Methyl Transfer from Methylcobalamin to Diaquocobinamide (Methylation/Vitamin B-12/Corrinoids) YUEH-TAI FANCHIANG, GERALD T

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 2698-2702, May 1984 Biochemistry

Methyl transfer from methylcobalamin to diaquocobinamide (methylation/vitamin B-12/corrinoids) YUEH-TAI FANCHIANG, GERALD T. BRATT, AND HARRY P. C. HOGENKAMP* Department of Biochemistry, The University of Minnesota, Minneapolis, MN 55455 Communicated by H. A. Barker, January 13, 1984

ABSTRACT The transfer of the methyl group from meth- EXPERIMENTAL PROCEDURES ylcobalamin to diaquocobinamide in aqueous solution has been Materials. Cyanocobalamin was purchased from Rhone- demonstrated by proton, carbon-13, and phosphorus-31 nu- Poulenc Industries (Paris). The corrinoids and their 13C-en- clear magnetic resonance spectroscopy. The products of this riched derivatives were prepared from cyanocobalamin by reaction are aquocobalamin and the methylaquocobinamides. published procedures: methylcobalamin (12), methylepico- Dicyanocobinamide and the cyanoaquocobinamides do not balamin (13), dicyanocobinamide, (cyanoaquo)cobinamide serve as methyl acceptors, while ligands such as pyridine and (14), diaquocobinamide, and (methylauo)cobinamide (15). histidine reduce the rate of the transfer reactions. The methyl Methods. Pulse Fourier-transform 3C (62.9-MHz), 31p transfer is not affected by oxidizing agents such as 02, N20, (101.3-MHz), and 1H (250.1-MHz) nuclear magnetic reso- and H202, suggesting that the reaction does not involve free nance spectra were obtained at 25°C with a Bruker WM250 Co(I) or Co(II) corrinoids. The pH dependence of the rate of spectrometer, locked to the resonance of internal 2H20. For the transfer reaction from methylcobalamin to diaquocobina- the 13C NMR spectra the transients resulting from the appli- mide demonstrates that methylcobalamin in the "base-on" cation of 90° pulses (25 ,sec) in a spectral width of 15,000 Hz form and diaquocobinamide are the most effective methyl do- were accumulated as 16,384 data points in the time domain nor and acceptor, respectively. The most plausible mechanism and transformed into an 8192-point spectrum. The data ac- for the transfer reaction involves the one-electron oxidation of quisition time was 541 msec with a 459-msec pulse delay. methylcobalamin by diaquocobinamide to a methylcobalamin For the 31p spectra the transients resulting from the applica- radical cation and cob(II)inamide. The very unstable methyl- tion of 900 pulses (27 ,sec) in a spectral width of 2000 Hz cobalamin radical cation releases a methyl radical, which re- were accumulated as 8192 data points in the time domain and acts with cob(II)inamide to generate the methylaquocobina- transformed into a 4096-point spectrum. The data acquisition mides. time was 2.048 sec without a pulse delay. For the 1H spectra the transients resulting from the application of 900 pulses (4 Methylcorrinoids, such as methylcobalamin and (5-meth- ,usec) in a spectral width of 3000 Hz were accumulated as oxybenzimidazolyl)-Co-methylcobamide, serve as cofactors 4096 data points in the time domain and transformed into a in several biochemical reactions. These reactions include the 2048-point spectrum. The data acquisition time was 684 methylation of homocysteine (1), the formation of methane msec with a 316-msec pulse delay. The 13C and 31p spectra (2), and the synthesis of acetate from CO2 (3). In addition, were obtained under conditions of simultaneous broad band methylcobalamin is involved in the biomethylation of heavy noise decoupling. Peak positions were determined by com- metals such as mercury(II) (4), arsenic(III), selenium(IV), puter examination of the final Fourier-transformed spectra. and