No abstract

The methyl transfer from methylcobalamin to thiols has been reinvestigated. By use of methylcobalamin selectively enriched with 13C in the methyl moiety, the methyl transfer to thiols was followed by 13C NMR. The methyl transfer occurs in aqueous mildly alkaline (pH 8-12) solution, even in the complete absence of oxygen. 31P NMR and EPR studies demonstrate that cob(II)alamin is the final corrinoid product. However, the pH dependence of the methyl-transfer reaction from methylcobalamin to beta-mercaptoethanol is consistent only with a nucleophilic displacement of the methyl group by a thiolate anion, resulting in the heterolytic cleavage of the carbon-cobalt bond. Difference visible spectroscopic measurements of the reaction mixture suggest that cob(I)alamin is formed as an intermediate.

Carbon-13 NMR spectroscopy and phosphorus-31 NMR spectroscopy have been used to study the reaction of several alkylcobalamins with 2-mercaptoethanol. At alkaline pH, when the thiol is deprotonated, the alkyl-transfer reactions involve a nucleophilic attack of the thiolate anion on the Co-methylene carbon of the cobalamins, yielding alkyl thioethers and cob(II)alamin. In these nucleophilic displacement reactions cob(I)alamin is presumably formed as an intermediate. The higher alkylcobalamins react more slowly than methylcobalamin. The lower reactivity of ethyl- and propylcobalamin is probably the basis of the inhibition of the corrinoid-dependent methyl-transfer systems by propyl iodide. The transfer of the upper nucleoside ligand of adenosylcobalamin to 2-mercaptoethanol is a very slow process; S-adenosyl-mercaptoethanol and cob(II)alamin are the final products of the reaction. The dealkylation of (carboxymethyl)cobalamin is a much more facile reaction. At alkaline pH S-(carboxymethyl)mercaptoethanol and cob(II)alamin are produced, while at pH values below 8 the carbon-cobalt bond is cleaved reductively to acetate and cob(II)alamin. The reductive cleavage of the carbon-cobalt bond of (carboxymethyl)cobalamin by 2-mercaptoethanol is extremely fast when the cobalamin is in the "base-off" form. Because we have been unable to detect trans coordination of 2-mercaptoethanol, we favor a mechanism that involves a hydride attack on the Co-methylene carbon of (carboxymethyl)cobalamin rather than a trans attack of the thiol on the cobalt atom.

The stoicheiometries and kinetics of the reactions of cis-diamminediaquaplatinum(li) with adenosylcobalamin and a series of al kylcobalamins have been examined. The reactions proceed with a 1.3: 1 and a 1 : 1 stoicheiometry (PP: cobalamin) for the interaction between cis-[Pt(NH3),(0H2),]2+ and adenosylcobalamin and the alkylcobalamins, respectively. In all cases the interactions generate the ' base-off ' form of the organocobalamins. Our kinetic studies indicate that the reactions are first order in organocobalamin and first order in C~S-[P~(NH,),(OH~)~]~+ at low concentrations of Pt", but that they approach zero order as the Pttl concentration is increased. The kinetic data are interpreted in terms of a fast equilibrium between the alkylcobalamin and cis-[Pt(NH3)2(OH2)2]2+ followed by a rate-determining ligand exchange between N3 of the 5,6-dimethylbenzimidazole ligand and a co-ordinated H 2 0 of the platinum complex. The mechanism of the ' base-on ' -' base-off ' conversion for the adenosylcobalamin-diamminediaquaplatinum(ii) complex is different from that for the alkylcobalamin-diamminediaquaplatinum(tt) complexes. The interaction between adenosylcobalamin and cis-[Pt(NH,)2(OH2)2]2+ dramatically reduces the light sensitivity of the carbon-cobalt bond. Prolonged exposure of the adenosylcobalamin-diamminediaquaplatinum(ii) complex to visible light in the presence of air yields 5'-deoxy-5'-oxoadenosine as the only nucleoside product. No photolysis occurs in the absence of air. These observations suggest that even in the presence of only stoicheiometric amounts of cis-[Pt(NH3)2(OH2)2]2+I the platinum complex interacts with the 5'-deoxyadenosyl ligand of the coenzyme. Carbon-1 3 and 3 l P n.m.r. spectroscopy studies using [5'-13C]adenosylcobalamin show that the adenosylcobalamin-diamminediaquaplatinum(i1) complex consists of at least four ' base-off ' conformers. These ' base-off ' conformers are probably the consequence of a downward distortion of the corrin ring resulting from the interaction between the 5'-deoxyadenosyl ligand, the corrin ring, and the c ~s -' base-on ' * ' base-off ' conversion for all the organocobalamins examined. The adenosylcobalamin-diamminediaquaplatinum(r1) complex is no longer light sensitive in the absence of oxygen, while irradiation of the complex with visible light in the presence of oxygen yields 5'-deoxy-5'-oxoadenosine as the only nucleoside product.

