Project Summary/Abstract Cytochromes c are crucial in methane-metabolizing archaea for the production and consumption of methane coupled to growth and energy conservation. Overall, methane-metabolizing archaea mediate the net flux of methane released into the atmosphere and thus, significantly impact the global methane cycle and climate change. Based on genomic studies, Cytochromes c have been hypothesized to play an important role in methane metabolism however the underlying molecular mechanisms remain elusive, primarily due to the absence of a well-developed genetic model system. Even attempts to study archaeal cytochrome c using an alternative approach such as heterologous expression in well-established bacterial systems have not proven successful so far. The main focus of the proposed research is to develop a genetically tractable methanogenic archaeon, Methanosarcina acetivorans, as a platform to functionally characterize archaeal cytochrome c and gain physiological insights into the role of cytochrome c in methane metabolism across different archaeal species. Cytochromes c are ubiquitous electron transfer proteins that require a covalent attachment to its heme co-factor, a process called cytochrome c biogenesis. Coordination between the cytochrome c biogenesis pathway and the cytochrome c of interest is critical for the successful production of a functional cytochrome c in a heterologous host. Using genetic and biochemical tools, I have recently characterized the cytochrome c biogenesis pathway in the model methanogenic archaeon, Methanosarcina acetivorans. Using this knowledge, the project aims to develop M. acetivorans as a genetic chassis to produce and functionally characterize archaeal cytochrome c from diverse archaeal species. Aim1 of the project will functionally characterize the crucial cytochromes c belonging to methane-producing archaea or methanogens, and Aim2 will study these proteins from methane-consuming archaea using both in vitro and in vivo analyses. This research will improve our understanding of methane metabolism and lead to the development of an archaeal host to study cytochrome c proteins from archaea. Ultimately, the knowledge garnered from these studies can be used to develop sustainable solutions for the global climate crisis and mitigate its harmful impacts on human health.