tellurium(IV) (5). The nonenzymatic methylation of sev- Chemical shifts were measured with respect to external neat eral metals by methylcobalamin has also been described. For tetramethylsilane for the 13C NMR spectra, external 85% instance, Agnes et al. (6, 7), Taylor and Hanna (8), and Fan- phosphoric acid for the 31P spectra, and internal 1H2HO set chiang et al. (9) showed that methylation of platinum by at 4.90 ppm for the 1H spectra. Reaction rates were deter- methylcobalamin required both platinum(II) and plati- mined by monitoring the decrease of the proton resonance of num(IV). In enzymic methyl transfer reactions the corrinoid the Co-CH3 group of methylcobalamin or the increase of cofactor serves alternately as an acceptor and as a donor of the corresponding resonance of the Co-CH3 group of meth- the methyl moiety, and thus a key feature in these reactions ylaquocobinamide. is formation and cleavage of the carbon-cobalt bond. Several cobalt chelates have been studied as models for RESULTS methyl donors and acceptors. For instance, Dodd et al. (10) have investigated the kinetics and mechanism of alkyl trans- In previous publications (16, 17) we have demonstrated that fer from alkylcobaloximes to cobaloxime(I), cobaloxime(II), the chemical shift of the [13C]methyl moiety of methylcorrin- and cobaloxime(III) acceptors. Costa et al. (11) studied the oids is markedly affected by the nature of the trans ligand. methyl transfer from the dimethyl derivative of 1,3-bis(bi- Thus the substitution of a strong field ligand by a weak one is acetylmonoximeimino)-propane cobalt(III) to aquocobala- accompanied by an upfield shift of the methyl resonance. min. Their results demonstrate that aquocobalamin can func- This property has allowed us to monitor the methyl transfer tion as a methyl carbanion acceptor from a suitably activated reaction from [13C]methylcobalamin, with 5,6-dimethylben- methyl donor. zimidazole as the trans ligand, to diaquocobinamide. Fig. 1 To date, to our knowledge no comparable studies using illustrates the 13C NMR spectra in the region of the just the naturally occurring corrinoids have been presented. Co-CH3 resonance for a 1 mM solution of [13C]methylco- In this paper we describe the results of experiments that balamin in the absence and in the presence of an equimolar demonstrate a facile methyl transfer from methylcobalamin amount of diaquocobinamide at pH 6.65, incubated at 25°C to diaquocobinamide. in the dark. The spectral changes with time in the presence of diaquocobinamide clearly demonstrate that the methyl The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: Bzm, 5,6-dimethylbenzimidazole. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2698 Downloaded by guest on September 29, 2021 Biochemistry: Fanchiang et al. Proc. NatL. Acad. Sci. USA 81 (1984) 2699 presence of diaquocobinamide the Co-CH3 1H resonance of methylcobalamin is broadened and shifted downfield from F 0.050 ppm to 0.088 ppm. To establish the stoichiometry of the reaction, 32 gmol of [13C]methylcobalamin and 20 gmol of diaquocobinamide in 5.5 ml of water, pH 7.0, were allowed to react at room temperature in the dark. After 24 hr the reaction mixture was applied to a 27 x 3 cm column of SP-Sephadex. The column was first washed with water to remove unreacted methylco- E balamin (14 ,umol). Methylaquocobinamide (18 gmol) was eluted with a 0.0-0.2 M NaCl gradient (in 0.025 M sodium phosphate, pH 8.0) as a prominent peak between fractions (6 ml) 55 and 80, establishing a 1:1 stoichiometry for the transfer reaction. The nature of the ligands coordinated to the cobalt atom of the cobinamide has a profound effect on the rate of the trans- D fer reaction. As shown in Table 1, dicyanocobinamide and

