UC Berkeley

The need for an urgent response to the climate crisis has become ever present. Both 2023 and2024 were the hottest recorded in Earth’s history with temperatures reaching 1.5 C above pre- industrial levels. If this level of warming continues over a 20-year period, it will violate the limit set by the Paris Climate Accord just a decade ago. The impacts of this warming are widely considered catastrophic to humankind, and the World Health Organization has categorized climate change as the single greatest threat to human health. To appropriately respond to and mitigate global warming, a detailed understanding of its specific mechanistic causes is paramount.Methane is considered one of the most critical greenhouse gasses, coming second only to carbon dioxide in terms of heat-trapping contributions in the atmosphere . The contributions of methane to the atmosphere consist of both biotic and abiotic sources, both of which are included in the anthropogenic sources of greenhouse gasses primarily driving global warming . It is estimated that about 75% of all atmospheric methane comes from biological sources which include, but are not limited to, agriculture, landfills, and natural wetlands. The class of organisms responsible for the biotic methane contribution is the methanogen, a type of Archaeon found in diverse and widespread anaerobic environments across the globe . Despite the ecological, evolutionary, and metabolic diversity of methanogens, the enzyme responsible for methane formation, methyl-coenzyme M reductase (MCR), is both ubiquitous throughout and unique to this class of organisms . The reaction catalyzed by MCR consists of the reduction of a methyl group from methyl-coenzyme M with reducing equivalents provided by coenzyme B which produces methane and a heterodisulfide of coenzyme M and coenzyme B. The subsequent reduction of this heterodisulfide couples this process to adenosine triphosphate (ATP) production and thus serves as the essential energy generating mechanism for these organisms .Given that MCR is of critical importance to global carbon cycling, it’s necessary to obtain adetailed understanding of the ways in which this enzyme functions in vivo. MCR’s discovery predates the classification of Archaea as a unique domain of life, and initial descriptions of the methyl-reducing cell fraction were obtained from a “methane bacteria”. Despite the discovery of this enzyme nearly fifty years ago, the majority of research surrounding it has been restricted to in vitro biochemical characterizations mainly due to the dearth of genetic tools available to manipulate methanogens until recently. The discoveries made surrounding MCR’s structure and activity have been critical to the development of methanogenesis inhibitors, like 2-bromoethanesulfunate (BES) and 3-nitrooxypropanol (3-NOP), which have proven to be useful for experimental perturbations of methanogenesis and the reduction of enteric methane emissions from cattle. Despite these strides made in understanding MCR biology, much remains to be uncovered about how this enzyme is assembled, regulated, and activated in vivo.MCR is a heterohexamer composed of a2, b2, and g2 subunits and two active sites, each of which coordinate the nickel containing tetrapyrrole, cofactor F430. The extreme oxygen sensitivity of MCR can be attributed to F430 because of the extraordinarily low redox potential of the Ni1+/Ni2+ pair (between -600 mV and -700 mV). The catalytic mechanism of MCR requires that F430 is initially in the reduced Ni1+ oxidation state, is oxidized during catalysis, and returns to Ni1+ upon reaction completion and product release. The installation of F430 and its reduction to Ni1+ are processes that require a suite of accessory proteins to accomplish . Furthermore, since the initial purification and characterization of MCR it has been known that a sub-stoichiometric amount of ATP is required for sustained activity in addition to several protein-containing cell fractions. As increased attention has been focused on MCR in light of its effects on the climate, so too has increased attention been paid to these accessory proteins which are required for the cell to have a mature and functional MCR. The work described in my thesis focuses on those proteins that are required for the ATP-based reductive activation of MCR, with particular focus on component A2, an ATP binding protein known to be associated with MCR through both genomic and biochemical evidence.Chapter 1 of this thesis provides necessary background by reviewing literature surrounding the cellular components needed for MCR function within methanogenic archaea. This chapter provides a description and overview of the “Methanogenesis Maker Proteins” (MMPs), a classification assigned to a group of genes that co-occur with MCR in genomes. Most of these MMPs have functions yet to be determined, but this chapter offers an overview of several MMPs with specifically characterized roles. It begins by describing the biosynthetic pathways for the coenzymes and cofactor used by MCR, coenzyme M, coenzyme B, and F430, and highlights which steps are performed by MMPs. Next, the current understanding of the MCR-associated proteins required for assembly and activation of the enzyme are summarized, including the installation of F430 by McrD and the McrC based activation complex. Finally, this chapter concludes by highlighting several new tools, both genomic and biochemical, that have been developed recently and could provide even more insight into MCR function. This chapter includes a table with all currently described MMPs, known functions, and associated annotations to ease comparisons between published work.Chapter 2 dives deep into role of component A2, an ATP-binding protein introduced in Chapter 1. Component A2 has long been associated with MCR activation, but its specific biochemical role has remained elusive. In this chapter we use a suite of genetic and biochemical experiments to prove that component A2 is a bona-fide ATPase whose activity is stimulated by the presence of MCR. We uncover that the ATPase activity of this enzyme is oxygen sensitive and can be interrupted by specific point mutations introduced into the nucleotide binding domains or into the zinc binding motif. Further analysis with these point mutants reveals that the interaction between A2 and MCR is dependent on the ability of the ATPase to bind ATP but is not dependent on hydrolysis or on zinc binding, leading to a model of interaction where A2 first binds ATP, interacts with MCR, and then hydrolyzes ATP, likely leading to conformational changes in MCR that allow the reductive activation of F430. Additionally, we perform a phylogenetic analysis to show that the zinc binding motif is unique to this class of remodeling ATPases and that component A2 has likely undergone horizontal gene transfer and clusters with a variant of MCR that performs the reverse reaction by oxidizing short chain alkanes. The work described in this chapter provides the first evidence that component A2 is an ATPase whose activity is directly modulated by interaction with MCR, a discovery that will be critical for further characterization of the full mechanism of reductive activation for MCR.Chapter 3 builds on a discovery outlined in chapter 2 that component A2 in M. acetivorans is part of a polycistronic “MCR activation operon” that contains six other MMPs. In this chapter we use CRISPR interference (CRISPRi) as a tool to study the effects of diminished gene expression of both the MCR operon and the MCR activation operon, given the essentiality of both. We find that the MCR operon behaves as a traditional polycistronic operon with a single transcription start site when perturbed with CRISPRi. Interestingly, we find that the MCR activation operon does not behave in the same way, indicating the possibility of multiple transcription start sites being present. We also perform growth analysis of each of these knockdown strains and find that, as expected, decreased expression of MCR has a significant effect on growth rate. In contrast, we determine that decreased expression of component A2 does not have a significant effect on growth rate, indicating that the amount of this enzyme required by the cell under standard laboratory conditions is very low.Together, the work described in this dissertation explores the role of the proteins associated with MCR function and specifically characterizes the ATP requirement for MCR activation. This work furthers our understanding of MCR function in vivo, allowing future studies to continue to draw a more detailed map of how this unique and complicated enzyme functions. Additionally, a clearer mechanistic understanding of MCR activation will allow for further applications towards the goal of methane emission mitigation. This work, combined with past and future experimentation, will continue to bring us closer to a full and complete picture of microbial methane production.