The membrane-bound hydrogenase of Rhodopseudomonas capsulata

Abstract

The hydrogenase of Rhodopseudomonas capsulata is an intrinsic membrane protein extractable from the membrane by detergents. Triton X-100 produces stable soluble extracts. Stability of solubilized hydrogenase depends drastically on two factors: temperature and gas-phase. The solubilised hydrogenase is more stable at 20°C than in the cold and is further stabilised under an H₂ atmosphere. The kinetic properties of the membrane-bound and Triton-solubilised forms of the enzyme have been compared. Both forms of the enzyme show a pH optimum for the reduction of benzyl or methyl viologen at 8.5–9.0, for H₂ production with methyl viologen semiquinone at 5.7 and for H²H exchange at 4.5. In vitro, the hydrogenase functions as a reversible enzyme although at a slower rate for H₂ evolution than for H₂ uptake. The apparent K_m for H₂ (uptake) is 0.25 μM. The artificial electron acceptors having the highest affinity for hydrogenase are methylene blue (K_m = 60 μM) and benzyl viologen (K_m = 100 μM). Methyl viologen has a higher affinity in the semiquinone form (K_m = 450 μM) than in the oxidized dicationic form (K_m = 3.6 mM). The Arrhenius plot of the activity of hydrogenase in the membrane and in the solubilised extract shows a break at 13°C. This transition temperature of 13°C is probably linked to a change of protein conformation. The activation energy is 110 kJ/mol (26.4 kcal/mol) adnd 38 kJ/mol (9.1 kcal/mol) below and above the transition temperature, respectively. While hydrogenase is a cold labile enzyme, it is remarkably resistant to heat inactivation, for example the membrane-bound form can withstand heating at 80°C for 3 h without loss of activity.