. . 15 10 5 0 -5 D

ppm

FIG. 1. Proton-decoupled carbon-13 NMR spectra of an aqueous solution of ['3C]methylcobalamin (1 mM) in the absence (spectrum A) and in the presence of an equimolar amount of diaquocobinamide C (spectra B-F). Spectrum A represents 1800 transients (541-msec acquisition time and 459-msec pulse delay). Spectra B-D represent 900, 1800, and 3600 transients, respectively, acquired during the first 15, 30, and 60 min of the reaction. Spectrum E represents 3600 transients acquired between the first and second hour of incubation. Spectrum F represents 3600 transients acquired after 48 hr of incu- B bation.

moiety is transferred from the cobalamin to the cobinamide and that the methyl transfer goes to completion. The 31p NMR spectrum of the final reaction mixture showed that A methylcobalamin (-58.3 Hz) is converted to aquocobalamin (-6.1 Hz). The exclusion of oxygen from the reaction mixtures did not affect the rate of the transfer reaction or the nature of the products. 1 0 -1 The methyl transfer reaction can also be followed by 1H ppm NMR. Fig. 2 shows the 1H NMR spectra in the region of the Co-CH3 resonance of a 1.87 mM solution of methylcobala- FIG. 2. Proton NMR spectra of the Co-CH3 region for an aqueous solution min in the absence and the presence of 10.2 mM diaquoco- of methylcobalamin (1.87 mM) in the absence (spectrum A) and in the presence of diaquocobinamide (10.2 mM) (spec- binamide (pH 7.5) incubated at 25°C in the dark. The spectral tra B-H) incubated at pH 7.5 and 25°C in the dark. Spectrum A changes with time not only demonstrate the methyl transfer represents 16 transients (684-msec acquisition time and 316-msec but also show that methylcobalamin and diaquocobinamide pulse delay). Spectra B-H, respectively, represent 16 transients ac- form a complex prior to the methyl transfer reaction. In the quired 1, 5, 10, 15, 30, 45, and 60 min after mixing of the reactants. Downloaded by guest on September 29, 2021 2700 Biochemistry: Fanchiang et al. Proc. NatL Acad Sci. USA 81 (1984) Table 1. Effect of ligands on the rate of the methyl transfer thermore, the addition of methylepicobalamin to a reaction reaction from ['3C]methylcobalamin mixture containing methylcobalamin and diaquocobinamide Relative rate, inhibits the transfer reaction from methylcobalamin. Methyl acceptor mM hr-' The methyl transfer from methylcobalamin to diaquocobinamide is irreversible. The addition of a large excess of Diaquocobinamide (1.9 mM) 0.54 aquocobalamin to a reaction mixture containing the products Diaquocobinamide (2.0 mM) + pyridine (10 mM) 0.28 of the transfer reaction, ['3C]methylaquocobinamide and Diaquocobinamide (1.0 mM) + pyridine (100 mM) 0.00 aquocobalamin, does not reverse the reaction. Incubation of Diaquocobinamide (2.0 mM) + histidine (10 mM) 0.08 3-[3C]methyl-a-aquocobinamide with aquocobalamin does Cyanoaquocobinamide (2.8 mM) 0.00 not generate ['3C]methylcobalamin. Incubation of a solution Dicyanocobinaroide (4.6 mM) 0.00 containing [13C]methylcobalamin and [12C]methylaquoco- ['3C]Methylcobalamin (0.98 mM) was allowed to react with the binamide in the dark and in the absence of oxygen for several indicated methyl acceptor at pH 7.5 and 25TC. days does not lead to methyl exchange. However, exposure of such a solution to visible light yields [13C]methylaquoco- the two (cyanoaquo)cobinamides do not serve as methyl ac- binamide. This methyl exchange reaction is reminiscent of ceptors. Furthermore, the addition of pyridine or histidine, the methyl transfer from methylcobaloxime to cob(II)alamin both of which are known to coordinate to corrinoids, to the in the light (18) and the methyl exchange between [14C]- reaction mixture greatly reduces the rate of the transfer reac- methylcobalamin and [12C]methylcobinamide at elevated tion. Pyridine not only reduces the rate of the reaction but temperatures (19). These reactions involve the homolytic also affects the nature of the products. Fig. 3 illustrates the cleavage of the carbon-cobalt bond followed by reaction of 3C NMR spectrum of a reaction mixture containing 1 mM the methyl radical with a Co(II) corrinoid. [13C]methylcobalamin, 2 mM diaquocobinamide, and 10 mM To determine the oxidation state of the reactive cobina- pyridine incubated at 25°C. The final spectrum (D) shows a mide species, the transfer reaction was repeated in solutions resonance at 0.40 ppm (integral 6.8) and a second, more saturated with 02 or N20 or in solutions containing 20 mM prominent one, at -0.02 ppm (integral 17.4). They presum- H202. None of these oxidizing agents affected the methyl ably correspond to a-methyl-3-pyridinatocobinamide and transfer reaction, suggesting that the methyl transfer in- methyl-a-pyridinatocobinamide, respectively. , volves Co(III) corrinoids: Methylepicobalamin, with the e-propionamide side chain projecting up from the corrin ring, also serves as a methyl CH3 OH2 2+ OH2 + CH3 donor in the transfer reaction. However, the rate of the reaction with methylepicobalamin is almost an order of magni- 1- -1o"' I+ [1] tude lower than that with methylcobalamin as a donor. Fur- Bzm OH2 Bzm OH2

