Vitamin B12, the ‘antipernicious anaemia factor’, is required for human and animal metabolism. It was discovered in the late 1940s and its unique corrin ligand was revealed approx. 10 years later by X-ray crystallography. The B12-coenzymes are cofactors in various important enzymatic reactions and are particularly relevant in the metabolism of anaerobic microorganisms. Microorganisms are the only natural sources of the B12-derivatives, whereas most spheres of life (except for the higher plants) depend on these cobalt corrinoids.
- bio-organometallic chemistry
- cobalt corrin
- coenzyme B12
- methyl transfer
- radical reaction
- vitamin B12
The cyanide-containing vitamin B12 (cyanocobalamin) is a relatively inert Co(III)-corrin, which has no direct cofactor role. The physiologically relevant B12-derivatives are the light sensitive and chemically more labile organometallic cofactors, coenzyme B12 (5′-deoxy-5′-adenosylcobalamin) and methylcobalamin. The natural B12-derivatives are either ‘complete’ or ‘incomplete’ corrinoids, which lack a nucleotide function. The natural ‘complete’ corrinoids may vary by the constitution of the ‘nucleotide base’ as well as by the ‘nucleotide linker’, in addition to their functional β-axial ligand. In cobalamins, such as vitamin B12, the most common of the ‘complete’ corrinoids, 5,6-dimethylbenzimidazole is the ‘nucleotide base’; in pseudovitamin B12, an adenine is found instead (see Figure 1) [1–3].
The ‘complete’ corrinoids are also unique due to the unusual α-configuration of their (pseudo)nucleotide appendage. The specific build-up of this function enables the heterocyclic base to bind in an intramolecular fashion to the corrin-bound cobalt centre. The nucleotide function steers the organometallic reactivity at the cobalt centre and is also relevant for recognition and tight binding by the B12-binding proteins .
B12: structure and reactivity
The structures of vitamin B12 and of coenzyme B12 were established by X-ray crystallography in the laboratory of D.C. Hodgkin. This work helped to clarify the nature of the corrin ligand and to discover the organometallic nature of coenzyme B12 (Figure 1). The crystal structures of methylcobalamin and other Co(III)cobalamins were analysed, in order to obtain structural details on the axial bonding at the corrin-bound cobalt centre and on the structure of the corrin ligand. Structural implications for B12-catalysed enzymatic reactions were derived from the data. In this latter context, the structure of the oxygen-sensitive Co(II)-corrin B12r [cob(II)alamin] was of particular interest, the corrinoid moiety resulting (in a formal sense) from (Co–C)-bond homolysis of coenzyme B12 . Detailed information on the structure of cob(I)alamin (B12s) is still lacking.
UV/visible and CD spectroscopy were used to study the coloured and chiral B12-derivatives in solution. NMR spectroscopy and MS helped to identify the diamagnetic Co(III)-form of corrinoids from a range of anaerobes and to characterize their solution structures. The Co(II)-forms, in turn, have been investigated by ESR spectroscopy, a technique used increasingly to analyse for paramagnetic intermediates in B12-catalysed enzymatic reactions [2,6].
B12-derivatives in electron transfer reactions
Under physiological conditions B12-derivatives are known to exist in three different oxidation states: Co(III), Co(II) or Co(I). Oxidation–reduction processes are thus of key importance for the chemistry and biology of B12. Axial co-ordination to the corrin-bound cobalt centre depends on the formal oxidation state of the cobalt ion: as a rule, the number of axial ligands decreases in parallel with the cobalt oxidation state. In the thermodynamically predominating forms of cobalt corrins, two axial ligands are bound to the Co(III)-centre, one axial ligand is bound to the Co(II)-centre and axial ligands are assumed to be absent from the Co(I)-centre. Electron transfer reactions involving B12-derivatives therefore are accompanied by changes in the number of axial ligands and depend upon the nature of axial ligands. Axial co-ordination of the nucleotide base and of strongly co-ordinating ligands stabilizes the corrin-bound cobalt centre against reduction. The reduction of alkyl-Co(III)corrins typically occurs at potentials more negative than that of the Co(II)/Co(I)-redox-pairs, such as B12r/B12s .
