Integrated thermodynamic analysis of electron bifurcating [FeFe]-hydrogenase to inform anaerobic metabolism and H-2 production

Electron bifurcating, [FeFe]-hydrogenases are recently described members of the hydrogenase family and catalyze a combination of exergonic and endergonic electron exchanges between three carriers (2 ferredoxin(red)(-) + NAD(P)H + 3 H+ = 2 ferredoxin(ox) + NAD(P)(+) + 2 H-2). A thermodynamic analysis of the bifurcating, [FeFe]-hydrogenase reaction, using electron path-independent variables, quantified potential biological roles of the reaction without requiring enzyme details. The bifurcating [FeFe]-hydrogenase reaction, like all bifurcating reactions, can be written as a sum of two non-bifurcating reactions. Therefore, the thermodynamic properties of the bifurcating reaction can never exceed the properties of the individual, non-bifurcating, reactions. The bifurcating [FeFe]-hydrogenase reaction has three competitive properties: 1) enabling NAD(P)H-driven proton reduction at pH(2) higher than the concurrent operation of the two, non-bifurcating reactions, 2) oxidation of NAD(P)H and ferredoxin simultaneously in a 1:1 ratio, both are produced during typical glucose fermentations, and 3) enhanced energy conservation (similar to 10 kJ mol(-1) H-2) relative to concurrent operation of the two, non-bifurcating reactions. Our analysis demonstrated ferredoxin E degrees' largely determines the sensitivity of the bifurcating reaction to pH(2), modulation of the reduced/oxidized electron carrier ratios contributed less to equilibria shifts. Hydrogenase thermodynamics data were integrated with typical and non-typical glycolysis pathways to evaluate achieving the 'Thauer limit' (4 H-2 per glucose) as a function of temperature and pH(2). For instance, the bifurcating [FeFe]-hydrogenase reaction permits the Thauer limit at 60 degrees C if pH (2)