High Reactivity of Supermolecular Nanoentities of a Vitamin B12 Derivative in Langmuir-Schaefer Films Toward Gaseous Toxins.

Recently, we have described the first supermolecular nanoentities (SMEs) of a vitamin B12 derivative, viz., a monocyano form of heptabutyl cobyrinate ((CN-)BuCby), unique nanoparticles with strong noncovalent intermolecular interactions, and emerging optical and redox properties. In this work, the fast response of thin films based on the SMEs of the B12 derivative to gaseous toxins (viz., hydrogen cyanide, ammonia, sulfur dioxide, and hydrogen sulfide) particularly dangerous for humans was demonstrated. The reaction between SMEs of (CN-)BuCby in Langmuir-Schaefer (LS) films and HCN generates dicyano species and proceeds ca. 5-fold more rapidly than the process involving drop-coated films that contain (CN-)BuCby in molecular form. The highest sensitivity toward HCN was achieved by using thicker LS films. The reaction proceeds reversibly: upon exposure to air, the dicyano complex undergoes partial decyanation. The decyanated complex retains reactivity toward HCN for at least four subsequent cycles. The processes involving SMEs of (CN-)BuCby and NH3, SO2, and H2S are irreversible, and the sensitivity of the films toward these gases is lower in comparison with HCN. Presented data provides mechanistic information on the reactions involving solid vitamin B12 derivatives and gaseous toxins. In the case of NH3, deprotonation of the coordinated Co(III)-ion water molecule occurs, and the generated hydroxocyano species exhibit high air stability. After binding of SO2, a mixture of sulfito and dicyano species is produced, and the regenerated film contains aquacyano and diaqua or aquahydroxo species, which possess high reactivity toward gaseous toxins. Reaction with H2S produces a mixture of the Co(III)-dicyano form and Co(II)-species containing sulfide oxidation products, which are resistant to aerobic oxidation. Our findings can be used for the development of naked-eye, electronic optic, and chemiresistive sensors toward gaseous toxins with improved reactivity for prompt cyanide detection in air, blood, and plant samples and for analysis of exhaled gases for the diagnosis of diseases.

meso-Bromination of cyano- and aquacobalamins facilitates their processing into Co(II)-species by glutathione.

Cyanocobalamin (CNCbl), a medicinal form of vitamin B12, is resistant to glutathione (GSH), and undergoes intracellular processing via reductive decyanation producing the Co(II)-form of Cbl (Cbl(II)) mediated by the CblC-protein. Alteration of the CblC-protein structure might inhibit CNCbl processing. Here, we showed that introducing a bromine atom to the C10-position of the CNCbl corrin ring facilitates its reaction with GSH leading to the formation of Cbl(II) and cyanide dissociation. In a neutral medium, the reaction between C10-Br-CNCbl and GSH proceeds via the complexation of the reactants further leading to dimethylbenzimidazole (DMBI) substitution and electron transfer from GSH to the Co(III)-ion. The reaction is accelerated upon the GSH thiol group deprotonation. The key factors explaining the higher reactivity of C10-Br-CNCbl compared with unmodified CNCbl towards GSH are increasing the electrode potential of CNCbl two-electron reduction upon meso-bromination and the substantial labilization of DMBI, which was shown by comparing their reactions with cyanide and the pKa values of DMBI protonation (pKa base-off). Aquacobalamin (H2OCbl) brominated at the C10-position of the corrin reacts with GSH to give Cbl(II) via GSH complexation and subsequent reaction of this complex with a second GSH molecule, whereas unmodified H2OCbl generates glutathionyl-Cbl, which is resistant to further reduction by GSH.

Aquacobalamin Accelerates Orange II Destruction by Peroxymonosulfate via the Transient Formation of Secocorrinoid: A Mechanistic Study.

Besides its use in medicine, vitamin B12 (cobalamin) and its derivatives have found in numerous applications as catalysts. However, studies related to the activation of oxidants via cobalamin are scant. In this work, we showed how the addition of aquacobalamin (H2OCbl) accelerates the destruction of azo-dye Orange II by peroxymonosulfate (HSO5-) in aqueous solutions. In neutral and weakly alkaline media, the process is initiated by the modification of the corrin macrocycle with HSO5-, which requires the preliminary deprotonation of the aqua-ligand in H2OCbl to give hydroxocobalamin, producing 5,6-dioxo-5,6-secocobalamin or its isomer (14,15-dioxo-14,15-secocobalamin). In acidic solutions, where the concentration of hydroxocobalamin is negligible, the formation of dioxo-seco-species is not observed, and the reaction between H2OCbl and HSO5- results in slow chromophore bleaching. Using terephthalic acid, we demonstrated the formation of hydroxyl radicals in the mixture of H2OCbl with HSO5-, whereas the generation of sulfate radicals was proved by comparing the effects of ethanol and nitrobenzene on Orange II destruction using the H2OCbl/HSO5- system. The reaction mechanism includes the binding of HSO5- to the Co(III) ion of dioxo-secocobalamin, which results in its deprotonation and the labilization of the O-O bond, leading to the formation of sulfate and hydroxyl radicals which further react with Orange II.

