N-carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 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 CO2. The rates of carbamate formation (v1) and cleavage (v2) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k1* of carbamate formation from 'MFR'(MFR + MFRH+) and 'CO2' (CO2 + H2CO3 + HCO3-+ CO32-) was found to be 7 M-1.s-1 (v1 = k1* ['MFR'] ['CO2']) while the pseudo first order rate constant k2* of carbamate cleavage was 12 s-1 (v2 = k2* [carbamate]). The equilibrium constant K* = k1*/k2* = [carbamate]/['MFR']['CO2'] was 0.6 M-1 at pH 7.0 corresponding to a free energy change DeltaG degrees ' of + 1.3 kJ.mol-1. The pH and temperature dependence of k1*, of k2* and of K* were determined. From the second order rate constant k1* 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 CO2 formation from HCO3- in methanogenic archaea, the latter being important as CO2 is mainly present as HCO3- which has to be converted to CO2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO2. 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 'CO2' and of 'MFR' determined by 1D-NMR spectroscopy and the pKa of H2CO3 and of MFRH+ the concentrations of CO2 and of MFR were obtained, allowing to calculate k1 (v1 = k1 [MFR] [CO2]). The second order rate constant k1 was found to be approximately 1000 M-1 x s-1 at 300 K and pH values between 7.0 and 8. 0 which is in the order of k1 values determined for other carbamate forming reactions by stopped flow.

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