JOURNAL OF BACTERIOLOGY, Jan. 1987, p. 87-92 Vol. 169, No. 1 0021-9193/87/010087-06$02.00/0 Copyright © 1987, American Society for Microbiology Inhibition by Corrins of the ATP-Dependent Activation and CO2 Reduction by the Methylreductase System in Methanobacterium bryantii WILLIAM B. WHITMAN'* AND RALPH S. WOLFE2 Department of Microbiology, University of Georgia, Athens, Georgia 30602,1 and Department of Microbiology, University ofIllinois, Urbana, Illinois 618012 Received 1 August 1986/Accepted 28 September 1986 Corrins inhibited the ATP-dependent activation of the methylreductase system and the methyl coenzyme M-dependent reduction of CO2 in extracts of Methanobacterium bryantii resolved from low-molecular-weight factors. The concentrations of cobinamides and cobamides required for one-half of maximal inhibition of the ATP-depen4ent activation were between 1 and 5 ,M. Cobinamides were more inhibitory at lower concentra- tiops than cobamides. Deoxyadenosylcobalamin was not inhibitory at concentrations up to 25 ,uM. The inhibition of CO2 reduction was competitive with respect to CO2. The concentration of methylcobalamin required for one-half of maximal inhibition was 5 ,M. Other cobamideg inhibited at similar concentrations, but diaquacobinami4e inhibited at lower concentrations. With respect to their affinities and specificities for corrins, inhibition of both the ATP-dependent activation'and CO2 reduction closely resembled the corrin- dependent activation of the methylreductase described in similar extracts (W. B. Whitman and R. S. Wolfe, J. Bacteriol. 164:165-172, 1985). However, whether the multiple effects of corrins are due to action at a single site is unknown. The effect of corrins (cobamides and cobinamides) on in CO2 reduction. However, the precise role of CH3-S-CoM is vitro methanogenesis is enigmatic. On one hand, small not known (9, 20). amounts of corrins stimulate the methyl coenzyme M (CH3- We described a corrin-dependent activation of the S-CoM) methylreductase system three- to fivefold (29). On methylreductase system in extracts of the mesophile the other hand, as reportfd in this communication, corrins Methanobacterium bryantii (29). This activation was unlike also inhibit CO2 reduction as well as ATP activation of the previously described effects of corrins in methanogenesis. It methylreductase system. This system (23) catalyzes the required catalytic amounts of corrins. Qther workers have reduction of CH3-S-CoM to form methane: shown that methylcobalamin (MeCbl) is a substrate for CH3-S-COM + H2 + CH4 methanogenesis in extracts of M. bryantii and Methanosar- Mg2+,2+HS-CoMATP, B, FAD cina barkeri (3, 4, 30). In our studies, corrins stimulated the demethylation of CH3-S-CoM. In methylotrophic Most of our information about the methylreductase system methanogens, a cobamide-containing protein is required for comes from studies of Methanobacterium thermoautotro- methylation of HS-CoM (2, 25-27). Furthermore, the phys- phicum. In M. thermoautotrophicum, the enzyme system iological significance of the coffin-dependent activation of contains at least four proteins (11, 17). Component C, which the methylreductase system was not certain. Although acti- contains the nickel tetrapyrrole factor F-430 and tightly vation required low concentrations of corrins, the coenzyme bound CoM, and the colorless A2 protein, which binds to and methyl forms were not required, and the greatest stim- N6-ATP agarose, have been purified to homogeneity (6, 7, ulation was obtained with n9nphysiological cobinamides. 14, 22). The methylreductase system also requires a low- Therefore, we also investigated the effects of cortins on molecular-weight factor of unknown function called compo- other reactions of the methylreductase system, including the nent B, Mg2+, flavin adenine dinucleotide (FAD), and cata- CH3-S-CoM-dependent reduction of C02, which may be the lytic amounts of ATP (10, 17, 18). physiological reaction of this system. The results of these Extracts of M. thermoautotrophicum also catalyze a CH3- studies are described in this report. S-CoM-dependent reduction of CO2 (9, 20): MF, MP, CH3-S-CoM MATERIALS AND METHODS Mg2+, ATP, B, FAD Methylreductase assays. Growth and anaerobic prepara- tion of cell extracts of M. bryantii M.o.H. were described In addition to the components of the methylreductase sys- previously (28, 29). Low-molecular-weight components of tem, this reaction requires two additional coenzymes, the extracts were removed by anaerobic chromatography on methanofuran (MF) and tetrahydromethanopterin (MP) (9, Sephadex G-25 (29). In a total volume of 0.2 ml, the 13, 15, 16, 21, 24). These coenzymes are additional C-1 methylreductase assay contained p.2 ml of Sephadex-treated carriers at the formyl, methine, methylene, and methyl extract (about 3 mg of protein), 2 ,umol of CH3-S-CoM, 2 levels of oxidation (8, 15), and some of the enzymes neces- ,umol of MgCl2, 10 ,ul of partially purified component B, 50 sary for their activity have been identified in extracts (5, 12). nmol of disodium ATP, and an H2 atmosphere (29). Methane Small amounts of CH3-S-CoM are also necessary to initiate was measured as described previously (28), and the velocity of the reaction was calculated from the linear portion of the * Corresponding author. time course of the reaction by linear regression analysis (28). 