in which Bzm represents 5,6-dimethylbenzimidazole. D The kinetics for the methyl transfer reaction from methylcobalamin to diaquocobinamide were determined by H NMR. An excess of diaquocobinamide over methylcobalamin was used in all the rate determinations sp that the diaquocobinamide concentration remained essentially constant. Plots of log(I, - I) vs. time (I = intensity) gave straight C lines for at least 70% of the reactions. The results described thus far suggest that methylcobalamin in the "base-on" form is an effective methyl donor, while corrinoids with weak ligands coordinated to cobalt are effective methyl acceptors. Thus the rate of the methyl transfer reaction should be markedly influenced by the pH of the reaction mixture. In dilute acid the 5,6-dimethylbenzimidazole ligand of methylcobalamin is protonated and no long- B er coordinated to cobalt (12). The pKa for this "base-on" "base-off' conversion is 2,7. In addition, the pH of the reaction mixture affects the ionization of the water ligands of diaquocobinamide (20). With increasing pH diaquocobinamide is deprotonated to the (aquohydroxy)cobinamides (PKa - 6.0), which in turn are the conjugate acids of dihydroxyco- binamide (pKa = 11). The dependence of the rate of methyl A transfer on the pH of the reaction mixture is summarized in Fig. 4. The optimal pH for the reaction lies exactly between the pKa values of the "base-on"= "base-off' conversion and of the deprotonation of diaquocobinamide (pH 4.4).

15 10 5 0 -5 DISCUSSION ppm Four mechanisms for transmethylation reactions between cobalt complexes can be formulated: (i) transfer of a methyl FIG. 3. Proton-decoupled '3C NMR spectra of an aqueous solu- radical to a Co(II) corrinoid, (ii) transfer of a methyl carboni- tion of ['3C]methylcobalamin (1 mM) in the presence of diaquoco- um ion to a nucleophilic Co(I) corrinoid, (iii) transfer of a binamide (2 mM) and pyridine (10 mM) incubated at 250C in the methyl carbanion to an electrophilic Co(III) corrinoid, and dark. Each spectrum represents' 3600 transients (541-msec acquisi- In the tion time and 459-msec, pulse delay) acquired for A-D, respectively, (iv) oxidative demethylation of methylcobalamin (21). 1, 5, 9, and 48 hr after miixing of the reactants. methyl transfer reaction from methylcobalamin to diaquoco- Downloaded by guest on September 29, 2021 Biochemistry: Fanchiang et aL Proc. NatL Acad. Sci. USA 81 (1984) 2701

10 ist as equilibrium mixtures of the five- and six-coordinate complexes. Diaquocobinamide and even the (cyanoaquo)cobinamides exist entirely as the six-coordinate complexes. Our results presented in Table 1 and Fig. 4 argue against a simple electrophilic substitution reaction. In an electrophilic substitution reaction the (hydroxyaquo)cobinamides and not 8 diaquocobinamide would generate the five-coordinate elec- trophile most readily and thus one would expect a pH opti- mum at 8.5, exactly between the pKa values of diaquocobinamide.

