Methyl transfer from methylcobalamin to diaquocobinamide (methylation/vitamin B-12/corrinoids)

Transcription

1 Proc. Nati. cad. Sci. US Vol. 81, pp , May 1984 Biochemistry Methyl transfer from methylcobalamin to diaquocobinamide (methylation/vitamin B-12/corrinoids) YUEH-TI FNCHING, GERL T. BRTT, N HRRY P. C. HOGENKMP* epartment of Biochemistry, The University of Minnesota, Minneapolis, MN Communicated by H.. Barker, January 13, 1984 BSTRCT The transfer of the methyl group from methylcobalamin to diaquocobinamide in aqueous solution has been demonstrated by proton, carbon-13, and phosphorus-31 nuclear magnetic resonance spectroscopy. The products of this reaction are aquocobalamin and the methylaquocobinamides. icyanocobinamide and the cyanoaquocobinamides do not serve as methyl acceptors, while ligands such as pyridine and histidine reduce the rate of the transfer reactions. The methyl transfer is not affected by oxidizing agents such as 02, N20, and H202, suggesting that the reaction does not involve free Co(I) or Co(II) corrinoids. The ph dependence of the rate of the transfer reaction from methylcobalamin to diaquocobinamide demonstrates that methylcobalamin in the "base-on" form and diaquocobinamide are the most effective methyl donor and acceptor, respectively. The most plausible mechanism for the transfer reaction involves the one-electron oxidation of methylcobalamin by diaquocobinamide to a methylcobalamin radical cation and cob(ii)inamide. The very unstable methylcobalamin radical cation releases a methyl radical, which reacts with cob(ii)inamide to generate the methylaquocobinamides. Methylcorrinoids, such as methylcobalamin and (5-methoxybenzimidazolyl)-Co-methylcobamide, serve as cofactors in several biochemical reactions. These reactions include the methylation of homocysteine (1), the formation of methane (2), and the synthesis of acetate from CO2 (3). In addition, methylcobalamin is involved in the biomethylation of heavy metals such as mercury(ii) (4), arsenic(iii), selenium(iv), and tellurium(iv) (5). The nonenzymatic methylation of several metals by methylcobalamin has also been described. For instance, gnes et al. (6, 7), Taylor and Hanna (8), and Fanchiang et al. (9) showed that methylation of platinum by methylcobalamin required both platinum(ii) and platinum(iv). In enzymic methyl transfer reactions the corrinoid cofactor serves alternately as an acceptor and as a donor of the methyl moiety, and thus a key feature in these reactions is formation and cleavage of the carbon-cobalt bond. Several cobalt chelates have been studied as models for methyl donors and acceptors. For instance, odd et al. (10) have investigated the kinetics and mechanism of alkyl transfer from alkylcobaloximes to cobaloxime(i), cobaloxime(ii), and cobaloxime(iii) acceptors. Costa et al. (11) studied the methyl transfer from the dimethyl derivative of 1,3-bis(biacetylmonoximeimino)-propane cobalt(iii) to aquocobalamin. Their results demonstrate that aquocobalamin can function as a methyl carbanion acceptor from a suitably activated methyl donor. To date, to our knowledge no comparable studies using just the naturally occurring corrinoids have been presented. In this paper we describe the results of experiments that demonstrate a facile methyl transfer from methylcobalamin to diaquocobinamide. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. EXPERIMENTL PROCEURES Materials. Cyanocobalamin was purchased from Rhone- Poulenc Industries (Paris). The corrinoids and their 13C-enriched derivatives were prepared from cyanocobalamin by published procedures: methylcobalamin (12), methylepicobalamin (13), dicyanocobinamide, (cyanoaquo)cobinamide (14), diaquocobinamide, and (methylauo)cobinamide (15). Methods. Pulse Fourier-transform 3C (62.9-MHz), 31p (101.3-MHz), and 1H (250.1-MHz) nuclear magnetic resonance spectra were obtained at 25 C with a Bruker WM250 spectrometer, locked to the resonance of internal 2H20. For the 13C NMR spectra the transients resulting from the application of 90 pulses (25,sec) in a spectral width of 15,000 Hz were accumulated as 16,384 data points