Vitamin B12

Dorothy Hodgkin
Nobel Laureate
We dedicate this 'Molecule of the Month to the Memory of Dorothy Crowfoot Hodgkin, Chancellor of the University of Bristol 1971-1988 Dorothy Hodgkin
Bristol Chancellor

Cobalt - An Essential Element for Life

Cobalt appears centrally in the periodic table, and with its neighbours, iron, manganese, nickel and copper, has a central rôle in a number of biochemical metalloenzyme reactions.

The disease, pernicious anæmia, was first described in 1821, and was invariably fatal. In 1926 Minot and Murphy, following Whipple's observation that anæmic dogs could be cured by feeding them raw liver, discovered that pernicious anæmia could also be treated by supplementing the human diet with liver. An intensive search for the "liver factor" was started. However cobalt's vital status was not proved until after World War II, in 1948, when the 'anti-pernicious anæmia factor', which became called Vitamin B12, was finally purified and isolated as crystals by Folkers and his co-workers at Merck Laboratories, and by Smith and Parker at Glaxo Laboratories. Small red crystals of Vitamin B12 were then grown by Lester Smith and given to Dorothy Hodgkin for crystal structure analysis. All that was known at this stage was that the approximate empirical formula was C61-64H84-90N14O13-14PCo.

A crystal structure on a molecule of this size and complexity had never been attempted before, it was a huge and complex task, since crystal structure determinations were not the routine tasks that they are today, and the techniques were still being developed, both the X-ray and the computer equipment were tedious and difficult to use. Thus the x-ray crystal structure which emerged from this study between 1950 the early 1960s was the first determination of a chemical formula by X-ray diffraction, and the first determination of the structure of a metalloenzyme. It was a triumph for Dorothy Hodgkin and her Oxford x-ray crystallography group, inspiring many young crystallographers, and pointing them to biochemistry as an exciting new subject for their endevours. The structure work also caused Woodward (at Harvard) and Eschenmoser (at the Swiss Federal Institute of Technology) to start synthetic work on Vitamin B12. The synthesis took 11 more years, and involved more than 90 separate reactions performed by over 100 co-workers. The sterochemical puzzles involved in the synthesis led to the Woodward-Hoffman rules. This all adds up to three Nobel prizes in chemistry and one in medicine!

  1. Whipple(California), Minot and Murphy (Massachusetts) : (Physiology and Medicine) : the discovery of "anti-pernicious anæmia factor", now called Vitamin B12
  2. Dorothy Crowfoot Hodgkin (Oxford) : (Chemistry) : determinations by X-ray techniques of the structures of important biochemical substances.
  3. R.B.Woodward (Harvard) : (Chemistry) : outstanding achievements in the art of organic synthesis
  4. K.Fukui (Kyoto) and R.Hoffman (Cornell) : (Chemistry) : quantum mechanical studies of chemical reactivity

    Vitamin B12 and Diet

    We only need minute traces of Vitamin B12 in our diet, as little as 1µ gram per day, and provided we absorb this in our diet, all is well. If the stomach does not secrete hydrochloric acid properly, and the intestine thus not absorb the vitamin, then pernicious anæmia results. In cattle, sheep and other ruminants, microorganisms present in their rumen, can synthesise the vitamin, which is then used particularly for the methylmalonic acid to succinic acid isomerisation step, one of the isomerization steps catalysed by Vitamin B12. Humans do not have such microorganisms in their digestive system, so must absorb the vitamin from food.

    Methanogens are a primitive type of bacteria, archeobacteria, capable of using methane at the end of their electron acceptor chain, they involve a nickel corrinoid (F430) and Vitamin B12 in the catalysis of the reaction :

    4H2 + CO2 = CH4 + 2H2O

    A cow produces about 40 litres of methane per day, and methane is a potent atmospheric 'greenhouse' gas!

    Structural Details

    Vitamin B12 Vitamin B12 is the only known biomolecule with a stable carbon-metal bond - it is an organometallic compound. The core of the molecule is a corrin ring with various attached sidegroups. The ring consists of 4 pyrrole subunits, joined on opposite sides by a C-CH3 methylene link, on one side by a C-H methylene link, and with the two of the pyrroles joined directly. It is thus like a porphyrin, but with one of the bridging methylene groups removed. The nitrogen of each pyrolle is coordinated to the central cobalt atom. The sixth ligand below the ring is a nitrogen of a 5,6-dimethylbenzimidazole. The other nitrogen is linked to a five-carbon sugar, which in turn connects to a phosphate group, and thence back onto the corrin ring via one of the seven amide groups attached to the periphery of the corrin ring. The base ligand thus forms a 'strap' back onto the corrin ring.

    An important aspect of the corrin ring, when compared to the porphyrin, is the relative flexibility of the corrin system, the corrin ring is also less flat when viewed from the side than is a porphyrin ring. This adds up to some considerable differences between the chemistry of a cobalt porphyrin and a cobalt corrin.