0 OH --6 x o+ I o Ia) OH2 OH 1i j2+ OH2 Co PKa = 6.0 p ,= 11 Co1 [2] -4 L 11C [1 ^4 OH2 OH2 OH _I _ + Co OH ,) _ Indeed, if the five-coordinate cobinamide is the electrophilic species the (cyanoaquo)cobinamides should be more effective as methyl acceptors than diaquocobinamide, because the higher electron donating ability of cyanide ion would pro- mote the formation of the five-coordinate cobinamide. The 1 0 i results presented in Table 1 show that the (cyanoaquo)cobin- 0 2 4 6 8 10 12 14 amides are ineffective as methyl acceptors and that the pH weaker ligands, pyridine and histidine, decrease the rate of FIG. 4. pH dependence of the rate constant (k') of the methyl the reaction. transfer reaction from methylcobalamin (1.9 mM) to diaquocobina- A mhcoalami ispending Schee mide (10.2 mM) at 25aC.aton of methylcobalamin IS presented In Scheme I. CH3 OH2 CH3 binamide, the first mechanism involves the homolytic cleavage of the carbon-cobalt bond of methylcobalamin and the C + []ol2l COv] + [Coll] + H20 [3] transfer of the methyl radical to a Co(II) cobinamide. Such a mechanism is unlikely because we have been unable to de- Bzm OH2 Bzm OH2 tect paramagnetic corrinoids by ESR spectroscopy even in reaction mixtures that were purged with helium. Further- CH3 OH2 CH3 more, a mechanism in which small traces of cob(II)alamin catalyze the methyl transfer from methylcobalamin to dia- CO|Ivl+ Co1l + H20 fast COli + [soil] [4 quocobinamide is also eliminated because the addition of H202 to the reaction mixture does not inhibit methyl trans- Bzm OH2 Bzm OH2 fer. The second mechanism, which involves Co(I) corrinoids, is unlikely because neither the rate of the methyl trans- Scheme I fer reaction nor the nature of the products is affected when the reaction mixtures are saturated with 02 or N20. Both In the first half-reaction (Eq. 3) methylcobalamin is oxidized agents are known to oxidize Co(I) corrinoids to the Co(II) by diaquocobinamide to a Co(IV) methylcobalamin radical and Co(III) forms (22). At first, our observations seemed to cation. Halpern et al. (24) have demonstrated that organobis- be in accord with the third mechanism, which involves an (dimethylglyoximato)cobalt(III) complexes undergo revers- electrophilic attack of diaquocobinamide on the Co-CH3 ible one-electron oxidations to the corresponding [CoR]+ moiety of methylcobalamin, the methyl group being formally radical cations. These radical cations are stable in aqueous transferred as a carbanion. Dodd et al. (10) have reported methanol solution at -78°C and they have been character- that a similar methyl exchange between methylbis(cyclohex- ized as cobalt(IV) complexes. At higher temperature some of anedionedioximato)pyridinecobalt(III) [CH3Co(chgH)2- the radical cations, such as those derived from the benzyl- pyr] and aquobis(dimethylglyoximato)hydroxideCo(III) and secondary alkyl cobalt complexes, undergo a nucleo- [(H20)Co(dmgH)20H] is extremely slow. They speculate philically induced heterolytic cleavage of the carbon-cobalt that the low electrophilicity of (H20)Co(dmgH)20H is due to bond to the corresponding alcohol and a cobalt(II) complex. the fact that it is substitution inert and thus does not generate We postulate that the actual methyl transfer in the second a reactive five-coordinate species. In contrast, the methyl half-reaction (Eq. 4) involves the attack of cob(II)inamide on transfer from methylcobalamin to diaquocobinamide is quite the Co(IV) methylcobalamin radical cation to yield aquoco- fast (9.6 x 10-2 M-' sec-1 at pH 4.5, 25°C), implying that balamin and the methylaquocobinamides. We postulate such diaquocobinamide is readily converted to the five-coordi- homolytic cleavage of the carbon-cobalt bond because we nate square pyramidal aquocobinamide. However, Pratt (23) were unable to detect cob(II)alamin or 13C-enriched metha- has pointed out that only the organoaquocobinamides, such nol, the products expected from a heterolytic cleavage. Fur- as (methylaquo)cobinamide and (vinylaquo)cobinamide, ex- thermore, the addition of 1 M NaCl to the reaction mixture Downloaded by guest on September 29, 2021 2702 Biochemistry: Fanchiang et aL Proc. NatL Acad Sd USA 81 (1984) did not generate CH3Cl nor did it significantly affect the rate corrin ring, is a very poor methyl donor because this side of the transfer reaction. A 1:1 stoichiometry was observed chain hinders the formation of a productive complex. for the methyl transfer from methylcobalamin to diaquoco- The results described in this manuscript are of relevance binamide, indicating that the latter