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Activation Mechanism of Methanol:5-Hydroxybenzimidazolylcobamide Methyltransferase from

1996; Elsevier BV; Volume: 271; Issue: 37 Linguagem: Inglês

10.1074/jbc.271.37.22346

1083-351X

Piet Daas, Wilfred R. Hagen, Jan T. Keltjens, Chris van der Drift, Godfried D. Vogels,

Heme Oxygenase-1 and Carbon Monoxide

Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes involved in the transmethylation reaction from methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 only binds the methyl group of methanol when the cobalt atom of its corrinoid prosthetic groups is present in the highly reduced Co(I) state. Formation of this redox state requires H2, hydrogenase, methyltransferase activation protein, and ATP. Optical and electron paramagnetic resonance spectroscopy studies were employed to determine the oxidation states and coordinating ligands of the corrinoids of MT1 during the activation process. Purified MT1 contained 1.7 corrinoids per enzyme with cobalt in the fully oxidized Co(III) state. Water and N-3 of the 5-hydroxybenzimidazolyl base served as the upper and lower ligands, respectively. Reduction to the Co(II) level was accomplished by H2 and hydrogenase. The cob(II)amide of MT1 had the base coordinated at this stage. Subsequent addition of methyltransferase activation protein and ATP resulted in the formation of base-uncoordinated Co(II) MT1. The activation mechanism is discussed within the context of a proposed model and compared to those described for other corrinoid-containing methyl group transferring proteins. Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes involved in the transmethylation reaction from methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 only binds the methyl group of methanol when the cobalt atom of its corrinoid prosthetic groups is present in the highly reduced Co(I) state. Formation of this redox state requires H2, hydrogenase, methyltransferase activation protein, and ATP. Optical and electron paramagnetic resonance spectroscopy studies were employed to determine the oxidation states and coordinating ligands of the corrinoids of MT1 during the activation process. Purified MT1 contained 1.7 corrinoids per enzyme with cobalt in the fully oxidized Co(III) state. Water and N-3 of the 5-hydroxybenzimidazolyl base served as the upper and lower ligands, respectively. Reduction to the Co(II) level was accomplished by H2 and hydrogenase. The cob(II)amide of MT1 had the base coordinated at this stage. Subsequent addition of methyltransferase activation protein and ATP resulted in the formation of base-uncoordinated Co(II) MT1. The activation mechanism is discussed within the context of a proposed model and compared to those described for other corrinoid-containing methyl group transferring proteins. INTRODUCTIONMethanosarcina barkeri can utilize methanol as sole source for methanogenesis and growth. The first step in the reduction of methanol to methane is the formation of an enzyme-bound methylcobamide catalyzed by methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) 1The abbreviations used are: MT1methanol:5-hydroxybenzimidazolylcobamide methyltransferaseCH3-S-CoM (methyl-coenzyme M)2-(methylthio)ethanesulfonic acidHS-CoM (coenzyme M)2-mercaptoethanesulfonic acidMT2Co-methyl-5-hydroxybenzimidazolylcobamide:HS-CoM methyltransferaseMAPmethyltransferase activation proteinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonatePAGEpolyacrylamide gel electrophoresisTESN-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acidHBI5-hydroxybenzimidazolylB12-HBI5-hydroxybenzimidazolylcobamideCAPS3(cyclohexylamino)-1-propanesulfonic acidkPakilopascal(s). (1van der Meijden P. e Brümmelstroet B.W. Poirot C.M. van der Drift C. Vogels G.D. J. Bacteriol. 1984; 160: 629-635Google Scholar). The methyl group of methylated MT1 is subsequently transferred to 2-mercaptoethanesulfonic acid (coenzyme M, HS-CoM) by Co-methyl-5-hydroxybenzimidazolylcobamide:HS-CoM methyltransferase (MT2) (2van der Meijden P. Heythuysen H.J. Pouwels A. Houwen F. van der Drift C. Vogels G.D. Arch. Microbiol. 