| Abstrakt: |
The archaebacterium, Pyrococcus furiosus, grows optimally at 100 °C by a fermentative type metabolism in which H2and CO2are the only detectable products. The organism also reduces elemental sulfur (S0) to H2S. Cells grown in the absence of S0contain a single hydrogenase, located in the cytoplasm, which has been purified 350-fold to apparent homogeneity. The yield of H2evolution activity from reduced methyl viologen at 80 °C was 40%. The hydrogenase has a Mrvalue of 185,000 ± 15,000 and is composed of three subunits of Mr46,000 (α), 27,000 (β), and 24,000 (γ). The enzyme contains 31 ± 3 g atoms of iron, 24 ± 4 g atoms of acid-labile sulfide, and 0.98 ± 0.05 g atoms of nickel/185,000 g of protein. The H2-reduced hydrogenase exhibits an electron paramagnetic resonance (EPR) signal at 70 K typical of a single [2Fe-2S] cluster, while below 15 K, EPR absorption is observed from extremely fast relaxing iron-sulfur clusters. The oxidized enzyme is EPR silent. The hydrogenase is reversibly inhibited by O2and is remarkably thermostable. Most of its H2evolution activity is retained after a 1-h incubation at 100 °C. Reduced ferredoxin from P. furiosusalso acts as an electron donor to the enzyme, and a 350-fold increase in the rate of H2evolution is observed between 45 and 90 °C. The hydrogenase also catalyzes H2oxidation with methyl viologen or methylene blue as the electron acceptor. The temperature optimum for both H2oxidation and H2evolution is > 95 °C. Arrhenius plots show two transition points at ∼ 60 and ∼ 80 °C independent of the mode of assay. That occurring at 80 °C is associated with a dramatic increase in H2production activity. The enzyme preferentially catalyzes H2production at all temperatures examined and appears to represent a new type of “evolution” hydrogenase. |
