occurred at a rate too fast to be measured. The thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin was detectable by alkylation of the cob(I)alamin nucleophile with iodoacetate. Detection of the product, caboxymethylcob(III)alamin, depended on the presence of FMNH(2) in the reaction mixture. FMNH(2) failed to substitute for potassium borohydride in in vitro assays for corrinoid adenosylation catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme, even under conditions where Fre and NADH were present in the reaction mixture to ensure that FMN was always reduced. These results were interpreted to mean that Fre was not responsible for the generation of cob(I)alamin in vivo. Consistent with this idea, a fre mutant displayed wild-type cobalamin biosynthetic phenotypes. It is proposed that S. enterica serovar Typhimurium LT2 may not have a cob(III)alamin reductase enzyme and that, in vivo, nonadenosylated cobalamin and other corrinoids are maintained as co(II)rrinoids by reduced flavin nucleotides generated by Fre and other flavin oxidoreductases.     

Modification of the heme·heme oxygenase system with iron and cobalt tetrasulfonated phthalocyanines

Modification of heme·heme oxygenase by iron(III) and cobalt(II) tetrasulfonated phthalocyanines has been performed. New compounds have been isolated and their properties have been investigated by difference spectroscopy, electrophoresis, molecular weight estimation, electron paramagnetic resonance (EPR) and carboxymethylation at histidyl groups. Spectrophotometric titration data indicate the ratio of the reagents in this process to be 1:1. The visible absorption spectra show the main peak at 650 nm for the iron compound and 682 nm for the cobalt one. Electrophoresis and molecular weight estimation show both complexes to be monomers. Cobalt(II) tetrasulfonated phthalocyanine, under aerobic conditions with heme oxygenase protein, undergoes autooxidation to the cobalt(III) complex, as has been proved by EPR and spectroscopic data. Iron and cobalt phthalocyanine modified heme·heme oxygenase with excess dithionite is reduced at the phthalocyanine ligand. In the presence of oxygen, the reduction product transforms into oxygenated Fe(III)Lheme oxygenase or Co(III)heme oxygenase, respectively. Reduction of the iron(III) model complex with ascorbic acid under anaerobic conditions leads to degradation of the phthalocyanine moiety, while Co(III)heme oxygenase with ascorbic acid is reduced to Co(II)Lheme oxygenase. As has been shown by carboxymethylation of the heme oxygenase protein at the histidine residues, the predominant binding site of both phthalocyanine complexes is the heme-binding histidyl residue. There is evidence that there is a second binding site with lower affinity towards Co(II)L on the heme oxygenase protein. Iron and cobalt tetrasulfonated phthalocyanines are not able to displace heme from the heme·heme oxygenase complex. In this reaction the iron complex undergoes degradation and the cobalt one gives a hybrid compound with heme·heme oxygenaseHeme oxygenase protein complexes with iron and cobalt tetrasulfonated phthalocyanines do not exhibit activity in their oxidative degradation.     

A novel reaction of S-adenosyl-L-methionine correlated with the activation of pyruvate formate-lyase

Conversion of the inactive form of pyruvate formate-lyase to the catalytically active enzyme is accomplished by the Fe-dependent ‘enzyme II’; reduced flavodoxin, S-adenosyl-L-methionine and the effector pyruvate are required. It was found that adenosylmethionine is reductively processed during activation of pyruvate formate-lyase to yield methionine, adenine and 5-deoxyribose. We suggest that transient adenosylation of enzyme II is required for its function as a converter enzyme.     