    In addition, the corrin only has a conjugated chain around part of the ring system, whereas a porphyrin is delocalised around the whole four pyrolle rings.


    The first type of Vitamin B12 to be isolated had a cyanide group attached to the cobalt, this was picked up during the purification of the vitamin, and is the form which is still referred to as "Vitamin B12". The commercial form of the vitamin is usually obtained as the cyanide, it metabolises easily to the coenzyme. Smokers should note that the cyanide absorbed from the smoke into the blood causes the replacement of the 5'-deoxyadenosyl group by a cyanide.
    The principal coenzymatic form of Vitamin B12 is 5'-deoxyadenosylcobalamin. The notable feature of the molecule is the cobalt-carbon bond between the 5' carbon of the 5'-deoxyadenosyl moiety (the sugar part) and the cobalt of cobalamin. In Vitamin B12 as it is extracted, a cyanide replaces this sugar link, this occurs during the final purification with active charcoal. Aquocobalamin and hydroxycobalamin, with water and hydroxide are also known, as is the methylated form, methylcobalamin. A number of other cobalt-carbon bonded have been prepared synthetically, but are not known to occur in vivo

    Vitamin B12 in its various forms, and in cooperation with its coenzymes and various different substrates is involved in three different forms of reaction:

    1. several mutases in which a hydrogen and some group on an adjacent carbon exchange places, This can be followed by an elimination of water or ammonia.
    2. Ribonucleotide reductase, a reductase whereby ribose is reduced to deoxyribose.
    3. Methyl group transfers
      • Methionine synthetase
      • Methane synthetase
      • Acetate synthetase

    The first two reactions involve a Co(II) oxidation state intermediate, the third probably involves a Co(I), and in both in its 'resting state' the cobalt is a Co(III). Central to the catalytic rôle of is the relative weakness of the cobalt-carbon bond, with a dissociation energy of around 120 kJmol-1. In several of the rearrangements, and in the reductase, EPR signals have been observed for the deoxyadenosyl radical and for a cobalt(II) corrin.

    In the hydrogen migrations in all of the Vitamin B12 coenzyme-dependent rearrangement reactions, there is no exchange of the hydrogen with water protons, and the 1,2 intramolecular shifts are stereospecific for both the hydrogen atom and the exchanging group. Different reactions also proceed with either retention or inversion of the configuration.

    The mechanistic picture which emerges from this is that the rearrangement involves a kind of 'reaction in a bottle', between the Vitamin B12 and the substrate, with the large coenzyme acting as the bottle. Co(II) and free radicals are very oxygen sensitive, so that the whole process must be kept anaerobic.

    Model Complexes

    The simplist model for Vitamin B12 is the [MeCo(CN)5]3- ion, and an early observation was that this complex will transfer its methyl group to an Hg(II), generating the highly poisonous [MeHg+] moiety. A mercury methylation forming this highly toxic ion, and also involving methylcobalamin, and the related F430 metalloenzyme F430 is believed to be behind the Minamata tragedy in Japan.

    The principle models used for Vitamin B12 are the alkylcobalt(III)dimethylglyoxinates, so called alkylcobaloximes, which have the advantages of easy preparation and simplicity. methyl cobaloxime The methyl complex photolyses readily to form ethane, and the methyl radicals can be trapped with spin-traps such as PBN. reaction with Hg(II) forms the highly toxic MeHg(II) ion, modeling what happens when Hg(II) attacks methyl Vitamin B12. Of course none of these will ever provide substitutes for Vitamin B12, but they do provide platforms on which various aspects of the mechanisms of the Vitamin B12 catalysed reactions can be studied.

    Other Vitamin B12 Information

    More information of the structure and component parts of vitamin B12 can be found as a Chime-enhanced page on the Oxford MOTM site.


    There is a huge literature about Vitamin B12. Here are some key references :
    1. G.Dodson, J.P.Glusker and D Sayre (Eds), "Structural studies on molecules of biological interest - A volume in honour of Dorothy Hodgkin", Clarendon, Oxford, 1981.
    2. D.Dolphin (Ed), B12.Vols I and II, John Wiley, New York, 1982.
    3. B.T.Goldring, "The B12 Mystery", Chemistry in Britain, 950-4, 1990.
    4. J.M.Pratt, Inorganic Chemistry of Vitamin B12, Academic Press, New York, 1972.
    5. Ei-Ichiro Ochiai, "Vitamin B12 and B12 Coenzymes", Chapter 12, Bioinorganic Chemistry, An Introduction, Allyn and Bacon, Boston, 1977
    6. J.J.R.Frausto da Silva and R.J.P.Williams, The Biological Chemistry of the Elements, The Inorganic Chemistry of Life, Chapter 16, Nickel and Cobalt : remnants of Early Life?, Clarendon, Oxford, 1991.
    7. G.Zubay, (Ed), Biochemistry, Macmillan, New York, 1988.
    8. S.J.Lippard and J.M.Berg, "Principles of Bioinorganic Chemistry", p336-343 University Science Books, Mill Valley, California, 1994