87 88 WHITMAN AND WOLFE J. BACTERIOL. greater than 50 puM with 50 nmol of ATP to S to 15 puM with 20 nmol of ATP and 5 ,uM with 10 nmol of ATP (Fig. 1). Because ATP is not required for activation by cobalamins (29), these results implied that CN-Cbl inhibited the activa- tion of the methylreductase by ATP. Methylreductase assays in extracts of M. bryantii contain a brief lag which is dependent on ATP (28). CN-Cbl in- creased the ATP-dependent lag early in the methylreductase assay (Fig. 2). Phosphoenolpyruvate, which acts like an ATP-generating system in these extracts (28), shortened the lag in the absence of CN-Cbl from 5 to 3 min. In the presence of CN-Cbl, phosphoenolpyruvate shortened the lag from 9 to 6 min, but it did not eliminate the effect of CN-Cbl (Fig. 2). Other cobalamins, cobamides, and cobinamides also length- ened the ATP-dependent lag (data not shown). This effect I 10 was especially dramatic for diaquacobinamnide (Aq2Cbi), E which activated the methylreductase system at very low concentrations (29). Even in the presence of high concentra- tions of ATP, 25 ,uM Aq2Cbi inhibited the methylreductase (Fig. 2). Phosphoenolpyruvate abolished the inhibition ofthe 20l nmo AT velocity of the methylreductase reaction. However, the ATP-dependent lag was only partially reduced (Fig. 2). The failure of phosphoenolpyruvate to abolish the inhibition by corrins also provided evidence that inhibition was not due to 0 nmol ATP the stimulation of an additional corrin-dependent reaction which competed with the methylreductase for ATP. 10 20 30 40 50 The inhibition by corrins appeared to be a direct effect on the ATP-dependent activation of the methylreductase. The [cobolamin] (EpLM) ATP-dependent activation is complete during the first few minutes of the methylreductase assay, and ATP is rapidly FIG. 1. Activation of the methylreductase by cobalamins at high degraded in these extracts (19, 28). If the activation is the and low concentrations of ATP. The cobalamin used was CN-Cbl. target of inhibition by corrins, corrins added after the No activity was obtained in the absence of ATP in the presence or activation was complete should no longer inhibit. MeCbl (25 absence of cobalamin. ,uM) inhibited the methylreductase only when it was added with low concentrations of ATP (Table 1). When MeCbl was The lag ofthe assay was equal to the intercept in the abscissa added after the activation by low concentrations of ATP, it of the linear portion of the reaction. The rate of activation activated. Like CN-Cbl (Fig. 1), when MeCbl was added was taken to be the reciprocal of the lag (1/lag). One unit of with high concentrations of ATP, activation was also ob- methylreductase activity is 1 nmol of CH4 produced per min. served. Therefore, the reversal of inhibition observed with CH3-S-CoM-dependent CO2 reduction. Assays were iden- ATP and phosphoenolpyruvate was not due to an indirect tical to the methylreductase assays except that the concen- tration of CH3-S-CoM was reduced to 200 nmol and 1 ,ug of MF (or carbon dioxide reduction factor), 2.5 ,ug of MP, 5% C02, and 50 mM potassium piperazine-N,N'-bis(2-ethane- sulfonate) (PIPES), pH 7.2, were added. Under these con- +PEP ditions, the addition of 5% CO2 to the gas atmosphere of the 800 assay vials caused the pH of the extract to decrease by less than 0.05. Assays were continued until there was no further E increase in the amount of CH4 (at least 60 min). The total 0" 600- CO2 reduced was determined as the total CH4 formed in the presence of CO2 minus the total CH4 formed in the absence of added CO2 (5). 400- Materials. Corrins, CH37S-CoM, and component B were prepared as described previously (29). MF and MP were the gift of J. Leigh. All common reagents were analytical grade or better. Biochemicals were obtained from Sigma Chemical 200- Co. (St. Louis, Mo.). RESULTS 10 20 30 10 20 30 Inhibition of the ATP-dependent activation. When the TIME (min) TIME (min) methylreductase was assayed at suboptimal concentrations FIG. 2. Effect of phosphoenolpyruvate on the corrin activation of ATP, the activation by cyanocobalamin (CN-Cbl) was of the methylreductase. ATP (50 nmol) was present in all assays. greatly reduced (Fig. 1). Furthermore, high concentrations Phosphoenolpyruvate (PEP; 2 ,umol) was added where indicated. of CN-Cbl were actually inhibitory, so that the concentration Symbols: 0, no corrins added; 0, 25 pLM CN-Cbl added; A, 25 ,uM of CN-Cbl required for maximal activity was reduced from Aq2Cbi added.