The problem of a solvent exposable disulfide when preparing Co(II)-substituted metallo-β-lactamase L1 from Stenotrophomonas maltophilia

In an effort to prepare Co(II)-substituted metallo-beta-lactamase L1 from Stenotrophomonas maltophilia for future spectroscopic and mechanistic studies, two methods for the preparation of Co(II)-L1 were tested. Method A involved adding CoCl2 directly to apo-L1 under anaerobic conditions. The resulting enzyme contained 1.9 moles of cobalt and exhibited very little activity, and UV-Vis, 1H NMR, and EPR studies indicated that most of the cobalt in this sample was Co(III). Method B involved reducing the single and unique disulfide bridge in L1 with tris(carboxyethyl)phosphine prior to adding CoCl2. The resulting enzyme was pink, contained 2.5 moles of cobalt per mole of enzyme, and exhibited kcat and Km values of 18+1 s(-1) and 10+/-1 microM, respectively, when using nitrocefin as the substrate. UV-Vis, 1H NMR, and EPR studies revealed that this enzyme sample contained high-spin Co(II). The UV-Vis spectra also provided evidence for Co(II) bound to one or both of the reduced cysteines. Efforts to block this non-specific Co(II) binding site using a chemical modification agent or site-directed mutagenesis were unsuccessful. The data presented here demonstrate the problem of solvent-exposed disulfides when preparing Co(II)-substituted enzymes and offers a convenient method to circumvent the problem.     

Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovine carbonic anhydrases. Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes

The reactions with N,N-diethyldithiocarbamate (DDC) of zinc, cobalt and copper carbonic anhydrase from bovine erythrocytes were investigated. The native zinc enzyme was inhibited by DDC, but no removal of zinc could be detected even at a very high [ligand]/[protein] ratio. At identical pH values a larger inhibitory effect was found for the cobalt enzyme. The metal was removed by DDC from the protein at pH less than 7.0. No cobalt removal occurred at pH 10, where a stable ternary complex with the enzyme-bound Co(II) was detected. Its optical and EPR spectra are indicative of five-coordinate Co(II). The reaction of the Cu(II) enzyme with stoichiometric chelating agent was marked by the appearance of an electronic transition at 390 nm (epsilon = 4300 M-1 X cm-1). Metal removal from the copper enzyme readily occurred as the ligand was in excess over the metal, with parallel appearance of a band at 440 nm, which was attributed to the free Cu(II)-DDC complex. Also, in the case of the copper enzyme an alkaline pH was found to stabilize the ternary adduct with the diagnostic 390 nm band. EPR spectra showed that the ternary adduct is a mixture of two species, both characterized by the presence in the EPR spectrum of a superhyperfine structure from two protein nitrogens and by a low g parallel value, indicative of coordination to sulfur ligands. It is suggested that the two species contain the metal as penta- and hexacoordinated, respectively. Measurements of the longitudinal relaxation time, T1, of the water protons suggested that water coordination is retained in the latter case. Hexacoordination with retention of water is also proposed for the Cu(II) derivatives with the bidentate oxalate and bicarbonate anions, unlike the corresponding Co(II) derivatives, which are pentacoordinated. Different coordination of Co(II) and Cu(II) adducts may be relevant to the difference of activity of the two substituted enzymes.     

Kinetic and magnetic properties of cobalt(III) ion in the active site of carbonic anhydrase.

Cobalt(III)bovine carbonic anhydrase B was prepared by the oxidation of the cobalt(II) enzyme with hydrogen peroxide and was purified by affinity chromatography. The oxidation reaction is inhibited by specific inhibitors of carbonic anhydrase. The inhibition is explained by the fact that the Co(II)-enzyme . inhibitor complex cannot be directly oxidized by hydrogen peroxide, but has to dissociate to give free Co(II) enzyme which is then oxidized. The Co(III) ion in Co(III) carbonic anhydrase cannot be directly substituted by zinc ions. It can be reduced by either dithionite or BH-4 ions to give, first, their complexes with the Co(II) enzyme, and upon their removal, a fully active Co(II) enzyme. Cyanide and azide bind to cobalt(III) carbonic anhydrase with similar rate constants of 0.060 +/- 0.005 and 0.070 +/- 0.007 M-1 S-1 respectively. These rates are faster than those found for Co(III) inorganic complexes. The Co(III) ion in both Co(III) carbonic anhydrase and Co(III) carboxypeptidase A was found to be diamagnetic, indicating a near octahedral symmetry.     

Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli.

Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II). It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site. The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.     

Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

The human-type ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri (LrPduO) catalyzes the adenosylation of Co(II)rrinoids to generate adenosylcobalamin (AdoCbl) or adenosylcobinamide (AdoCbi⁺). This process requires the formation of “supernucleophilic” Co(I)rrinoid intermediates in the enzyme active site which are properly positioned to abstract the adeonsyl moiety from co-substrate ATP. Previous magnetic circular dichroism (MCD) spectroscopic and X-ray crystallographic analyses revealed that LrPduO achieves the thermodynamically challenging reduction of Co(II)rrinoids by displacing the axial ligand with a non-coordinating phenylalanine residue to produce a four-coordinate species. However, relatively little is currently known about the interaction between the tetradentate equatorial ligand of Co(II)rrinoids (the corrin ring) and the enzyme active site. To address this issue, we have collected resonance Raman (rR) data of Co(II)rrinoids free in solution and bound to the LrPduO active site. The relevant resonance-enhanced vibrational features of the free Co(II)rrinoids are assigned on the basis of rR intensity calculations using density functional theory to establish a suitable framework for interpreting rR spectral changes that occur upon Co(II)rrinoid binding to the LrPduO/ATP complex in terms of structural perturbations of the corrin ring. To complement our rR data, we have also obtained MCD spectra of Co(II)rrinoids bound to LrPduO complexed with the ATP analogue UTP. Collectively, our results provide compelling evidence that in the LrPduO active site, the corrin ring of Co(II)rrinoids is firmly locked in place by several amino acid side chains so as to facilitate the dissociation of the axial ligand.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Metal--glutathione interaction in aqueous solution. Nickel(II), cobalt(II) and copper(II) complexes with oxidized glutathione.

The interaction of copper(II), nickel(II) and cobalt(II) ions with oxidized glutathione in aqueous solutions have been examined by spectroscopic methods. Cu(II) is the only ion which interacts with disulphide bridge and forms dimeric species containing the Cu(II)-S-S-Cu(II) unit. Ni(II) and Co(II) bind mainly with the terminal NH2 and COO- groups of glutamic acid, and the complexes formed are of nearly octahedral symmetry. At high pH, in the Co(II)-GSSG solution Co(II) is oxidized to Co(III) with the concomitant reduction of GSSG to GSH. Considerable differences were observed between the oxidized and reduced form of glutathione in the coordination ability towards metal ions.     

Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt-beta-lactamase catalyzed hydrolysis of beta-lactams

Metallo-beta-lactamases are native zinc enzymes that catalyze the hydrolysis of beta-lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted beta-lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pK(a) = 8.9 +/- 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H(2)O/D(2)O) of 5.2 +/- 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt beta-lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pK(a) 6.32 +/- 0.1 and 7.47 +/- 0.1 being in their deprotonated basic forms and one group of pK(a) 9.48 +/- 0.1 being in its protonated form.     

Preparation and spectroscopic characterization of a coupled binuclear center in cobalt(II)-substituted hemocyanin.

A binuclear cobalt derivative of arthropod hemocyanin (Hc) has been prepared by the reaction of apo-Hc with Co(II) in the presence of thiocyanate. The crude product of the reaction contains specifically and adventitiously bound metal, the latter being removable by EDTA treatment. The specifically bound Co(II) constitutes a binuclear metal center that exhibits optical and CD spectra typical in their absorption maxima and extinction coefficients of Co(II) complexes with near-tetrahedral geometry. The EPR spectrum of the binuclear Co(II) derivative contains a resonance at g approximately 13, which is characteristic of integer spin systems and indicates coupled metal ions; the excess Co(II) bound to crude products exhibits an EPR signal at g approximately 4. The time course of derivative formation was followed by EPR, optical and atomic absorption techniques, and by fluorimetry. The intensity of the optical absorption in the visible region due to Co(II) increases with increasing stoichiometry of specifically bound metal [up to 2 Co(II) per protein monomer], but the intensity of the Co(II) EPR signal increases only during the formation of a mononuclear derivative. As the reaction proceeds over approximately 100 h to the formation of the binuclear derivative, the EPR signal intensity decreases to 10% of the value expected for 2 mol of EPR-active Co(II)/mol of protein. The binuclear cobalt derivative cannot be reconstituted to native Hc with Cu(I), indicating the stable loading of Co(II) in the active site. EPR and optical spectroscopic evidence is presented showing that the binuclear derivative does not bind oxygen.     

