Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air. Hydrogenase was located in the 140,000 x g supernatant in cell-free extracts. The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with a yield of 19%, resulting in a 85% pure preparation with a specific activity of 6.0 U (mg protein)-1. With native PAGE, a mol. mass of 100 and 200 kDa was determined. With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa (HoxK) were observed. Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD. The optimum of enzyme activity was at pH 9 and at 49 degrees C. Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme. The relationship of the T. ferrooxidans hydrogenase to other proteins was examined. A 9.5-kb EcoRI fragment of T. ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase. Antibodies against this enzyme did not react with the T. ferrooxidans hydrogenase in Western blot analysis. The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase. The N-terminal sequence of 20 amino acids of HoxG of T. ferrooxidans was 83.3% identical to that of the 60-kDa subunit. HupL, of the hydrogenase of Anabaena sp. Sequences of ten internal peptides of HoxG were 50-100% identical to the respective sequences of HupL of the Anabaena sp. hydrogenase.