in the time domain and transformed into an 8192-point spectrum. The data acquisition time was 541 msec with a 459-msec pulse delay. For the 31p spectra the transients resulting from the application of 900 pulses (27,sec) in a spectral width of 2000 Hz were accumulated as 8192 data points in the time domain and transformed into a 4096-point spectrum. The data acquisition time was sec without a pulse delay. For the 1H spectra the transients resulting from the application of 900 pulses (4,usec) in a spectral width of 3000 Hz were accumulated as 4096 data points in the time domain and transformed into a 2048-point spectrum. The data acquisition time was 684 msec with a 316-msec pulse delay. The 13C and 31p spectra were obtained under conditions of simultaneous broad band noise decoupling. Peak positions were determined by computer examination of the final Fourier-transformed spectra. Chemical shifts were measured with respect to external neat tetramethylsilane for the 13C NMR spectra, external 85% phosphoric acid for the 31P spectra, and internal 1H2HO set at 4.90 for the 1H spectra. Reaction rates were determined by monitoring the decrease of the proton resonance of the Co- group of methylcobalamin or the increase of the corresponding resonance of the Co- group of methylaquocobinamide. RESULTS In previous publications (16, 17) we have demonstrated that the chemical shift of the [13C]methyl moiety of methylcorrinoids is markedly affected by the nature of the trans ligand. Thus the substitution of a strong field ligand by a weak one is accompanied by an upfield shift of the methyl resonance. This property has allowed us to monitor the methyl transfer reaction from [13C]methylcobalamin, with 5,6-dimethylbenzimidazole as the trans ligand, to diaquocobinamide. Fig. 1 illustrates the 13C NMR spectra in the region of the Co- resonance for a 1 mm solution of [13C]methylcobalamin in the absence and in the presence of an equimolar amount of diaquocobinamide at ph 6.65, incubated at 25 C in the dark. The spectral changes with time in the presence of diaquocobinamide clearly demonstrate that the methyl bbreviation: Bzm, 5,6-dimethylbenzimidazole. *To whom reprint requests should be addressed. 2698

2 Biochemistry: Fanchiang et al. Proc. NatL. cad. Sci. US 81 (1984) 2699 F E presence of diaquocobinamide the Co- 1H resonance of methylcobalamin is broadened and shifted downfield from to 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. fter 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 methylcobalamin (14,umol). Methylaquocobinamide (18 gmol) was eluted with a M NaCl gradient (in 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 transfer reaction. s shown in Table 1, dicyanocobinamide and H G F E FIG. 1. Proton-decoupled carbon-13 NMR spectra of an aqueous solution of ['3C]methylcobalamin (1 mm) in the absence (spectrum ) and in the presence of an equimolar amount of diaquocobinamide (spectra B-F). Spectrum represents 1800 transients (541-msec acquisition time and 459-msec pulse delay). Spectra B- 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 incubation. 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 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. The methyl transfer reaction can also be followed by 1H NMR. Fig. 2 shows the 1H NMR spectra in the region of the Co- resonance of a 1.87 mm solution of methylcobalamin in the absence and the presence of 10.2 mm diaquocobinamide (ph 7.5) incubated at 25 C in the dark. The spectral changes with time not only demonstrate the methyl transfer but also show that methylcobalamin and diaquocobinamide form a complex prior to the methyl transfer reaction. In the C B FIG. 2. Proton NMR spectra of the Co- region for an aqueous solution of methylcobalamin (1.87 mm) in the absence (spectrum ) and in the presence of diaquocobinamide (10.2 mm) (spectra B-H) incubated at ph 7.5 and 25 C in the dark. Spectrum represents 16 transients (684-msec acquisition time and 316-msec pulse delay). Spectra B-H, respectively, represent 16 transients acquired 1, 5, 10, 15, 30, 45, and 60 min after mixing of the reactants.