Electronic and steric effects in cobalt Schiff bases complexes: Synthesis, characterization and catalytic activity of some cobalt(II) tetra-halogens-dimethyl salen complexes

The synthesis, characterization and catalytic activity of a series of tetra-halogeno-dimethyl salen cobalt (II) complexes are reported in this paper. The investigated complexes of cobalt (II) with Schiff bases are: αα′-di-methyl Salen cobalt (II) [Co(dMeSalen)], 3,3′,5,5′-tetra chloro α,α′-di-methyl Salen cobalt (II), [Co(tCldMeSalen)], 3,3′-di-bromo 5,5′-di-chloro α,α′-di-methyl Salen cobalt (II), [Co(tBrdMeSalen)], 3,3′,5,5′-tetra bromo α,α′-di-methyl Salen cobalt (II), [Co(tBrdMeSalen)] and 3,3′,5,5′-tetra iodo α,α′-di-methyl Salen cobalt (II), [Co(tIdMeSalen)] (where Salen is bis(salicylaldehyde)ethylenediamine). The characterization of the complexes was performed by elemental analysis, cyclic voltammetry, UV-Vis, IR and EPR spectroscopies. The study was made in DMF, and pyridine was used for coordination as axial base. The redox potential is influenced by the substituent grafted on aromatic ring and in the azomethynic position and also by the molecules coordinating in axial position (solvent, DMF, or pyridine). The catalytic oxygenation of 2,6-di-tert-butylphenol by these complexes leads to the obtention of benzoquinone and diphenoquinone products. The cobalt (II) complexes form reversible adducts with molecular oxygen.     

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.     

Folding strategy to prepare Co(II)-substituted metallo-beta-lactamase L1

In an effort to overcome previous problems with the preparation of Co(II)-substituted metallo-β-lactamase L1, two strategies were undertaken. Attempts to prepare Co(II)-substituted L1 using biological incorporation resulted in an enzyme that contained only 1 Eq of cobalt and exhibited no catalytic activity. Co(II)-substituted L1 could be prepared by refolding metal-free L1 in the presence of Co(II), and the resulting enzyme contained 1.8 Eq of cobalt, yielded a UV-Vis spectrum consistent with 5-coordinate Co(II), and exhibited a k_cat of 63 s⁻¹ and K_m of 20 μM when using nitrocefin as the substrate. Pre-steady-state fluorescence and UV-Vis studies demonstrated that refolded, Co(II)-substituted L1 uses the same kinetic mechanism as Zn(II)-containing L1, in which a reaction intermediate is formed when using nitrocefin as substrate. The described refolding strategy can be used to prepare other Co(II)-substituted Zn(II)-metalloenzymes, particularly those that contain a solvent-exposable disulfide, which often causes oxidation of Co(II) to Co(III).     

Cobalt substitution of mouse R2 ribonucleotide reductase as a model for the reactive diferrous state: spectroscopic and structural evidence for a ferromagnetically coupled dinuclear cobalt cluster

