NASA Astrobiology Institute (NAI)

The research addresses how anaerobic Archaea cope with oxidative stress, with the long-term view of how anaerobic life evolved to adapt to rising oxygen levels before, during and after the evolution of oxygenic photosynthesis. The research also addresses ancient enzymes involved in metabolic pathways with a focus on energy conservation in methanogenic Archaea.

Research this reporting period addressed how anaerobic Archaea cope with oxidative stress, with the long-term view of how anaerobic life evolved to adapt to rising oxygen levels before, during and after the evolution of oxygenic photosynthesis. The work focuses on the physiological function and catalytic mechanism of novel enzymes from both methanogenic and non-methanogenic Archaea. One of the enzymes is a novel hexameric protein disulfide reductase (MdrA) from Methanosarcina acetivorans containing an iron-sulfur cluster that when oxidized under aerobic conditions dissociates causing the hexamer to also dissociate exposing the active site and initiating activity. The composition of the iron-sulfur cluster has continued in collaboration with Dr. Steve Cramer at UC Davis. Conditions were established at PSU for incorporation of ⁵⁷Fe into the enzyme for spectroscopic studies that are currently underway at UC Davis. Further, paralogs of MdrA from other Archaea were heterologously overexpressed and will be characterized to further define this novel family. The other enzyme under investigation is a quinone reductase (WrbA) from the sulfate-reducing archaeon Archaeoglobus fulgidus. We hypothesize that the enzyme reduces quinones to the two-electron reduced state when the organism is under oxidative stress to avoid the one-electron semiquinone from reacting with oxygen and producing the superoxide radical. Crystals of the protein were generated that will be analyzed to determine the atomic structure, the basis for understanding evolution of mechanism. Finally, our research on ancient pathways continued with an investigation into the physiology of energy conservation in acetate-utilizing methanogens with a focus on membrane-bound electron transport in M. acetivorans. Thus far, a role for ferredoxin was uncovered wherein this electron carrier was shown to accept electrons from the CO dehydrogenase/acetyl-CoA, an ancient enzyme complex that is key to the pathway of acetate conversion to methane. In addition, an electron transport role for cytochrome C was established. Research is continuing towards investigating the role of a membrane-bound complex, Rnf, and biochemical properties of the subunits.