Versatile enzymology and heterogeneous phenotypes in cobalamin complementation type C disease.

Nutritional deficiency and genetic errors that impair the transport, absorption, and utilization of vitamin B12 (B12) lead to hematological and neurological manifestations. The cblC disease (cobalamin complementation type C) is an autosomal recessive disorder caused by mutations and epi-mutations in the MMACHC gene and the most common inborn error of B12 metabolism. Pathogenic mutations in MMACHC disrupt enzymatic processing of B12, an indispensable step before micronutrient utilization by the two B12-dependent enzymes methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). As a result, patients with cblC disease exhibit plasma elevation of homocysteine (Hcy, substrate of MS) and methylmalonic acid (MMA, degradation product of methylmalonyl-CoA, substrate of MUT). The cblC disorder manifests early in childhood or in late adulthood with heterogeneous multi-organ involvement. This review covers current knowledge on the cblC disease, structure-function relationships of the MMACHC protein, the genotypic and phenotypic spectra in humans, experimental disease models, and promising therapies.

Mechanism of cyanocobalamin chlorination by hypochlorous acid.

Hypochlorous acid (HOCl) is a strong oxidant produced by myeloperoxidase. Previous work suggested that HOCl modifies the corrin ring of cobalamins to yield chlorinated species via mechanisms that are incompletely understood. Herein, we report a mechanistic study on the reaction between cyanocobalamin (CNCbl, vitamin B12) and HOCl. Under weakly acidic, neutral and weakly alkaline conditions, the reaction produces the c-lactone derivative of CNCbl chlorinated at the C10-position of corrin ring (C10-Cl-CNCbl-c-lactone). Formation of C10-Cl-CNCbl-c-lactone was not observed at pH ≥ 9.9. The chlorination of CNCbl by HOCl proceeds via two pathways involving one and two HOCl molecules: the reaction is initiated by the very fast formation of a complex between CNCbl and HOCl, which either undergoes slow transformation to chlorinated species, or rapidly reacts with a second HOCl molecule to produce C10-Cl-CNCbl. Subsequent reaction of C10-Cl-CNCbl with HOCl proceeds rapidly toward lactone ring formation by H-atom abstraction at position C8. This work uncovered mechanisms and products of the reaction of a biologically active and therapeutically used cobalamin, CNCbl and the endogenous oxidant HOCl. Binding and reactivity studies of C10-Cl-CNCbl and C10-Cl-CNCbl-c-lactone with relevant proteins of the cobalamin pathway and with cultured cells are necessary to elucidate the potential physiological effects of these species.

Catalytic effect of riboflavin on electron transfer from NADH to aquacobalamin.

Reduction of cobalamin by non-dedicated cellular reductases has been reported in earlier work, however, the sources of reducing power and the mechanisms are unknown. This study reports results of kinetic and mechanistic investigation of the reaction between aquacobalamin, H2OCbl, and reduced ß-nicotinamide adenine dinucleotide, NADH. This interaction leads to the formation of one-electron reduced cobalamin, cob(II)alamin, and proceeds via water substitution on aquacobalamin by NADH and further decomposition of NADH-Co(III) complex to cob(II)alamin and NADH·+. Riboflavin catalyzes the reduction of aquacobalamin by NADH both in free form and with aquacobalamin bound to the cobalamin processing enzyme CblC. The rate-determining step of this catalytic reaction is the interaction between riboflavin and NADH to produce a charge transfer complex that reacts with aquacobalamin. Aquacobalamin quenches the fluorescence of NADH and riboflavin predominantly via a static mechanism.

Characterization of the complex between native and reduced bovine serum albumin with aquacobalamin and evidence of dual tetrapyrrole binding.

