Mechanism of oxygen detoxification by the surprisingly oxygen-tolerant hyperthermophilic archaeon, Pyrococcus furiosus.

The anaerobic archaeon Pyrococcus furiosus grows by fermenting carbohydrates producing H(2), CO(2), and acetate. We show here that it is surprisingly tolerant to oxygen, growing well in the presence of 8% (vol/vol) O(2). Although cell growth and acetate production were not significantly affected by O(2), H(2) production was reduced by 50% (using 8% O(2)). The amount of H(2) produced decreased in a linear manner with increasing concentrations of O(2) over the range 2-12% (vol/vol), and for each mole of O(2) consumed, the amount of H(2) produced decreased by approximately 2 mol. The recycling of H(2) by the two cytoplasmic hydrogenases appeared not to play a role in O(2) resistance because a mutant strain lacking both enzymes was not more sensitive to O(2) than the parent strain. Decreased H(2) production was also not due to inactivation of the H(2)-producing, ferredoxin-dependent membrane-bound hydrogenase because its activity was unaffected by O(2) exposure. Electrons from carbohydrate oxidation must therefore be diverted to relieve O(2) stress at the level of reduced ferredoxin before H(2) production. Deletion strains lacking superoxide reductase (SOR) and putative flavodiiron protein A showed increased sensitivity to O(2), indicating that these enzymes play primary roles in resisting O(2). However, a mutant strain lacking the proposed electron donor to SOR, rubredoxin, was unaffected in response to O(2). Hence, electrons from sugar oxidation normally used to produce H(2) are diverted to O(2) detoxification by SOR and putative flavodiiron protein A, but the electron flow pathway from ferredoxin does not necessarily involve rubredoxin.|

Structural and functional studies of MinD ATPase: implications for the molecular recognition of the bacterial cell division apparatus.

Proper placement of the bacterial cell division site requires the site-specific inactivation of other potential division sites. In Escherichia coli, selection of the correct mid-cell site is mediated by the MinC, MinD and MinE proteins. To clarify the functional role of the bacterial cell division inhibitor MinD, which is a membrane-associated ATPase that works as an activator of MinC, we determined the crystal structure of a Pyrococcus furiosus MinD homologue complexed with a substrate analogue, AMPPCP, and with the product ADP at resolutions of 2.7 and 2.0 A, respectively. The structure reveals general similarities to the nitrogenase iron protein, the H-Ras p21 and the RecA-like ATPase domain. Alanine scanning mutational analyses of E.coli MinD were also performed in vivo. The results suggest that the residues around the ATP-binding site are required for the direct interaction with MinC, and that ATP binding and hydrolysis play a role as a molecular switch to control the mechanisms of MinCDE-dependent bacterial cell division.