Date of Graduation

12-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Lessner, Daniel J.

Committee Member

Millett, Francis S.

Second Committee Member

Ivey, D. Mack

Third Committee Member

Barabote, Ravi D.

Keywords

Molecular biology; Cell biology; Reactive oxygen species; M. acetivorans; NADPH

Abstract

All cells have a reduced intracellular environment. In the presence of oxygen, the non-specific oxidation of intracellular components leads to the production of reactive oxygen species (ROS) within cells leading to oxidative stress. During oxidative stress labile cofactors (e.g. Fe-S clusters) are lost and deleterious disulfide bonds are formed within proteins. Intracellular redox maintenance systems are used to direct reducing equivalents towards the repair of oxidatively-damaged proteins. The thioredoxin system is the ubiquitous intracellular redox system, found in virtually all species. The canonical thioredoxin system is comprised of a NADPH-dependent thioredoxin reductase (TrxR) that functions to reduced thioredoxin (Trx). Although the thioredoxin system is well understood in many bacteria and eukaryotes, it is far less understood in archaea, in particular strictly anaerobic methane-producing archaea (methanogens). Methanogens are the only organisms capable of methane production. Biologically produced methane is essential for the global carbon cycle, but is also a byproduct of agriculture and farming of ruminants thus exacerbating the extent of anthropogenic climate change. The ability of methanogens to produce methane requires a large number of oxygen-sensitive metalloenzymes. However, methanogens can survive oxygen exposure, indicating that they possess intracellular redox maintenance systems. Methanogens use the deazaflavin F420 and the Fe-S cluster protein ferredoxin as primary electron carriers, instead of NADPH. Results presented here reveal that Methanosarcina acetivorans, and likely the majority of methanogens, use NADPH-dependent thioredoxin systems. NADPH is produced through the oxidation of the primary electron carriers F420 and ferredoxin. M. acetivorans contains multiple Trx homologs (MaTrx1-7) that serve alternative purposes within M. acetivorans. In particular, MaTrx3 and MaTrx6 are membraned associated where they likely function in the oxidation/reduction of extracellular proteins. MaTrx7 is the primary intracellular Trx, as it is the only MaTrx reduced by MaTrxR, and it is capable of reducing several hundred M. acetivorans proteins. Enzyme assays reveal that M. acetivorans can produce NADPH in the presence of oxygen, supporting a role for the NADPH system in response to oxidative stress. Overall, these results provide insight into the roles of a thioredoxin system in M. acetivorans, which may lead to methods to control methane production in methanogens.

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