Hydrogenase from the hyperthermophilic archaeon, Pyrococcus furiosus, catalyzes the reversible activation of H-2 gas and the reduction of elemental sulfur (S degrees) at 90 degrees C and above. The pure enzyme, modified with polyethylene glycol (PEG), was soluble (> 5 mg/mL) in toluene and benzene with t(1/2) values of more than 6 h at 25 degrees C. At 100 degrees C the PEG-modified enzyme was less stable in aqueous solution (t(1/2) similar to 10 min) than the native (unmodified) enzyme (t(1/2) similar to 1 h), but they exhibited comparable H-2 evolution, H-2 oxidation, and S degrees reduction activities at 80 degrees C. The H-2 evolution activity of the modified enzyme was twice that of the unmodified enzyme at 25 degrees C. The PEG-modified enzyme did not catalyze S degrees reduction (at 80 degrees C) in pure toluene unless H2O was added. The mechanism by which hydrogenase produces H2S appears to involve H2O as the proton source and H-2 as the electron source. The inability of the modified hydrogenase to catalyze S degrees reduction in a homogeneous nonaqueous phase complicates potential applications of this enzyme.