The R2 dimer of mouse ribonucleotide reductase contains a dinuclear iron-oxygen cluster and tyrosyl radical/subunit. The dinuclear diferrous form reacts with dioxygen to generate the tyrosyl radical essential for the catalytic reaction that occurs at the R1 dimer. It is important to understand how the reactivity toward oxygen is related to the crystal structure of the dinuclear cluster. For the mouse R2 protein, no structure has been available with a fully occupied dinuclear metal ion site. A cobalt substitution of mouse R2 was performed to produce a good model for the very air-sensitive diferrous form of the enzyme. X-band EPR and light absorption studies (epsilon(550 nm) = 100 mm(-1) cm(-1)/Co(II)) revealed a strong cooperative binding of cobalt to the dinuclear site. In perpendicular mode EPR, the axial signal from mouse R2 incubated with Co(II) showed a typical S = 3/2 Co(II) signal, and its low intensity indicated that the majority of the Co(II) bound to R2 is magnetically coupled. In parallel mode EPR, a typical integer spin signal (M(s) = +/-3) with g approximately 12 is observed at 3.6 K and 10 K, showing that the two Co(II) ions (S = 3/2) in the dinuclear site are ferromagnetically coupled. We have solved the 2.4 A crystal structure of the Co(II)-substituted R2 with a fully occupied dinuclear cluster. The bridging Co(II) carboxylate ligand Glu-267 adopts an altered orientation compared with its counterpart Glu-238 in Escherichia coli R2. This might be important for proper O(2) activation of the more exposed native diferrous site in mouse R2 compared with E. coli R2.     

Syntheses and crystal structures of mono-, di- and trinuclear cobalt complexes of a salen type ligand

Three cobalt complexes containing the salen type ligand, bis(salicylidene)-meso-1,2-diphenylethylenediaminato (mdpSal²⁻), are reported. The complexes differ in nuclearity and include the mononuclear, Co(mdpSal) (1), which contains a Co(II) metal center bound to one mdpSal⁻² ligand frame in a square planar geometry. The second complex is the dinuclear [Co(mdpSal)Cl]₂ (2) in which both cobalt ions have been oxidized to the +3 oxidation state. The overall geometry of complex 2 is an edge-sharing bioctahedron with the coordination sphere around each cobalt metal center consisting of one mdpSal⁻² ligand and one Cl⁻ ion. The shared edge between the Co(III) ions contains two bridging phenolate groups, one from each ligand frame. Complex 3 is a linear, mixed valence, trinuclear species, [Co(mdpSal)(OAc)(μ-OAc)]₂Co, with the oxidation states of the metal centers assigned as Co(III)-Co(II)-Co(III). The terminal Co(III) centers are equivalent with the central Co(II) lying on the inversion center of the molecule. Each cobalt ion in 3 adopts an octahedral geometry with the terminal Co(III) ions being bound to one mdpSal²⁻ ligand each. All phenolate groups bridge to the central Co(II). The coordination sphere about each metal center in the trinuclear complex is completed by four acetate groups, two of which bind in a μ-fashion bridging from the terminal Co(III) metal centers to the central Co(II). The complexes have been characterized by X-ray crystallography as well as UV-Vis and IR spectroscopy.     

The R-state proinsulin and insulin hexamers mimic the carbonic anhydrase active site

The cobalt(II)-substituted proinsulin and insulin hexamers have been studied in solution via electronic absorption spectroscopy. Hexameric proinsulin is shown to undergo the phenol-induced T6 to R6 conformational transition in a manner analogous to that previously established for insulin. In the absence of coordinating anions, the coordination spheres of the Co(II) ions in the proinsulin and insulin R6 hexamers comprise identical pseudotetrahedral arrangements of 3 histidine residues and 1 hydroxide ion. At alkaline pH, the visible absorption spectrum of the phenol-induced R6 Co(II) center is strikingly similar to the distinctive spectrum of the alkaline form of Co(II)-carbonic anhydrase. Exogenous ligands may coordinate to the Co(II) ions of the R6 proinsulin and insulin hexamers via replacement of the hydroxide ion, forming pseudotetrahedral adducts possessing characteristic spectra. The binding affinity of such ligands is shown to be strongly pH-dependent. The data presented establish that, although the Co(II)-substituted proinsulin and insulin R6 hexamers lack enzyme-like activity, these species duplicate spectrochemical characteristics of the Co(II)-carbonic anhydrase active site that are believed to be important signatures of carbonic anhydrase catalytic function.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.