Theodore Tran

Archaea are single-celled organisms that make up the third domain of life. However, these organisms remain understudied. Methanogenic archaea (methanogens) are the only organisms known to reduce carbon dioxide to methane, also known as methanogenesis. Methanogens produce around seventy percent of the methane on Earth and are crucial in the breakdown of organic materials in environments that lack oxygen. Methanogenesis consists of a series of oxidoreductive steps, which involve the movement of electrons from one product to another. At least one of these reactions depends on the protein complex Heterodisulfide reductase and its specific subunit, HdrA. This subunit plays a central role in this protein complex by carrying out a process known as electron bifurcation. However, while electron bifurcation is known to occur, how HdrA accomplishes this reaction is unknown. To investigate the roles of iron-sulfur clusters in electron bifurcation, I constructed and expressed a mutant gene encoding for the HdrA protein using the model methanogen Methanococcus maripaludis. I engineered a variant HdrA protein where certain cysteines are mutated to serine residues. To test its electron bifurcation activity, the purified mutant Hdr complex was subjected to an in-vitro ferredoxin (Fd) reduction assay. Should the HdrA mutants be sufficient for disrupting the Hdr complex bifurcation activity, it would indicate that my modified iron-sulfur cluster in HdrA is essential for electron bifurcation activity. In contrast, should bifurcation activity not be affected, it would suggest that other iron-sulfur clusters are required for the electron transfer and that the iron-sulfur cluster that I targeted in HdrA is not essential for electron bifurcation activity. This result would also indicate that HdrA does not utilize the entire iron-sulfur cluster chain in order to generate its reduced product and would suggest a novel role for iron-sulfur clusters in stabilizing this protein.