FIG. 6.

HupUV hydrogenase activities in the soluble cytosolic fraction of JP91(pAC206) cells as a function of pH. JP91(pAC206) cells grown photoheterotrophically in MN medium were broken by passage through a French pressure cell. The soluble cytoplasmic fraction obtained by centrifugation at 100,000 × g for 70 min was used to determine HupUV hydrogenase activities. (A) H₂ production linked to MV oxidation at pH 4.0. The phosphate-citrate buffer (1.25 ml, final concentration of 100 mM) in the reaction chamber was first sparged with argon to remove O₂, and then the reaction vessel was closed, and 0.25 ml of soluble cytosolic fraction (0.9 mg of protein) was added. Two minutes later, MV⁺ (50 μl, final concentration of 120 mM) was injected into the reaction vessel. (B) pH dependence of MV⁺-mediated H₂ production and H₂ and HD formation in exchange with D₂. Initial rates determined for the first minute of H₂ (○) and HD (●) production (in 1.5 ml, 0.8 mg of protein) are plotted versus pH. To measure the H-D exchange, the reaction vessel was sparged first with D₂. H₂ (■) was formed by proton reduction with MV⁺. The buffers used (final concentration of 100 mM) were phosphate-citrate (pH 2.9 to 7.0), phosphate-Tris (pH 6.6 to 8.5), phosphate-glycine-NaOH (pH 7.5 to 10), and glycine-NaOH (pH 9.0 to 12.8).