Serum albumin binds to a variety of endogenous ligands and drugs. Human serum albumin (HSA) binds to heme via hydrophobic interactions and axial coordination of the iron center by protein residue Tyr161. Human serum albumin binds to another tetrapyrrole, cobalamin (Cbl), but the structural and functional properties of this complex are poorly understood. Herein, we investigate the reaction between aquacobalamin (H2OCbl) and bovine serum albumin (BSA, the bovine counterpart of HSA) using Ultraviolet-Visible and fluorescent spectroscopy, and electron paramagnetic resonance. The reaction between H2OCbl and BSA led to the formation of a BSA-Cbl(III) complex consistent with N-axial ligation (amino). Prior to the formation of this complex, the reactants participate in an additional binding event that has been examined by fluorescence spectroscopy. Binding of BSA to Cbl(III) reduced complex formation between the bound cobalamin and free cyanide to form cyanocobalamin (CNCbl), suggesting that the ß-axial position of the cobalamin may be occupied by an amino acid residue from the protein. Reaction of BSA containing reduced disulfide bonds with H2OCbl produces cob(II)alamin and disulfide with intermediate formation of thiolate Cbl(III)-BSA complex and its decomposition. Finally, in vitro studies showed that cobalamin binds to BSA only in the presence of an excess of protein, which is in contrast to heme binding to BSA that involves a 1:1 stoichiometry. In vitro formation of BSA-Cbl(III) complex does not preclude subsequent heme binding, which occurs without displacement of H2OCbl bound to BSA. These data suggest that the two tetrapyrroles interact with BSA in different binding pockets.

Studies on reaction of glutathionylcobalamin with hypochlorite. Evidence of protective action of glutathionyl-ligand against corrin modification by hypochlorite.

Glutathionylcobalamin (GSCbl), a tight complex of glutathione (GSH) with cobalamin(III), is readily oxidized to aquacobalamin by hypochlorite. Corrin macrocycle remains unmodified in the presence of threefold excess of hypochlorite, whereas aqua- and cyanocobalamins are partially transformed to chlorinated species under the same conditions. The suggested mechanism of reaction between GSCbl and hypochlorite involves subsequent oxidation of thiol and amino groups and dissociation of oxidized glutathione from Co(III)-ion.

Comparative studies of reaction of cobalamin (II) and cobinamide (II) with sulfur dioxide.

The kinetics of reactions of cobalamin (II) and cobinamide (II) with sulfur dioxide was studied by UV-visible (UV-vis) spectroscopy. Reaction results in oxidation of Co(II) center and involves two aquated SO2 moieties. The final product is suggested to be complex Co(III)-S2O 4•- . The absence of corrin ring modifications during the reactions was proved.

Reactions of aquacobalamin and cob(II)alamin with chlorite and chlorine dioxide.

Reactions of aquacobalamin (H2O-Cbl(III)) and its one-electron reduced form (cob(II)alamin, Cbl(II)) with chlorite (ClO2-) and chlorine dioxide (ClO 2• ) were studied by conventional and stopped-flow UV-Vis spectroscopies and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). ClO2- does not react with H2O-Cbl(III), but oxidizes Cbl(II) to H2O-Cbl(III) as a major product and corrin-modified species as minor products. The proposed mechanism of chlorite reduction involves formation of OCl- that modifies the corrin ring during the course of reaction with Cbl(II). H2O-Cbl(III) undergoes relatively slow destruction by ClO 2• via transient formation of oxygenated species, whereas reaction between Cbl(II) and ClO 2• proceeds extremely rapidly and leads to the oxidation of the Co(II)-center.

Kinetics and mechanism of oxidation of super-reduced cobalamin and cobinamide species by thiosulfate, sulfite and dithionite.

We studied the kinetics of reactions of cob(I)alamin and cob(I)inamide with thiosulfate, sulfite, and dithionite by UV-Visible (UV-Vis) and stopped-flow spectroscopy. We found that the two Co(I) species were oxidized by these sulfur-containing compounds to Co(II) forms: oxidation by excess thiosulfate leads to penta-coordinate complexes and oxidation by excess sulfite or dithionite leads to hexa-coordinate Co(II)-SO2(-) complexes. The net scheme involves transfer of three electrons in the case of oxidation by thiosulfate and one electron for oxidation by sulfite and dithionite. On the basis of kinetic data, the nature of the reactive oxidants was suggested, i.e., HS2O3(-) (for oxidation by thiosulfate), S2O5(2-), HSO3(-), and aquated SO2 (for oxidation by sulfite), and S2O4(2-) and SO2(-) (for oxidation by dithionite). No difference was observed in kinetics with cob(i)alamin or cob(i)inamide as reductants.

Comparative study of reaction of cobalamin and cobinamide with thiocyanate.

The interaction of Co(III) and Co(II) cobalamin (Cbl) and cobinamide (Cbi) with thiocyanate was examined with UV-vis and EPR spectra. S/N-linkage isomerism was explored on Co(III) and Co(II) Cbl and Cbi models using density functional theory (DFT; BP86, B3LYP). Performed calculations suggest the prevalence of isothiocyanato isomers over thiocyanato complexes on both Co(III) and Co(II) centers. The formation of Cbl(II) complex with thiocyanate was observed at high ligand concentrations which was proposed to be hexacoordinated. DFT data maintain the possibility of hexacoordinated Co(II) complexes with thiocyanate in which one of extra-ligands is weakly coordinated. It is found that high thiocyanate concentrations could retard cyanide binding to cobalamin but not to cobinamide.