Identification of an unusual paramagnetic species and of three [2Fe-2S] clusters in the iron-only hydrogenase from the hyperthermophilic bacterium Thermotoga maritima.

Thermotoga maritima is a H2-producing, fermentative anaerobe and is one of the most thermophilic bacteria known. Its iron-only (Fe-)hydrogenase was previously shown to be a homotetramer and to contain two [4Fe-4S] and two [2Fe-2S] clusters per monomer, but the enzyme lacked the characteristic EPR signal of the oxidized H cluster, the proposed site of H2 catalysis in mesophilic Fe-hydrogenases (Juszczak, A., Aono, S. and Adams, M.W.W. (1991) J. Biol. Chem. 266, 13834-13841). The two types of cluster were shown by spectroelectrochemistry to have reduction potentials (Em) of -390 and -440 mV, respectively. We have now identified two additional redox centers in the enzyme, a [2Fe-2S] center with a higher reduction potential (Em = -365 mV) and an unusual paramagnetic species (Em > -200 mV). The higher potential [2Fe-2S] center can be reduced by sodium dithionite at pH 6.0 and exhibits an axial-type EPR signal with gz = 2.026 and gy = gx = 1.940. The two lower potential [2Fe-2S] centers are fully reduced by sodium dithionite only at pH 10.0. Both of these clusters in their reduced states exhibit rhombic-type EPR signals with gz = 2.005, gy = 1.955, and gx = 1.921. This hydrogenase is therefore thought to contain three [2Fe-2S] clusters, as well as two [4Fe-4S] clusters. In addition, a nearly isotropic EPR signal (g = 2.01) was observed when the enzyme was anaerobically oxidized by organic dyes such as thionine (E alpha = 64 mV) or 2,6-dichlorophenolindophenol (E alpha = 217 mV). This resonance was not observed at 20 K due to relaxation broadening and therefore did not arise from a conventional organic radical. The oxidized enzyme was fully active in an H2 production assay, and also reacted directly with H2. In contrast, the air-oxidized enzyme was inactive and did not exhibit the g = 2.01 EPR signal. This resonance was assigned to a novel paramagnetic species with an approximate Em value of -70 mV. It is thought to be associated with the H2 activating site of this atypical Fe-hydrogenase.