Mutational analyses of the enzymes involved in the metabolism of hydrogen by the hypertherrnophilic archaeon Pyrococcus furiosus

Pyrococcus furiosus grows optimally near 100 degrees C by fermenting carbohydrates to produce hydrogen (H-2) or, if elemental sulfur (S-0) is present, hydrogen sulfide instead. It contains two cytoplasmic hydrogenases, SHI and SHII, that use NADP(H) as an electron carrier and a membrane-bound hydrogenase (MBH) that utilizes the redox protein ferredoxin. We previously constructed deletion strains lacking SHI and/or SHII and showed that they exhibited no obvious phenotype. This study has now been extended to include biochemical analyses and growth studies using the Delta SHI and Delta SHII deletion strains together with strains lacking a functional MBH (Delta mbhL). Hydrogenase activity in cytoplasmic extracts of various strains demonstrate that SHI is responsible for most of the cytoplasmic hydrogenase activity. The Delta mbhL strain showed no growth in the absence of S-0, confirming the hypothesis that, in the absence of S-0, MBH is the only enzyme that can dispose of reductant On the form of H-2) generated during sugar oxidation. Under conditions of limiting sulfur, a small but significant amount of H-2 was produced by the Delta mbhL strain, showing that SHI can produce H-2 from NADPH in vivo, although this does not enable growth of Delta mbhL in the absence of S-0. We propose that the physiological function of SHI is to recycle H-2 and provide a link between external H-2 and the intracellular pool of NADPH needed for biosynthesis. This likely has a distinct energetic advantage in the environment, but it is clearly not required for growth of the organism under the usual laboratory conditions. The function of SHII, however, remains unknown.