Oxygen Uptake and Antioxidant Responses of the Free-Living Diplomonad Hexamita sp.

ABSTRACT. The free-living anaerobic flagellate Hexamita sp. was observed to actively consume O₂ with a K_m O₂ of 13 μM. Oxygen consumption increased lineraly with O₂ tension up to a threshold level of 100 μM, above which it was inhibited. Oxygen uptake was supported by a number of substrates but probably not coupled to energy conservation as cytochromes could not be detected spectro-photometrically. In addition, inhibitors specific for respiratory chain components did not significantly affect O₂ uptake. Respiration was however, partially inhibited by flavoprotein and iron-sulfur protein inhibitors. NAD(P)H supported O₂ consumption was measured in both particulate and soluble fractions; this activity was partially inhibited by quinacrine. A chemosensory response was observed in cells exposed to air, however no response was observed in the presence of superoxide dismutase plus catalase. Catalase and nonspecific peroxidase activity could not be detected, but superoxide dismutase activity was present. Superoxide dismutase was sensitive to NaN₃ and H₂O₂ but not KCN, suggesting a Fe prosthetic group. Flow cytometric analysis revealed that thiol levels in live cells were depleted in the presence of t-butyl H₂O₂. The observed NADPH-driven glutathione reductase activity is believed to recycle oxidized thiols in order to re-establish reduced thiol levels in the cell. The corresponding thiol cycling enzyme glutathione peroxidase could not be detected. The ability to withstand high O₂ tensions (100 μM) would enable Hexamita to spend short periods in a wider range of habitats. Prologed exposure to O₂ tensions higher than 100 μM leads to irreversible damage and cell death.