Hydrogenase II of Clostridium pasteurianum is a monomeric protein of Mr = 53,000 containing 8 iron and 8 acid-labile sulfide atoms/mol. It is distinct from hydrogenase I from the same organism (Mr = 60,000 12 Fe and 12 S2-/mol). Metal analyses showed that neither hydrogenase contains nickel or any other metals in significant amounts. The iron atoms of hydrogenase II resisted chelation by 2,2'-bipyridyl but all were susceptible when the enzyme was treated with ferricyanide. Core extrusion indicated the presence of two [4Fe-4S] clusters in hydrogenase II and EPR spectroscopy showed two distinct paramagnetic species which could be interpreted as one [4Fe-4S]2+(2+,1+) and one [4Fe-4S]2+(2+,3+) per molecule. The absorption coefficient of H2-reduced hydrogenase II at 420 nm was 23,000 M-1 cm-1 with a A420 / A275 ratio of 0.27. There were large differences between hydrogenase I and hydrogenase II in the absorption spectra of the air-oxidized, H2-reduced, and dithionite-reduced forms of the enzymes. Hydrogenase II catalyzed H2 evolution with methyl viologen or ferredoxin as the electron carrier, and H2 oxidation with methylene blue or methyl viologen as the electron acceptor. Apparent Km values were determined for all these reactions with both hydrogenases. Hydrogenase II is a relatively inactive enzyme, except in the reduction of methylene blue by H2. The pH dependencies of H2 oxidation were similar for both hydrogenases but were very different in H2 evolution. The activation energy values were much higher for H2 catalysis by hydrogenase II than for hydrogenase I. The two hydrogenases have the same sensitivity to inactivation by O2 but differ in their sensitivity to metal-chelating reagents and to CO. Hydrogenase I is more readily inhibited by CO but hydrogenase II binds CO irreversibly. From the above data, a mechanism is proposed to account for the observed differences in the catalytic activities of hydrogenase I and hydrogenase II.