N-Carboxymethanofuran (carbamate) formation from methanofuran and CO2 in methanogenic archaea - Thermodynamics and kinetics of the spontaneous reaction

N-Carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 to methane in m ethanogenic archaea. The reaction proceeds spontaneously. We address here t he question whether the rate of spontaneous carbamate formation is high eno ugh to account for the observed rate of methanogenesis from CO2. The rates of carbamate formation (v(1)) and cleavage (v(2)) were determined under equ ilibrium 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 fr om 'MFR'(MFR + MFRH+) and 'CO2' (CO2 + H2CO3 + HCO3- + CO32-) was found to be 7 m(-1).s(-1) (v(1) = k(1)* ['MFR'] ['CO2']) while the pseudo first orde r rate constant k(2)* of carbamate cleavage was 12 s(-1) (v(2) = k(2)* [car bamate]). The equilibrium constant K* = k(1)*/k(2)* = [carbamate]/['MFR'][' CO2'] was 0.6 m(-1) at pH 7.0 corresponding to a free energy change Delta G degrees' 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 sponta neous carbamate formation is of the same order as the maximal rate of metha ne formation and as the rate of spontaneous CO2 formation from HCO3- in met hanogenic archaea, the latter being important as CO2 is mainly present as H CO3- which has to be converted to CO2 before it can react with MFR. An enzy me catalyzed carbamate formation thus appears not to be required for methan ogenesis from CO2. Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosar cina barkeri and Methanobacterium thermoautotrophicum or by purified formyl methanofuran dehydrogenase which catalyzes the reduction of N-carboxymethan ofuran to N-formylmethanofuran. From the concentrations of 'CO2' and of 'MFR' determined by 1D-NMR spectros copy and the pK(a) of H2CO3 and of MFRH+ the concentrations of CO2 and of M FR were obtained, allowing to calculate k(1) (v(1) = k(1) [MFR] [CO2]). 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.