The transfer of the methyl group from methylcobalamin to diaquocobinamide in aqueous solution has been demonstrated by proton, carbon-13, and phosphorus-31 nuclear magnetic resonance spectroscopy. The products of this reaction are aquocobalamin and the methylaquocobinamides. Dicyanocobinamide and the cyanoaquocobinamides do not serve as methyl acceptors, while ligands such as pyridine and histidine reduce the rate of the transfer reactions. The methyl transfer is not affected by oxidizing agents such as 02, N20, and H202, suggesting that the reaction does not involve free Co(I) or Co(II) corrinoids. The pH dependence of the rate of the transfer reaction from methylcobalamin to diaquocobinamide demonstrates that methylcobalamin in the "base-on" form and diaquocobinamide are the most effective methyl donor and acceptor, respectively. The most plausible mechanism for the transfer reaction involves the one-electron oxidation of methylcobalamin by diaquocobinamide to a methylcobalamin radical cation and cob(II)inamide. The very unstable methylcobalamin radical cation releases a methyl radical, which reacts with cob(II)inamide to generate the methylaquocobinamides.Methylcorrinoids, such as methylcobalamin and (5-methoxybenzimidazolyl)-Co-methylcobamide, serve as cofactors in several biochemical reactions. These reactions include the methylation of homocysteine (1), the formation of methane (2), and the synthesis of acetate from CO2 (3). In addition, methylcobalamin is involved in the biomethylation of heavy metals such as mercury(II) (4), arsenic(III), selenium(IV), and tellurium(IV) (5). The nonenzymatic methylation of several metals by methylcobalamin has also been described. For instance, Agnes et al. (6, 7), Taylor and Hanna (8), and Fanchiang et al. (9) showed that methylation of platinum by methylcobalamin required both platinum(II) and platinum(IV). In enzymic methyl transfer reactions the corrinoid cofactor serves alternately as an acceptor and as a donor of the methyl moiety, and thus a key feature in these reactions is formation and cleavage of the carbon-cobalt bond.Several (14), diaquocobinamide, and (methylauo)cobinamide (15).Methods. Pulse Fourier-transform 3C (62.9-MHz), 31p (101.3-MHz), and 1H (250.1-MHz) nuclear magnetic resonance spectra were obtained at 25°C with a Bruker WM250 spectrometer, locked to the resonance of internal 2H20. For the 13C NMR spectra the transients resulting from the application of 90°pulses (25 ,sec) in a spectral width of 15,000 Hz were accumulated as 16,384 data points in the time domain and transformed into an 8192-point spectrum. The data acquisition time was 541 msec with a 459-msec pulse delay. For the 31p spectra the transients resulting from the application of 900 pulses (27 ,sec) in a spectral width of 2000 Hz were accumulated as 8192 data points in the time domain and transformed into a 4096-point spectrum. The data acquisition time was 2.048 sec without a pulse delay. For the 1H spectra the transients resulting from the application of 900 pulses (4 ,usec) ...