Organometallic reactions of B12-derivatives
Much of the biological activity of the B12-dependent enzymes can be traced back directly to the organometallic reactivity of B12-derivatives. Formation and cleavage of the (Co–C)-bond in organometallic B12-derivatives was studied on all relevant oxidation levels of the cobalt-centre. Two basic reaction modes are essential steps of the reactions catalysed by most B12-dependent enzymes and are of particular interest: (i) the homolytic mode of formation/cleavage of the organometallic axial bond at the cobalt centre (formally a one-electron reduction/oxidation of the metal centre, see Figure 2) is of particular importance for the role of coenzyme B12 as a cofactor: coenzyme B12 is considered a ‘reversible carrier of an alkyl radical’. The (Co–C)-bond of coenzyme B12 has been determined to be approx. 30 kcal/mol (1 cal=4.184 J) strong and was affected only slightly by the co-ordination of the nucleotide. The reactions of B12r with alkyl radicals (such as the 5′-deoxy-5′-adenosyl radical) are very fast. Indeed, the radicaloid B12r is an efficient ‘radical trap’ and its reactions with radicals occur with minimal restructuring of the cobalt corrin moiety [4,8]; (ii) the heterolytic mode of formation, nucleophile-induced cleavage of the (Co–C)-bond at the cobalt centre (formally a two-electron reduction/oxidation of the metal-ion) involves the formation/cleavage of two axial bonds (see Figure 2). This mode is particularly important in enzyme-catalysed methyl-transfer reactions and is represented by the reaction of Co(I)-corrins with alkylating agents and by the nucleophile-induced demethylation of methyl-Co(III)-corrins. Alkylation at the (‘supernucleophilic’) Co(I)-centre usually occurs via ‘classical’ bimolecular nucleophilic substitution (SN2). The intramolecular co-ordination of dimethylbenzimidazole stabilizes ‘base-on’ methylcobalamin by about 4 kcal/mol and has a notable thermodynamic effect on heterolytic reactions of methylcobalamin .
Alkyl-Co(III)-corrins are rather resistant to proteolytic cleavage of the (Co–C)-bond under physiological conditions, crucial for the cofactor role of the B12-coenzymes. However, (visible) light induces the cleavage of the (Co–C)-bond of organometallic B12-derivatives.
B12-dependent methyl transferases
B12-dependent enzymatic methyl group transfer is relevant in many organisms. Indeed, B12-derivatives are well suited as cofactors in enzymatic methyl group transfer reactions (see above). Anaerobic acetogenesis, methanogenesis and catabolism of acetic acid to methane and carbon dioxide depend on B12-catalysed enzymatic methyl transfer reactions. Various substrates act as sources of methyl groups, such as methanol, aromatic methyl ethers, methyl amines or N5-methyltetrahydropterins (such as N5-methyltetrahydrofolate). Thiols are the methyl group acceptors in methanogenesis and in methionine synthesis. In the anaerobic biosynthesis of acetyl-CoA from one-carbon precursors the nickel-centre of an Fe/Ni-cluster appears to be the methyl group acceptor.
B12-dependent methionine synthesis is one of the two known B12-dependent enzymes in mammals. B12-dependent MetH (methionine synthase) of Escherichia coli catalyses methyl transfer by a sequential mechanism in which homocysteine and N5-methyltetrahydrofolate act as methyl group acceptors and donors, and tetrahydrofolate and methionine are formed (see Figure 3). MetH is a modular protein, where the B12-binding domain is bound to N5-methyltetrahydrofolate binding, homocysteine binding and reactivating modules (the latter binds S-adenosylmethionine). During turnover MetH catalyses two methyl group transfer steps, which occur with an overall retention of configuration (consistent with two SN2 steps) with heterolytic cleavage/formation of the (Co–CH3)-bond .
X-ray crystal analysis of the B12-binding domain of MetH provided the first insight into the structure of a B12-binding protein [9,10]. It revealed the cobalt-co-ordinating nucleotide of bound methylcobalamin to be displaced by a histidine residue and to be anchored in a pocket of the protein. Accordingly, in MetH, the corrinoid cofactor is bound in a ‘base-off/His-on’ constitution. The crucial cobalt-ligating histidine residue is part of a GXXHXD sequence, a conserved motif in the proteins that bind B12 in the ‘base-off/His-on’ mode.
Coenzyme B12-dependent enzymes
Ten coenzyme B12-dependent enzymes are now known: four carbon skeleton mutases, diol dehydratase, glycerol dehydratase, ethanolamine ammonia lyase, two amino mutases and B12-dependent ribonucleotide reductase. MMCM (methylmalonyl-CoA mutase) is the only human adenosylcobamide-dependent enzyme .
Coenzyme B12-dependent enzymes perform transformations that are difficult to achieve by typical ‘organic’ reactions. With the exception of the enzymatic ribonucleotide reduction, the results of the coenzyme B12-catalysed enzymatic reactions correspond to isomerizations with vicinal exchange of a hydrogen atom and of a group with heavy atom centres. Homolytic cleavage of the (Co–C)-bond of the protein-bound coenzyme B12 provides a 5′-deoxy-5′-adenosyl radical and B12r. The coenzyme B12-dependent enzymes then rely upon the reactivity of bound organic radicals, which are formed (directly or indirectly) by a H-atom abstraction by the 5′-deoxy-5′-adenosyl radical. The substrate radicals rearrange rapidly to the product radicals with little participation of the bound B12r. The major tasks of the enzymes thus concern the enhancement of the critical radical reactions, the reversible generation of the radical intermediates and the protection of the proteins from non-specific radical chemistry [11,12].
The homolysis of the (Co–C)-bond of coenzyme B12 is much faster in the protein-bound state than in aqueous solution: the means of activation are still enigmatic. A model according to which the protein binds well with the separated fragments of the homolysed coenzyme B12 has broad experimental support. Indeed, analogues of coenzyme B12, where the organometallic attachment of the adenine portion is a flexible and extended chain, bind to the apoenzymes intact and are inhibitors [12,13].
