Cobalamin reactivity

Most of the examples listed are pentacyanide, corrinoid, or DMG complexes. The axial ligands are not identified in the tables, but are as follows corrinoids, 5,6-dimethylbenziminazole (cobalamins), H O or none (cobinamides), (DMG)2, usually pyridine or H2O, less frequently NHj, imidazole, benzimidazole, PBuj, etc. The nature of the axial and equatorial ligands may have a striking effect on reactivity, but few direct comparisons are available these are discussed in the next section. 

On the other hand, lactamization occurs at C-8 in aqueous alkali. The 7-hydroxyethyl derivative reacts similarly to give a cyclic ether. The reaction does not necessarily require 02 and is inhibited by excess CN". For B12, some ligand dependence was observed, and the order of reactivity is cyano > aqua > alkyl, sulfonato cobalamin (no reaction). In addition, colour changes during the reaction imply participation of the cobalt atom, but the mechanism is not clear (Scheme 93). 

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.

As cobalt(III) complexes (d6), cobalamins strictly adhere to an octahedral geometry, with an axial benzimidazole nitrogen ligand. It appears that in protein-bound B12 this axial ligand is replaced by a histidine provided by the peptide chain, with consequent configurational changes that influence the reactivity of the... 

Cobalt accepts a methyl group from methyl-tetrahydrofolate, forming methyl Co +-cobalamin. Transfer of the methyl group onto homocysteine results in the formation of Co+-cobalamin, which can accept a methyl group from methyl-tetrahydrofolate to reform methyl Co +-cobalamin. However, except under strictly anaerobic conditions, demethylated Co+-cobalamin is susceptible to oxidation to Co +-cobalamin, which is catalyticaUy inactive. Reactivation of the enzyme requires reductive methylation, with S-adenosyl methionine as the methyl donor, and a flavoprotein linked to NADPH. For this reductive reactivation to occur, the dimethylbenzimidazole group of the coenzyme must be displaced from the cobalt atom by a histidine residue in the enzyme (Ludwig and Matthews, 1997). 

The biologically relevant reactivity of coenzyme B12 is associated with the homolysis of its Co-C bond, as well as with the recombination of the products of homolysis (Figure 8). The remarkable structural similarity between the corrinoid homolysis fragment Co"-cobalamin and the Co "-corrin part of coenzyme B12 contributes to lowering... 

The half-wave potential for the enzyme-bound Co VCo cobalamin couple of the methionine synthase from E. coli at 526 mV versus SHE is about 80 mV lower than that of the Co /Cokcobalamin couple in neutral aqueous solution. Access to the catalytic cycle of the enzyme by one-electron reduction of Co kcobalamin (and reactivation upon occasional adventitious formation of Co -cobalamin) is indicated to be accomplished by a unique mechanism. The (thermodynamically unfavorable) reduction with intermediate formation of the enzyme-bound Cokcobalamin is driven by a rapid methylation of the highly reduced Co -center of the reduced corrin with Y-adenosyhnethionine. The modular nature of methionine synthase allows for the control of the methyl-group transfer processes by modulating and alternating conformational equilibria. ... 

The active LCo complexes indicated above can be used to test this theory. Porphyrins and phthalocya-nines have an O-shaped system which has a more extended -system than that in cobalamins, but it does not provide a substantial increase in reactivity. It should be noted that the hydrogen bonds of the cobaloxime catalysts are essentially as effective as 7r-bonds in continuing the effects of delocalization around the macrocyclic ring. This effect has been noted elsewhere.142 Catalyst 11 comprises an O-shaped -system. Replacement of one jr-bond with a a-bond in the analogue 13 significantly affects the catalytic properties since both complexes retain their O-shape with -conjugation. Additional replacement of "T-bonds with o-bonds leads to a complete loss of catalytic properties as chelates 13, 20, or 21 indicate. Chelate 22, cannot be a CCT catalyst because of the absence of interaction between the two jr-systems. Chelate 34 is an exception its molecular structure is similar to 21 and 13, but it catalyzes chain transfer with a measurable rate. A possible explanation of this phenomenon will be provided in section 3.7. 

Methionine synthase is composed of five structural domains that provide for binding of its substrate HCY, the methyl donor 5-methyItetrahydrofolate, cobal-amin, and SAM (Fig. 4). In most tissues SAM is utilized to methylate oxidized cobalamin, in conjunction with electron donation by methionine synthase reductase, thereby restoring methylcobalamin and allowing resumption of activity. This mode of reactivation is required approximately every 100-1,000 turnovers, even under strictly anaerobic laboratory conditions (Bandarian et al., 2003). Under physiological conditions, oxidation of cobalamin is undoubtedly much more common, illustrating how vitamin B12 serves as a sensor of redox status. During oxidative stress, cobalamin is more frequently oxidized and more HCY is diverted toward cysteine and GSH synthesis. 

While four domains of methionine synthase bind reaction components (HCY, SAM, cobalamin, and methylfolate), the fifth domain, known as the Cap domain, hovers above cobalamin while it is in its readily oxidized Cob(I) state, limiting access of reactive oxygen species or electrophilic substances. As such, the Cap domain restricts inactivation of methionine synthase and consequently promotes methylation over transsulfuration. In rt-PCR smdies using RNA from cultured human neuroblastoma cells, we found that the Cap sequence, corresponding to... 

All non-heme iron containing ribonucleotide reductases are also inhibited by hydroxyurea and related hydroxamates, while the adenosyl-cobalamin-dependent reductases are not affected (27, 156). The inhibition by these reagents can be partially reversed by excess Fe+2 or dithiols. Reaction of ribonucleotide reductase of E. coli with [14C]hydro-xyurea inactivated only the B2 subunit and this inactivation was not reversed by removal of the radioactivity (157). Inactivation by hydroxyurea does not affect the iron content of protein B2, but involves the destruction of the stable free radical (66,67). Reactivation can be accomplished by removal of the iron and reconstitution of apoprotein B2 with Fe+2. Hydroxyurea has been demonstrated to be a powerful radical scavenger in another system (158). 

Cleavage of the Co—C bond in carboxymethylcobalamin by thiols is more complex, involving both a nucleophilic attack by thiolate and a reductive cleavage of the Co—C bond. At low pH, cobalamin is in the base-off form, facile reductive cleavage gives acetate and cob(II)alamin as products. At high pH (pH > 8), S-(carboxyme-thyl)mercaptoethanol and cob(II)alamin from . The presence of base-on (pH > 8) and base-off (pH < 7) forms may be responsible for the different pH dependent reactivities . 

In those cases where the methyl group donor is chemically less reactive, as with iV -methyl-THF and methanol, retention of configuration is observed (Table XII). For the cobalamin-dependent methionine synthase, retention of configuration is consistent with a postulated mechanism for this enzyme involving two sequential transfers of the methyl group, one from fV -methyI-THF to cobalt to generate methylcobalamin and a second from cobalt to the sulfur of homocysteine (349) [Eq. (66)] ... 

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