N ‐Carboxymethanofuran (carbamate) formation from methanofuran and CO 2 in methanogenic archaea

Abstract

N ‐Carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO 2 is the first reaction in the reduction of CO 2 to methane in methanogenic archaea. The reaction proceeds spontaneously. We address here the question whether the rate of spontaneous carbamate formation is high enough to account for the observed rate of methanogenesis from CO 2 . The rates of carbamate formation ( v 1 ) and cleavage ( v 2 ) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k 1 * of carbamate formation from ‘MFR’(MFR + MFRH + ) and ‘CO 2’ (CO 2 + H 2 CO 3 + HCO 3 − + CO 3 2– ) was found to be 7 m −1 ·s −1 ( v 1 = k 1 *[‘MFR’][‘CO 2 ’]) while the pseudo first order rate constant k 2 * of carbamate cleavage was 12 s −1 ( v 2 = k 2 *[carbamate]). The equilibrium constant K * = k 1 */ k 2 * = [carbamate]/[‘MFR’][‘CO 2 ’] was 0.6 m −1 at pH 7.0 corresponding to a free energy change Δ G °′ of + 1.3 kJ·mol −1 . The pH and temperature dependence of k 1 *, of k 2 * and of K * were determined. From the second order rate constant k 1 * it was calculated that under physiological conditions the rate of spontaneous carbamate formation is of the same order as the maximal rate of methane formation and as the rate of spontaneous CO 2 formation from HCO 3 − in methanogenic archaea, the latter being important as CO 2 is mainly present as HCO 3 − which has to be converted to CO 2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO 2 . Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosarcina barkeri and Methanobacterium thermoautotrophicum or by purified formylmethanofuran dehydrogenase which catalyzes the reduction of N ‐carboxymethanofuran to N ‐formylmethanofuran. From the concentrations of ‘CO 2 ’ and of ‘MFR’ determined by 1D‐NMR spectroscopy and the p K a of H 2 CO 3 and of MFRH + the concentrations of CO 2 and of MFR were obtained, allowing to calculate k 1 ( v 1 = k 1 [MFR][CO 2 ]). The second order rate constant k 1 was found to be approximately 1000 m −1 ·s −1 at 300 K and pH values between 7.0 and 8.0 which is in the order of k 1 values determined for other carbamate forming reactions by stopped flow.