1983; 134: 238-242Google Scholar). As a result methyl-coenzyme M (CH3-S-CoM) is produced, which is the substrate for the final step in methanogenesis in all methanogens studied so far (3Keltjens J.T. van der Drift C. FEMS Microbiol. Rev. 1986; 39: 259-303Google Scholar).The corrinoid prosthetic group of MT1 can only be methylated by methanol when the central cobalt atom of the cobamide is present in the highly reduced Co(I) state (4van der Meijden P. van der Lest C. van der Drift C. Vogels G.D. Biochem. Biophys. Res. Commun. 1984; 118: 760-766Google Scholar, 5van der Meijden P. Jansen L.P.J.M. van der Drift C. Vogels G.D. FEMS Microbiol. Lett. 1983; 19: 247-251Google Scholar). Since this state is extremely sensitive toward oxidation, MT1 readily inactivates upon manipulation and even during catalysis. Reactivation is possible and requires participation of a reducing system, methyltransferase activation protein (MAP), and ATP (4van der Meijden P. van der Lest C. van der Drift C. Vogels G.D. Biochem. Biophys. Res. Commun. 1984; 118: 760-766Google Scholar, 6Daas P.J.H. Gerrits K.A.A. Keltjens J.T. van der Drift C. Vogels G.D. J. Bacteriol. 1993; 175: 1278-1283Google Scholar, 7van der Meijden P. Heythuysen H.J. Sliepenbeek H.T. Houwen F.P. van der Drift C. Vogels G.D. J. Bacteriol. 1983; 153: 6-11Google Scholar, 8Daas P.J.H. Wassenaar R.W. Willemsen P. Theunissen R.J. Keltjens J.T. van der Drift C. Vogels G.D. J. Biol. Chem. 1996; 172: 22339-22345Google Scholar). The reducing system consists of hydrogen, hydrogenase, and ferredoxin. Ferredoxin is not absolutely required, though it stimulates the apparent reaction rate of methyl group transfer (6Daas P.J.H. Gerrits K.A.A. Keltjens J.T. van der Drift C. Vogels G.D. J. Bacteriol. 1993; 175: 1278-1283Google Scholar).Here, we report the UV-visible absorbance and electron paramagnetic resonance properties of the corrinoid prosthetic groups of MT1 under various additions of the reducing system, MAP, and ATP. From these results, the sequence of events leading to the formation of the cob(I)amide of MT1 is deduced. The activation of MT1 proceeds by a novel mechanism, which is presented in a model and compared to those described for other corrinoid-containing methyl group-transferring proteins.DISCUSSIONOptical and EPR spectroscopical studies were employed to determine the oxidation state of the central cobalt atom and the coordination of the ligands in the corrinoid protein MT1 under various additions of MAP, ATP, and a reducing system. After isolation MT1 contained somewhat less then 2 mol of B12-HBI/mol of α2β protein. The UV-visible light spectrum (Fig. 2) indicated that the prosthetic group was present in the hexa-coordinated Co(III) oxidation state with the nucleotide 5-hydroxybenzimidazole and water as the lower and upper ligands, respectively. Purified MT1 is inactive, and reactivation apparently requires the reduction of Co(III), which is brought about by a reducing system (H2, hydrogenase, and ferredoxin), MAP, and ATP (4van der Meijden P. van der Lest C. van der Drift C. Vogels G.D. Biochem. Biophys. Res. Commun. 1984; 118: 760-766Google Scholar, 6Daas P.J.H. Gerrits K.A.A. Keltjens J.T. van der Drift C. Vogels G.D. J. Bacteriol. 1993; 175: 1278-1283Google Scholar, 8Daas P.J.H. Wassenaar R.W. Willemsen P. Theunissen R.J. Keltjens J.T. van der Drift C. Vogels G.D. J. Biol. Chem. 1996; 172: 22339-22345Google Scholar) (Fig. 5). Incubation of MT1 with hydrogen, hydrogenase, and ferredoxin resulted in the reduction to the Co(II) state. EPR spectroscopy demonstrated that the HBI-base was still coordinated at this stage (base-on). Subsequent addition of both MAP and ATP induced a conversion of base-on into base-off Co(II) MT1. We previously showed MAP to be autophosphorylated by ATP; MAP-phosphate is able to substitute for the requirement of MAP and ATP (8Daas P.J.H. Wassenaar