functions as an oxidizing to the enzymatic methyl transfer reactions. Our results dem- agent and that cob(II)inamide acts as a radical acceptor. onstrate that methylcobalamin in the "base-on" form is an Using cyclic voltammetry, Lexa et al. (25) determined that effective methyl donor, while aquocobalamin in the "base- the standard potential ofthe Co(III)/Co(II) couple ofdiaquo- off' form is an effective methyl acceptor. Thus in the enzy- cobinamide at pH values below 6 is +0.265 V vs. SCE. In matic methyl transfer reaction the enzyme is able to control acidic solutions the Co(III)/Co(II) wave is well separated the reaction by positioning the lower ligand near the cobalt from the Co(II)/Co(I) wave, but as the pH is increased the atom or by replacing the dimethylbenzimidazole moiety by a first wave shifts to the negative and finally merges at alkaline weaker field ligand. pH with the second wave at -0.74 V vs. SCE. These observations demonstrate that diaquocobinamide is an effective This work was supported by Grants GM-23773 and GM-27423 oxidizing agent. However, the substitution of one or both of from the National Institutes of Health. the water ligands with stronger-field ligands such as OH-, CN-, or dimethylbenzimidazole lowers the potential of the 1. Taylor, R. T. (1982) in B,2, ed. Dolphin, D. (Wiley, New first Co(III)/Co(II) wave. The results presented in Table 1 York), Vol. 2, pp. 307-355. and Fig. 4 clearly demonstrate that diaquocobinamide is the 2. Poston, J. M. & Stadtman, T. C. (1975) in Cobalamin, Bio- chemistry and Pathophysiology, ed. Babior, B. H. (Wiley, most effective methyl acceptor, completely in accord with New York), pp. 111-140. its role as an oxidizing agent. 3. Ljungdahl, L. G. & Wood, H. G. (1982) in B,2, ed. Dolphin, D. The 1H NMR spectra in the Co-CH3 region (Fig. 2) as (Wiley, New York), Vol. 2, pp. 165-202. well as the 31P NMR spectra (data not shown) demonstrate 4. Wood, J. M. (1982) in B,2, ed. Dolphin, D. (Wiley, New York), that methylcobalamin and diaquocobinamide form a com- Vol. 2, pp. 151-164. plex prior to the methyl transfer reaction and thus the oxida- 5. McBride, B. C. & Wolfe, R. S. (1971) Biochemistry 10, 4312- tion and methyl radical transfer probably occur in this com- 4317. plex as outlined in Scheme II. 6. Agnes, G., Bendle, N., Hill, H. A. O., Williams, F. R. & Wil- liams, R. J. P. (1971) Chem. Commun., 850-851. 7. Agnes, G., Hill, H. A. O., Ridsdale, S. C., Kennedy, F. S. & -OH2 Williams, R. J. P. (1971) Biochim. Biophys. Acta 252, 207-211. 8. Taylor, R. T. & Hanna, M. L. (1976) Bioinorg. Chem. 6, 281- CH3 OH2 [ coiii] 293. I 2+ 9. Fanchiang, Y.-T., Ridley, W. P. & Wood, J. M. (1979) J. Am. coll + OH2 Chem. Soc. 101, 1442-1447. I1 10. Dodd, D., Johnson, M. & Lockman, B. L. (1977) J. Am. ,Iizm OH2 CH3 Chem. Soc. 99, 3664-3673. 11. Costa, G., Mestroni, G. & Cocevar, C. (1971) Chem. Coin- mun., 706-707. JII 12. Hogenkamp, H. P. C., Rush, J. E. & Swenson, C. A. (1965)J. Bzm Biol. Chem. 240, 3641-3644. 13. Tkachuck, R. D., Grant, M. E. & Hogenkamp, H. P. C. OH2 (1974) Biochemistry 13, 2645-2650. OH2 2+ 14. Friedrich, W. & Bernhauer, K. (1956) Chem. Ber. 89, 2507- I OH2 2512. 15. Pailes, W. H. & Hogenkamp, H. P. C. (1968) Biochemistry 7, CH3 Ll 4160-4166. CH3 16. Hogenkamp, H. P. C., Tkachuck, R. D., Grant, M. E., Fuen- tes, R. & Matwiyoff, N. A. (1975) Biochemistry 14, 3707-3714. 17. Needham, T. E., Matwiyoff, N. A., Walker, T. E. & Hogen- OH2 OH2 CH3 kamp, H. P. C. (1973) J. Am. Chem. Soc. 95, 5019-5024. 18. Schrauzer, G. N., Sibert, J. W. & Windgassen, R. J. (1968) J. lolll] coll Am. Chem. Soc. 90, 6681-6688. I 19. Friedrich, W. & Nordmeyer, J. P. (1969) Z. Naturforsch. 24b, ILm 4zm tzm 588-5%. 20. Pratt, J. M. (1972) in Inorganic Chemistry ofVitamin B,2 (Aca- demic, London), pp. 140-147. 21. Halpern, J. (1982) in B,2, ed. Dolphin, D. (Wiley, New York), Scheme I Vol. 1, pp. 501-541. 22. Pratt, J. M. (1972) in Inorganic Chemistry ofVitamin B,2 (Aca- demic, London), pp. 202-204. 23. Pratt, J. M. (1972) in Inorganic Chemistry ofVitamin B,2 (Aca- In the complex the methyl radical is immediately trapped by demic, London) pp. 130-138. cob(II)inamide and thus reaction with oxygen is not possi- 24. Halpern, J., Chan, M. S., Roche, T. S. & Tan, G. M. (1979) ble. Indeed, we have been unable to detect "3C-enriched Acta Chem. Scand. Ser. A 33, 141-148. formaldehyde by NMR spectroscopy. Methylepicobalamin, 25. Lexa, D., Saveant, J.-M. & Zickler, J. (1980) J. Am. Chem. with the e-propionamide side chain projecting up from the Soc. 102, 4851-4852. Downloaded by guest on September 29, 2021