3 2700 Biochemistry: Fanchiang et al. Table 1. Effect of ligands on the rate of the methyl transfer reaction from ['3C]methylcobalamin Relative rate, Methyl acceptor mm hr-' iaquocobinamide (1.9 mm) 0.54 iaquocobinamide (2.0 mm) + pyridine (10 mm) 0.28 iaquocobinamide (1.0 mm) + pyridine (100 mm) 0.00 iaquocobinamide (2.0 mm) + histidine (10 mm) 0.08 Cyanoaquocobinamide (2.8 mm) 0.00 icyanocobinaroide (4.6 mm) 0.00 ['3C]Methylcobalamin (0.98 mm) was allowed to react with the indicated methyl acceptor at ph 7.5 and 25TC. the two (cyanoaquo)cobinamides do not serve as methyl acceptors. Furthermore, the addition of pyridine or histidine, both of which are known to coordinate to corrinoids, to the reaction mixture greatly reduces the rate of the transfer reaction. Pyridine not only reduces the rate of the reaction but also affects the nature of the products. Fig. 3 illustrates the 3C NMR spectrum of a reaction mixture containing 1 mm [13C]methylcobalamin, 2 mm diaquocobinamide, and 10 mm pyridine incubated at 25 C. The final spectrum () shows a resonance at 0.40 (integral 6.8) and a second, more prominent one, at (integral 17.4). They presumably correspond to a-methyl-3-pyridinatocobinamide and, methyl-a-pyridinatocobinamide, respectively. Methylepicobalamin, with the e-propionamide side chain projecting up from the corrin ring, also serves as a methyl donor in the transfer reaction. However, the rate of the reaction with methylepicobalamin is almost an order of magnitude lower than that with methylcobalamin as a donor. Fur- C B FIG. 3. Proton-decoupled '3C NMR spectra of an aqueous solution of ['3C]methylcobalamin (1 mm) in the presence of diaquocobinamide (2 mm) and pyridine (10 mm) incubated at 250C in the dark. Each spectrum represents' 3600 transients (541-msec acquisition time and 459-msec, pulse delay) acquired for -, respectively, 1, 5, 9, and 48 hr after miixing of the reactants. Proc. NatL cad Sci. US 81 (1984) thermore, the addition of methylepicobalamin to a reaction mixture containing methylcobalamin and diaquocobinamide inhibits the transfer reaction from methylcobalamin. The methyl transfer from methylcobalamin to diaquocobinamide is irreversible. The addition of a large excess of aquocobalamin to a reaction mixture containing the products of the transfer reaction, ['3C]methylaquocobinamide and aquocobalamin, does not reverse the reaction. Incubation of 3-[3C]methyl-a-aquocobinamide with aquocobalamin does not generate ['3C]methylcobalamin. Incubation of a solution containing [13C]methylcobalamin and [12C]methylaquocobinamide in the dark and in the absence of oxygen for several days does not lead to methyl exchange. However, exposure of such a solution to visible light yields [13C]methylaquocobinamide. This methyl exchange reaction is reminiscent of the methyl transfer from methylcobaloxime to cob(ii)alamin in the light (18) and the methyl exchange between [14C]- methylcobalamin and [12C]methylcobinamide at elevated temperatures (19). These reactions involve the homolytic cleavage of the carbon-cobalt bond followed by reaction of the methyl radical with a Co(II) corrinoid. To determine the oxidation state of the reactive cobinamide species, the transfer reaction was repeated in solutions saturated with 02 or N20 or in solutions containing 20 mm H202. None of these oxidizing agents affected the methyl transfer reaction, suggesting that the methyl transfer involves Co(III) corrinoids: 1- -1o"' 2+ I+ + Bzm Bzm in which Bzm represents 5,6-dimethylbenzimidazole. The kinetics for the methyl transfer reaction from methylcobalamin to diaquocobinamide were determined by H NMR. n 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 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 longer 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 dihydroxycobinamide (pka = 11). The dependence of the rate of methyl 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). ISCUSSION Four mechanisms for transmethylation reactions between cobalt complexes can be formulated: (i) transfer of a methyl radical to a Co(II) corrinoid, (ii) transfer of a methyl carbonium ion to a nucleophilic Co(I) corrinoid, (iii) transfer of a methyl carbanion to an electrophilic Co(III) corrinoid, and (iv) oxidative demethylation of methylcobalamin (21). In the methyl transfer reaction from methylcobalamin to diaquoco- [1]