R.W. Willemsen P. Theunissen R.J. Keltjens J.T. van der Drift C. Vogels G.D. J. Biol. Chem. 1996; 172: 22339-22345Google Scholar). The phosphorylated protein, thus effects MT1 in such a way that the HBI-base becomes dissociated. In the experiment shown in Fig. 4, an estimated 38% of the corrinoids became base-uncoordinated when MT1 (15.8 µM) was incubated with 7.5 µM MAP and excess ATP (5 mM). From this, it follows that the concentration of base-off MT1 amounted to 6.0 µM, which is about equimolar with respect to MAP added. In agreement with this conclusion, lower amounts of base-off cob(II)amide were obtained when the MAP concentration was decreased in the EPR experiments.The conversion of base-on into base-off cob(II) amide has an important implication. In non-protein bound B12-HBI, such base-off conversion causes the midpoint redox potential of the Co(II)/Co(I) couple to increase from -592 to -500 mV (21Daas P.J.H. Keltjens J.T. Hagen W.R. van der Drift C. Arch. Biochem. Biophys. 1995; 319: 244-249Google Scholar). In a similar way, the action of MAP and ATP may facilitate the reduction in MT1 of cob(II)amide to the catalytically active species, the powerful nucleophile cob(I)amide. Although we could not detect the direct formation of cob(I)amide in our experiments, the findings that (i) the species is produced upon demethylation of methylated MT1 (5van der Meijden P. Jansen L.P.J.M. van der Drift C. Vogels G.D. FEMS Microbiol. Lett. 1983; 19: 247-251Google Scholar) and (ii) methyl-B12-HBI bound to MT1 is formed during the activation of the enzyme in the presence of H2, hydrogenase, MAP, ATP and methanol (this paper; Refs. 4van der Meijden P. van der Lest C. van der Drift C. Vogels G.D. Biochem. Biophys. Res. Commun. 1984; 118: 760-766Google Scholar, 5van der Meijden P. Jansen L.P.J.M. van der Drift C. Vogels G.D. FEMS Microbiol. Lett. 1983; 19: 247-251Google Scholar, 6Daas P.J.H. Gerrits K.A.A. Keltjens J.T. van der Drift C. Vogels G.D. J. Bacteriol. 1993; 175: 1278-1283Google Scholar), demonstrate that cob(I)amide must play a role in the catalytic and reductive activation cycles. The cob(II)amide/cob(I)amide midpoint redox potentials are strongly influenced by the protein environment. In the corrinoid/iron-sulfur proteins involved in acetyl-CoA synthesis and degradation that have been isolated from Clostridium thermoaceticum (24Ragsdale S.W. Crit. Rev. Biochem. Mol. Biol. 1991; 26: 261-300Google Scholar) and from Methanosarcina thermophila (25Jablonski P.E. Lu W.-P. Ragsdale S.W. Ferry J.G. J. Biol. Chem. 1993; 268: 325-329Google Scholar), reduction to the catalytically active Co(I) state occurred at midpoint redox potentials of -504 mV (26Harder S.A. Lu W.-P. Feinberg B.F. Ragsdale S.W. Biochemistry. 1989; 28: 9080-9087Google Scholar) and -486 mV (25Jablonski P.E. Lu W.-P. Ragsdale S.W. Ferry J.G. J. Biol. Chem. 1993; 268: 325-329Google Scholar), respectively. Here, reduction has to be performed by electrons derived from the carbonyl (CO)/CO2 oxidation (E0′ = -520 mV). It is important to note that the enzymes do not require ATP for the activation; in the purified, inactive cob(II)amide state, the corrinoids are already contained in the (at neutral pH) thermodynamically unfavorable base-off state by the protein backbone. Here, the observed midpoint redox potentials about equaled the E0′ = -500 mV of the free base-off cob(II)amide/cob(I)amide couple. For a number of corrinoid-containing methyltransferases, midpoint potentials have been measured that were significantly higher than found for the corresponding B12 derivatives in solution (25Jablonski P.E. Lu W.-P. Ragsdale S.W. Ferry J.G. J. Biol. Chem. 1993; 268: 325-329Google Scholar, 26Harder S.A. Lu W.-P. Feinberg B.F. Ragsdale S.W. Biochemistry. 1989; 28: 9080-9087Google Scholar, 27Banerjee R.V. Harder S.R. Ragsdale S.W. Matthews R.G. Biochemistry. 1990; 29: 1129-1135Google Scholar, 28Lu W.-P. Becher B. Gottschalk G. Ragsdale S.W. J. Bacteriol. 