Abstract
Pyrococcus furiosus grows optimally near 100°C by fermenting carbohydrates to produce hydrogen (H2) or, if elemental sulfur (S0) 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 ΔSHI and ΔSHII deletion strains together with strains lacking a functional MBH (ΔmbhL). Hydrogenase activity in cytoplasmic extracts of various strains demonstrate that SHI is responsible for most of the cytoplasmic hydrogenase activity. The ΔmbhL strain showed no growth in the absence of S0, confirming the hypothesis that, in the absence of S0, MBH is the only enzyme that can dispose of reductant (in the form of H2) generated during sugar oxidation. Under conditions of limiting sulfur, a small but significant amount of H2 was produced by the ΔmbhL strain, showing that SHI can produce H2 from NADPH in vivo, although this does not enable growth of ΔmbhL in the absence of S0. We propose that the physiological function of SHI is to recycle H2 and provide a link between external H2 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.
Highlights
Hydrogen gas (H2) plays an important role in anaerobic metabolism as the majority of anaerobes contain the enzyme hydrogenase responsible for the reversible interconversion of molecular hydrogen, protons, and electrons
These data indicate that SHI is responsible for the majority of hydrogenase activity in the cytoplasm and confirms that the activity of the membrane-bound hydrogenase (MBH) is strictly associated with the membrane (Sapra et al, 2000; Silva et al, 2000)
With sufficient S0 (2 g/L) only a very small amount of H2 was produced in the parental strains and in ΔmbhL (
Summary
Hydrogen gas (H2) plays an important role in anaerobic metabolism as the majority of anaerobes contain the enzyme hydrogenase responsible for the reversible interconversion of molecular hydrogen, protons, and electrons. The enzyme responsible for H2 formation is a unique membrane-bound hydrogenase (MBH) complex, which uses the energy from this exergonic reaction to create an ion gradient across the membrane that drives ATP synthesis, resulting in the generation of an estimated 0.3 mol of ATP per mol H2 (Sapra et al, 2003). This system is one of the simplest forms of respiration. SHI and SHII are proposed to be involved in H2 recycling to provide NADPH for biosynthesis (Ma and Adams, 2001b; van Haaster et al, 2008)
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