Methylcobalamin reduction

Figure 2-13 (a) Methylcobalamin. (b) Mechanism of methylcobalamin reduction in nonaqueous solution. 

In 1933 Challenger et al. discovered that trimethylarsine was synthesized from inorganic arsenic compounds by molds (93). Recently, McBride and Wolfe (94), have reported the synthesis of dimethylarsine from arsenate by cell extracts of the methanogenic bacterium M. O. H. Methylcobalamin is the alkylating coenzyme for this synthesis which requires reduction of arsenate to arsenite, methylation of arsenite to methylarsonic acid, reduction and methylation of methylarsonic acid to dimethylarsinic acid, and finally a four electron reduction of dimethylarsinic acid to dimethylarsine (Fig. 13). 

The mechanisms of transmethylation involving methylcobalamin have been extensively discussed by Wood and co-workers (189-191). They proposed a classification based on the standard reduction potentials for the elements (Table IV) and having three types of reaction ... 

A metal-carbon bond splitting is the first step in the sequence leading from methylcobalamin Im-Co(corrin)-CH3 to acetylcobalamin Im-Co(corrin)-COCH3 [117]. The radical CH3 formed in the primary photoredox step, associated with the reduction of Co(III) to Co(II), is trapped by a CO molecule and the redox addition of the radical CH3CO to the reduced pentacoordinated complex Co(II) results in the final Co(III) acetyl complex. 

Figure 21-3. The methionine synthase reaction. Methionine synthase catalyzes the remethylation of homocysteine to methionine. In the first half reaction (1), a methyl group is transferred from 5-methyl tetrahydrofolate (5-MTHF) to the reduced form of cobalamin [Cob(I)], generating methyl-cobalamin [Methyl-Cob(III)] and tetrahydrofolate (THF). During the second half reaction (2), the methyl group is transferred from methylcobalamin to homocysteine, generating methionine. During the catalytic reaction, Cob(I) occasionally becomes oxidized, producing an inactive form of cobalamin, cob(II)alamin [Cob(II)]. The enzyme methionine synthase reductase (MTRR) then reactivates Cob(II) through reductive methylation, producing methyl-Cob(III). SAM, 5-adenosylmethionine SAH, 5-adeno-sylhomocysteine.

A significant observation for cobaloximes as B12 models was the reduction of MeCo L4(B) by Cr(aq) +, which gave Co L4(B) and Cr(Me)(aq) + (equation 37). These products are indicative of an inner-sphere (see Inner-sphere Reaction) electron transfer with Me as the bridging ligand. This result has promoted further studies, notably methyl transfer between cobaloximes and nickel tetraazacycle complexes, which provides a possible model for the methylcobalamin alkylation of CO hydrogenase. ... 

The first evidence for cobalamin involvement in the conversion of methanol to methane was provided by Blaylock and Stadtman [196,216-218] with extracts of methanol-grown M. barkeri they demonstrated enzymatic formation of methylcobalamin from methanol, and subsequent reduction of methylcobalamin to methane. Later Blaylock [196] showed that conversion of methanol to methylcobalamin requires a heat-stable cofactor and at least three proteins, a 100-200 kDa Bi2-enzyme (methyltransferase), a ferredoxin, and an unidentified protein. Blaylock speculated that the role of hydrogen and ferredoxin in the conversion of methanol to methylcobalamin was in the reduction of the Bi2-protein. This work led to the proposal that methylcobalamin was the direct precursor of methane in methanogenesis from various substrates [196,218]. 

In reaction mixtures which contained arsenate the reaction mixture turned brown, indicating the transfer of the methyl group from methylcobalamin. Methane formation was inhibited. Of the various methyl donors tested as substrates only methylcobalamin could form an alkyl arsine of the arsenic derivatives tested as substrates only cacodylic acid was reduced directly to an alkyl arsine without the addition of methylcobalamin. However, ATP and a hydrogen atmosphere were required, and the final alkyl arsine derivative was identified as dimethylarsine. The reductive pathway is as follows ... 

In biological systems the two-electron reduction may be accomplished by NADH and flavin adenine dinucleotide (FAD). The methyl donor is N -methylletrahydrofo(ate (CH -THF). The Co(Ill) corrinoid (methylcobalamin) can then partake in biomethyla-tion reactions ... 

Our laboratory was the first to demonstrate the ability of thimerosal to potently inhibit methionine synthase activity in cultured human neuronal cells (Waly et al., 2004). Subsequent research has shown that inhibition results from a reduction in GSH levels and impaired methylcobalamin synthesis, under conditions where methionine synthase activity is absolutely dependent upon methylcobalamin (M. Waly, unpublished observation). The fact that autistic subjects exhibit lower GSH levels and lower methionine synthase activity lends credence to the mercury... 

Methylcobalamin (2 c) can be isolated from microorganisms. Its largely covalent Co—CH3 bond undergoes all three possible types of reactions, namely homolysis, carbonium-ion transfer, and carbanion transfer (Scheme 1), thus including reduction and oxidation of cobalt, respectively. Thermal degradation of cobalamin preferentially yields methane and ethane as radical-type reaction products (cf. 

Scheme 1 Reaction of methylcobalamin in aqueous media (a) and (b) reduction of cobalt, (c) redox-neutral reaction (CH transfer Co(lll) —> Hg(ll)).

The roles of methylcobalamin in biological processes are still being discovered and are bound to become increasingly diverse. The story of methionine synthase and MetH has been enlightening and elegant. However, it is very likely the beginning of the story of methylcobalamin. The other methylcobalamin enzymes are involved in many different chemical processes, from methylation of non-nucleophilic atoms to reduction in methanogenesis. The elucidation of the roles of methylcobalamin will usher in new chemistry for the field of vitamin Bj2. 

The reactions described above all involve the displacement of a carbanion from the cobalt atom of methylcobalamin. These reactions occur under aerobic conditions with rate constants in the order of milliseconds. It is apparent that metals which react by electrophilic attack on the Co-C bond (SE2 mechanism) occur with the more oxidized state of the metal, i.e., Pb, Tl, Hg, Pd, which have standard reduction potentials greater than 0.8 volts. Because of the "base on - base off" equilibrium these reactions are pH dependent. 

Homolytic cleavage of the Co-C bond of methylcobalamin leads to methyl-radical transfer. For this reaction to occur, the attacking species must be a free radical, and so the generation of such a radical intermediate is necessary either by the one equivalent oxidation of a metal ion in the reduced state of a redox couple (e.g., > SnIII), or by a one equivalent reduction of a... 

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