1995; 177: 2245-2250Google Scholar). For example, the reduction of base-on cob(II)amide to cob(I)amide in the membrane-bound methyltetrahydromethanopterin:HS-CoM methyltransferase complex from Methanosarcina mazei showed an E0′ as high as -426 to -450 mV, which is about 150 mV more positive than the analogous reduction of free B12-HBI (28Lu W.-P. Becher B. Gottschalk G. Ragsdale S.W. J. Bacteriol. 1995; 177: 2245-2250Google Scholar). In comparing the UV-visible light spectra of MT1 with aqueous solutions of B12-HBI, we noticed some differences in the 400 nm and 500-600 nm regions (Fig. 2, Fig. 3). Since MT1 does not contain Fe-S clusters (this paper) or other chromophoric groups (results not shown), this had to be caused by a conformational distortion of the corrin ring structure by the protein (29Gianotti C. Dolphin D. B12. John Wiley & Sons, New York1982: 393Google Scholar). Such distortion is likely to change the reduction potential of the prosthetic group. Incubation of methyltetrahydromethanopterin:HS-CoM methyltransferase with ATP and the methyl donor (methyltetrahydromethanopterin) raised the apparent midpoint potential another 200 mV (E0′ = -245 mV) (28Lu W.-P. Becher B. Gottschalk G. Ragsdale S.W. J. Bacteriol. 1995; 177: 2245-2250Google Scholar), i.e. to a level where reduction becomes feasible at even very low hydrogen concentrations (E0′ = -414 mV). Remarkably, the 200 mV shift was not observed when the methyl donor was omitted, and the authors (28Lu W.-P. Becher B. Gottschalk G. Ragsdale S.W. J. Bacteriol. 1995; 177: 2245-2250Google Scholar) proposed that the simultaneous action of ATP and the methylating substrate in a ternary enzyme complex is required for raising the redox potential. This may also apply to MT1. As pointed out above, we never observed the characteristic UV-visible light spectral features of cob(I)amide upon incubation under even high (100 kPa) hydrogen partial pressure of MT1 with MAP and ATP. However, the additional presence of methanol results in the formation of methyl-B12-HBI in MT1, suggesting a cooperative action of MAP-phosphate and methanol in the reductive activation (Fig. 5). The EPR experiments outlined above indicated that the amount of base-off cob(II) amide formed was dependent on the amount of MAP added. Yet, MT1 may be fully activated in the presence of substoichiometric amounts of MAP provided ATP and methanol is present (6Daas P.J.H. Gerrits K.A.A. Keltjens J.T. van der Drift C. Vogels G.D. J. Bacteriol. 1993; 175: 1278-1283Google Scholar, 8Daas P.J.H. Wassenaar R.W. Willemsen P. Theunissen R.J. Keltjens J.T. van der Drift C. Vogels G.D. J. Biol. Chem. 1996; 172: 22339-22345Google Scholar). This may be explained by assuming that MAP becomes dephoshorylated after completion of the activation cycle of the MT1 molecule (8Daas P.J.H. Wassenaar R.W. Willemsen P. Theunissen R.J. Keltjens J.T. van der Drift C. Vogels G.D. J. Biol. Chem. 1996; 172: 22339-22345Google Scholar) (Fig. 5). Rephosphorylation by ATP then yields MAP-phosphate for activation of another molecule. Future investigations have to clarify questions with respect to the corrinoid midpoint redox potentials in MT1 and the effects hereon of MAP-phosphate and methanol.In order to be active, corrinoid-dependent methyltransferases often require an ATP-dependent reductive activation (3Keltjens J.T. van der Drift C. FEMS Microbiol. Rev. 1986; 39: 259-303Google Scholar, 30Matthews R.G. Banerjee R.V. Ragsdale S.W. Biofactors. 1990; 2: 147-152Google Scholar). As yet, only the activation mechanism of methionine synthase has been elucidated (31Banerjee R.V. Matthews R.G. FASEB J. 1990; 4: 1450-1459Google Scholar). Here, ATP is the substrate in the formation of the potent methylating agent, S-adenosyl methionine, which traps Co(I) out of the thermodynamic unfavorable Co(II) to Co(I) reduction equilibrium (31Banerjee R.V. Matthews R.G. FASEB J. 1990; 4: 1450-1459Google Scholar). In this paper, we have presented evidence that nature developed another approach to facilitate the generation of the active enzymes, notably by inducing in an ATP-dependent process the conformational change of the prosthetic group. Perhaps other corrinoid-containing methyltransferases from methanogens (3Keltjens J.T. van der Drift C. FEMS Microbiol. Rev. 1986; 39: 259-303Google Scholar) and other obligate anaerobic organisms (32Diekert G. Wohlfarth G. Antonie van Leeuwenhoek. 