4 x Ia) -4^4,) _ Biochemistry: 0 1 Fanchiang et al i Proc. NatL cad. Sci. US 81 (1984) 2701 ist as equilibrium mixtures of the five- and six-coordinate complexes. iaquocobinamide 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 diaquocobinamide would generate the five-coordinate electrophile most readily and thus one would expect a ph optimum at 8.5, exactly between the pka values of diaquocobinamide. 1i j2+ Co PKa = 6.0 L 11C I o+ o _I _ + Co p,= 11 Co1 [2] [1 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 promote the formation of the five-coordinate cobinamide. The results presented in Table 1 show that the (cyanoaquo)cobin 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- mhcoalami ispending Schee mide (10.2 mm) at25ac.aton of methylcobalamin IS presented In Scheme I. binamide, the first mechanism involves the homolytic cleavage of the carbon-cobalt bond of methylcobalamin and the transfer of the methyl radical to a Co(II) cobinamide. Such a mechanism is unlikely because we have been unable to detect paramagnetic corrinoids by ESR spectroscopy even in reaction mixtures that were purged with helium. Furthermore, a mechanism in which small traces of cob(ii)alamin catalyze the methyl transfer from methylcobalamin to diaquocobinamide is also eliminated because the addition of H202 to the reaction mixture does not inhibit methyl transfer. The second mechanism, which involves Co(I) corrinoids, is unlikely because neither the rate of the methyl transfer reaction nor the nature of the products is affected when the reaction mixtures are saturated with 02 or N20. Both agents are known to oxidize Co(I) corrinoids to the Co(II) and Co(III) forms (22). t first, our observations seemed to be in accord with the third mechanism, which involves an electrophilic attack of diaquocobinamide on the Co- moiety of methylcobalamin, the methyl group being formally transferred as a carbanion. odd et al. (10) have reported that a similar methyl exchange between methylbis(cyclohexanedionedioximato)pyridinecobalt(iii) [Co(chgH)2- pyr] and aquobis(dimethylglyoximato)hydroxideco(iii) [(H20)Co(dmgH)20H] is extremely slow. They speculate that the low electrophilicity of (H20)Co(dmgH)20H is due to the fact that it is substitution inert and thus does not generate a reactive five-coordinate species. In contrast, the methyl transfer from methylcobalamin to diaquocobinamide is quite fast (9.6 x 10-2 M-' sec-1 at ph 4.5, 25 C), implying that diaquocobinamide is readily converted to the five-coordinate square pyramidal aquocobinamide. However, Pratt (23) has pointed out that only the organoaquocobinamides, such as (methylaquo)cobinamide and (vinylaquo)cobinamide, ex- C + []ol2l COv] + [Coll] + H20 Bzm Bzm CO Ivl+ Co1l + H20 fast COli + [soil] [4 Bzm Bzm Scheme I In the first half-reaction (Eq. 3) methylcobalamin is oxidized by diaquocobinamide to a Co(IV) methylcobalamin radical cation. Halpern et al. (24) have demonstrated that organobis- (dimethylglyoximato)cobalt(iii) complexes undergo reversible one-electron oxidations to the corresponding [CoR]+ radical cations. These radical cations are stable in aqueous methanol solution at -78 C and they have been characterized as cobalt(iv) complexes. t higher temperature some of the radical cations, such as those derived from the benzyland secondary alkyl cobalt complexes, undergo a nucleophilically induced heterolytic cleavage of the carbon-cobalt bond to the corresponding alcohol and a cobalt(ii) complex. We postulate that the actual methyl transfer in the second half-reaction (Eq. 4) involves the attack of cob(ii)inamide on the Co(IV) methylcobalamin radical cation to yield aquocobalamin and the methylaquocobinamides. We postulate such homolytic cleavage of the carbon-cobalt bond because we were unable to detect cob(ii)alamin or 13C-enriched methanol, the products expected from a heterolytic cleavage. Furthermore, the addition of 1 M NaCl to the reaction mixture [3]

