Understanding Intracellular Redox Regulation in Methanogens: Characterization of the Components and Targets of the NADPH-dependent Thioredoxin System from Methanosarcina acetivorans

All cells have a reduced intracellular environment. In the presence of oxygen, the nonspecific 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 oxidativelydamaged 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. Acknowledgements The work contained within this dissertation was made possible through the advice, guidance, and input of Drs. Daniel Lessner, Mack Ivey, Frank Millet, and Ravi Barabote. Additionally, Drs. Andy Alverson, Yuchun Du, and Bill Durham provided their expertise in the respective fields of bioinformatics, proteomics, and biochemistry. Dr. Faith Lessner provided critical training of laboratory techniques that ultimately allowed this degree to be obtained in a reasonable time frame. The help each of you provided was and will be much appreciated. Dedication Acquisition of a doctoral degree is no short or easy task, and those who supported me along the way are due credit. Thank you, family and friends, for your advice, education, and support along the way. In particular, thanks to my maternal grandparents Jimmy and Judi Conway for the financial support and constant reminders to complete the task after accepting an additional full-time job near the end of my degree. Jimmy and his father W.R. Conway both spoke frequently about the importance of achieving the highest form of education possible, and while both passed away during the course of obtaining this degree both will be remembered for their love and support. To my paternal grandparents Robert and Charlotte McCarver, thank you for constantly reminding me that both of you hold degrees, one a doctorate of practical theology and the other a RN, and for your financial assistance. After nine and a half years I can finally say my degree was harder, at least in my opinion. To my uncle Heath McCarver, thank you for being a father to me after moving from home to begin school nearly a decade ago. Your advice and discussions helped me to look past the immediate hurdles and onto the goal of finishing. To my friend John Gomez, thank you for showing me throughout our friendship that advanced education is for anyone who applies themselves. To my fiancé and future wife Melissa Welch, thank you for dealing with my mood swings after long hours of seemingly wasted time in lab and for supporting my numerous hobbies that helped to maintain my sanity. I look forward to many years of scuba diving and a lower stress lifestyle with you now that my degrees are complete.