1994; 66: 209-221Google Scholar) are activated in a similar fashion. INTRODUCTIONMethanosarcina barkeri can utilize methanol as sole source for methanogenesis and growth. The first step in the reduction of methanol to methane is the formation of an enzyme-bound methylcobamide catalyzed by methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) 1The abbreviations used are: MT1methanol:5-hydroxybenzimidazolylcobamide methyltransferaseCH3-S-CoM (methyl-coenzyme M)2-(methylthio)ethanesulfonic acidHS-CoM (coenzyme M)2-mercaptoethanesulfonic acidMT2Co-methyl-5-hydroxybenzimidazolylcobamide:HS-CoM methyltransferaseMAPmethyltransferase activation proteinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonatePAGEpolyacrylamide gel electrophoresisTESN-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acidHBI5-hydroxybenzimidazolylB12-HBI5-hydroxybenzimidazolylcobamideCAPS3(cyclohexylamino)-1-propanesulfonic acidkPakilopascal(s). (1van der Meijden P. e Brümmelstroet B.W. Poirot C.M. van der Drift C. Vogels G.D. J. Bacteriol. 1984; 160: 629-635Google Scholar). The methyl group of methylated MT1 is subsequently transferred to 2-mercaptoethanesulfonic acid (coenzyme M, HS-CoM) by Co-methyl-5-hydroxybenzimidazolylcobamide:HS-CoM methyltransferase (MT2) (2van der Meijden P. Heythuysen H.J. Pouwels A. Houwen F. van der Drift C. Vogels G.D. Arch. Microbiol. 1983; 134: 238-242Google Scholar). As a result methyl-coenzyme M (CH3-S-CoM) is produced, which is the substrate for the final step in methanogenesis in all methanogens studied so far (3Keltjens J.T. van der Drift C. FEMS Microbiol. Rev. 1986; 39: 259-303Google Scholar).The corrinoid prosthetic group of MT1 can only be methylated by methanol when the central cobalt atom of the cobamide is present in the highly reduced Co(I) state (4van der Meijden P. van der Lest C. van der Drift C. Vogels G.D. Biochem. Biophys. Res. Commun. 1984; 118: 760-766Google Scholar, 5van der Meijden P. Jansen L.P.J.M. van der Drift C. Vogels G.D. FEMS Microbiol. Lett. 1983; 19: 247-251Google Scholar). Since this state is extremely sensitive toward oxidation, MT1 readily inactivates upon manipulation and even during catalysis. Reactivation is possible and requires participation of a reducing system, methyltransferase activation protein (MAP), and ATP (4van der Meijden P. van der Lest C. van der Drift C. Vogels G.D. Biochem. Biophys. Res. Commun. 1984; 118: 760-766Google Scholar, 6Daas P.J.H. Gerrits K.A.A. Keltjens J.T. van der Drift C. Vogels G.D. J. Bacteriol. 1993; 175: 1278-1283Google Scholar, 7van der Meijden P. Heythuysen H.J. Sliepenbeek H.T. Houwen F.P. van der Drift C. Vogels G.D. J. Bacteriol. 1983; 153: 6-11Google Scholar, 8Daas P.J.H. Wassenaar R.W. Willemsen P. Theunissen R.J. Keltjens J.T. van der Drift C. Vogels G.D. J. Biol. Chem. 1996; 172: 22339-22345Google Scholar). The reducing system consists of hydrogen, hydrogenase, and ferredoxin. Ferredoxin is not absolutely required, though it stimulates the apparent reaction rate of methyl group transfer (6Daas P.J.H. Gerrits K.A.A. Keltjens J.T. van der Drift C. Vogels G.D. J. Bacteriol. 1993; 175: 1278-1283Google Scholar).Here, we report the UV-visible absorbance and electron paramagnetic resonance properties of the corrinoid prosthetic groups of MT1 under various additions of the reducing system, MAP, and ATP. From these results, the sequence of events leading to the formation of the cob(I)amide of MT1 is deduced. The activation of MT1 proceeds by a novel mechanism, which is presented in a model and compared to those described for other corrinoid-containing methyl group-transferring proteins.