In coenzyme B12-dependent carbon skeleton mutases, such as MMCM (which interconverts R-methylmalonyl-CoA and succinyl-CoA), binding of the substrate triggers the homolysis of the (Co–C)-bond of the bound coenzyme B12. The radical carbon skeleton rearrangement reaction then proceeds as outlined in Figure 3.
All coenzyme B12-dependent carbon skeleton mutases [MMCM, GM (glutamate mutase), methyleneglutarate mutase, isobutyryl-CoA mutase] feature the B12-binding motif (GXXHXD) and a ‘base-off/His-on’-form of the bound B12-cofactor. Sequence homology, as exhibited by the B12-binding domains of MMCM, GM and MetH, is not found for the substrate binding domains of these enzymes. The crystal structures of MMCM (from Propionibacterium shermanii) and GM (from Clostridium cochlearium) were determined. In GM with bound coenzyme B12, the (Co–C)-bond of the cofactor is stretched to lengths of approx. 3.2 and 4.2 Å (1 Å=0.1 nm) (or broken) and the ribose part of the 5′-deoxyadenosyl moiety is present in two conformations, related to each other by a pseudo-rotation of the furanose ring. In this way, a probable structural basis for the operation of the radical mechanism in this coenzyme B12-dependent mutases is revealed. In contrast with MMCM and GM, in coenzyme B12-dependent diol dehydratase from Klebsiella oxytoca the corrinoid is bound ‘base-on’ [1,2,11,12,14].
Analysis of the solution structure of the B12-binding subunit of GM from Cl. tetanomorphum by heteronuclear NMR provided a structure of a cofactor-free B12-binding protein. These studies also indicated the B12-binding subunit to be largely preorganized for B12-binding and provided structural evidence of how coenzyme B12 would be recognized and bound ‘base-off/His-on’ .
Ribonucleotide reductases catalyse a key step in the biosynthesis of (all) DNA , the reduction of nucleoside di- or triphosphates to the corresponding 2′-deoxynucleotides. The RNR-Ll (ribonucleotide reductase from Lactobacillus leichmanii) uses coenzyme B12 as cofactor and nucleoside triphosphates as substrates, whereas 2′-deoxynucleoside triphosphates are allosteric effectors. ESR spectroscopy and crystallography showed ‘base-on’ binding of the B12-cofactor in RNR-Ll . In RNR-Ll, a protein centred thiyl-radical is generated from the homolysis of bound coenzyme B12 and then induces the radical steps that lead to the reductive removal of the 2′-hydroxy group of the ribonucleotide [1,2,16].
Other B12-dependent enzymatic transformations
Methanogens and acetogens dechlorinate chloromethanes by reduction with metal cofactors. The anaerobe Sulfurospirillum multivorans uses tetrachloroethene as the terminal electron acceptor. Its tetrachloroethene reductive dehalogenase is a corrinoid enzyme with norpseudo-B12, a novel corrinoid, as cofactor .
Interactions of B12 with nucleotides
The recent discovery of the so-called B12-riboswitches provided evidence for a new mechanism of genetic control and showed the direct interaction between coenzyme B12 and mRNAs to be biologically relevant . In view of the consideration of a pre-enzymatic origin of the basic elements of the B12-structure , the revelation of the existence of B12-responsive ribo-switches  and the successful in vitro-evolution of B12-binding RNA (‘B12-aptamers’)  all pointed (among other evidence) to the possibility of a functional role of B12-derivatives in the ‘RNA-world’. In this context, we very recently provided the complementary concept of ‘retro-riboswitching’ in covalent B12-nucleotide conjugates . These are of particular interest as models for B12-ribozymes  and point to a possible functional interaction of nucleotides with ‘complete’ B12-cofactors (which themselves are ‘molecular switches’).
B12: medical aspects
B12-deficiency is the cause for ‘pernicious anaemia’ and results (in most cases) from impaired uptake from food. In the human body, B12 is metabolically active as coenzyme B12 and as methylcobalamin [1,23].
Three soluble B12-binding proteins are known to be involved in the uptake and transport of cobalamins in humans: IF (intrinsic factor), TC (transcobalamin) and HC (haptocorrin). IF, TC and HC are genetically related B12-binders (apparent binding constants of >1012 l/mol). These proteins ensure that B12 reaches the two enzymes, MetH (in the cytosol) and methylmalonyl-CoA mutase (in the mitochondria). Intracellular B12-trafficking depends upon a complex interplay between the B12-binders and cellular surface receptors that recognize complexes between B12 and the B12-binding proteins .
I am grateful to the Austrian National Science Foundation (FWF, project P13595) for financial support.
Coenzymology: the biochemistry of vitamin biogenesis and cofactor-containing enzymes: Independent Meeting held at King's College, Cambridge, U.K., 4–7 April 2005. Organized and Edited by A.G. Smith (Cambridge, U.K.) and A.W. Munro (Leicester, U.K.).
Abbreviations: B12r, cob(II)alamin; GM, glutamate mutase; MetH, methionine synthase; MMCM, methyl-malonyl CoA mutase; RNR-Ll, ribonucleotide reductase of Lactobacillus leichmanii
- © 2005 The Biochemical Society