5 2702 Biochemistry: Fanchiang et al did not generate Cl nor did it significantly affect the rate of the transfer reaction. 1:1 stoichiometry was observed for the methyl transfer from methylcobalamin to diaquocobinamide, indicating that the latter functions as an oxidizing agent and that cob(ii)inamide acts as a radical acceptor. Using cyclic voltammetry, Lexa et al. (25) determined that the standard potential of the Co(III)/Co(II) couple of diaquocobinamide at ph values below 6 is V vs. SCE. In acidic solutions the Co(III)/Co(II) wave is well separated from the Co(II)/Co(I) wave, but as the ph is increased the first wave shifts to the negative and finally merges at alkaline ph with the second wave at V vs. SCE. These observations demonstrate that diaquocobinamide is an effective oxidizing agent. However, the substitution of one or both of the water ligands with stronger-field ligands such as -, CN-, or dimethylbenzimidazole lowers the potential of the first Co(III)/Co(II) wave. The results presented in Table 1 and Fig. 4 clearly demonstrate that diaquocobinamide is the most effective methyl acceptor, completely in accord with its role as an oxidizing agent. The 1H NMR spectra in the Co- region (Fig. 2) as well as the 31P NMR spectra (data not shown) demonstrate that methylcobalamin and diaquocobinamide form a complex prior to the methyl transfer reaction and thus the oxidation and methyl radical transfer probably occur in this complex as outlined in Scheme II. coll + I1,Iizm lolll] I ILm I 2+ I Ll coll 4zm Scheme I 2+ - [ coiii] JII Bzm tzm In the complex the methyl radical is immediately trapped by cob(ii)inamide and thus reaction with oxygen is not possible. Indeed, we have been unable to detect "3C-enriched formaldehyde by NMR spectroscopy. Methylepicobalamin, with the e-propionamide side chain projecting up from the Proc. NatL cad Sd US 81 (1984) corrin ring, is a very poor methyl donor because this side chain hinders the formation of a productive complex. The results described in this manuscript are of relevance to the enzymatic methyl transfer reactions. Our results demonstrate that methylcobalamin in the "base-on" form is an effective methyl donor, while aquocobalamin in the "baseoff' form is an effective methyl acceptor. Thus in the enzymatic methyl transfer reaction the enzyme is able to control the reaction by positioning the lower ligand near the cobalt atom or by replacing the dimethylbenzimidazole moiety by a weaker field ligand. This work was supported by Grants GM and GM from the National Institutes of Health. 1. Taylor, R. T. (1982) in B,2, ed. olphin,. (Wiley, New York), Vol. 2, pp Poston, J. M. & Stadtman, T. C. (1975) in Cobalamin, Biochemistry and Pathophysiology, ed. Babior, B. H. (Wiley, New York), pp Ljungdahl, L. G. & Wood, H. G. (1982) in B,2, ed. olphin,. (Wiley, New York), Vol. 2, pp Wood, J. M. (1982) in B,2, ed. olphin,. (Wiley, New York), Vol. 2, pp McBride, B. C. & Wolfe, R. S. (1971) Biochemistry 10, gnes, G., Bendle, N., Hill, H.. O., Williams, F. R. & Williams, R. J. P. (1971) Chem. Commun., gnes, G., Hill, H.. O., Ridsdale, S. C., Kennedy, F. S. & Williams, R. J. P. (1971) Biochim. Biophys. cta 252, Taylor, R. T. & Hanna, M. L. (1976) Bioinorg. Chem. 6, Fanchiang, Y.-T., Ridley, W. P. & Wood, J. M. (1979) J. m. Chem. Soc. 101, odd,., Johnson, M. & Lockman, B. L. (1977) J. m. Chem. Soc. 99, Costa, G., Mestroni, G. & Cocevar, C. (1971) Chem. Coinmun., Hogenkamp, H. P. C., Rush, J. E. & Swenson, C.. (1965) J. Biol. Chem. 240, Tkachuck, R.., Grant, M. E. & Hogenkamp, H. P. C. (1974) Biochemistry 13, Friedrich, W. & Bernhauer, K. (1956) Chem. Ber. 89, Pailes, W. H. & Hogenkamp, H. P. C. (1968) Biochemistry 7, Hogenkamp, H. P. C., Tkachuck, R.., Grant, M. E., Fuentes, R. & Matwiyoff, N.. (1975) Biochemistry 14, Needham, T. E., Matwiyoff, N.., Walker, T. E. & Hogenkamp, H. P. C. (1973) J. m. Chem. Soc. 95, Schrauzer, G. N., Sibert, J. W. & Windgassen, R. J. (1968) J. m. Chem. Soc. 90, Friedrich, W. & Nordmeyer, J. P. (1969) Z. Naturforsch. 24b, 588-5%. 20. Pratt, J. M. (1972) in Inorganic Chemistry of Vitamin B,2 (cademic, London), pp Halpern, J. (1982) in B,2, ed. olphin,. (Wiley, New York), Vol. 1, pp Pratt, J. M. (1972) in Inorganic Chemistry ofvitamin B,2 (cademic, London), pp Pratt, J. M. (1972) in Inorganic Chemistry ofvitamin B,2 (cademic, London) pp Halpern, J., Chan, M. S., Roche, T. S. & Tan, G. M. (1979) cta Chem. Scand. Ser. 33, Lexa,., Saveant, J.-M. & Zickler, J. (1980) J. m. Chem. Soc. 102,