Sulfur reduction is central to life as a vestigial reminder of Earth before the emergence of an oxygen-based energy currency. Organic sulfur in the form of sulfide remains an essential nutrient in all organisms, but higher eukaryotes are unable to reduce inorganic sulfur(IV) so rely on bacteria, archaea, yeast, and plants to produce useable sulfur. The six-electron reduction of sulfite to sulfide is central to the bio-geo sulfur cycle and is catalyzed by a single enzyme, sulfite reductase (SiR). This "high-volume" electron transfer is unique to sulfur and nitrogen cycles and represents one powerful example of a biochemical reaction where nature is more versatile than human chemical engineers. Together, the centrality of SiR to nutrient cycles and its unique biochemistry make a compelling case that research into its mechanism and structure lays important groundwork for understanding our natural environment.
The goals of this project are to experimentally dissect the chemistry and biology of electron transfer in sulfur reduction by the oxidoreductase SiR and to broaden the impact of this research through integrated educational outreach in the laboratory and classroom. Experimental results will advance knowledge about mechanisms of sulfur reduction by combining information at multiple resolutions from x-ray crystallography and single particle cryogenic electron microscopy (cryo-EM) with biochemical analysis. Specifically, two areas are under investigation. First, the role of specific amino acids in SiR activity will be probed with mutagenesis, SiR activity assays, and x-ray crystallographic structural analysis. Second, the SiR subunit assembly will be explored, with multi-angled light scattering, cryo-EM imaging, and single particle 3DEM analysis, to understand inter-protein chemistry that drives sulfur reduction,.
Broader Impacts Applying structural techniques to answer fundamental questions about environmental bacteriology benefits society at large by providing insight into the mechanisms that regulate the biogeological cycling of sulfur. In addition, exploring sulfur reduction pathways provides a unique platform for integrating research and teaching. The proposed outreach component uses multifaceted classroom exercises based on hypothesis-driven research to engage a range of students. Specifically, students from high school to postdoctoral researchers will be targeted to join the laboratory where they will be exposed to research in structural biology. Florida State University is home to one of approximately 20 Titan Krios cryogenic transmission electron microscopes worldwide and training on this unique instrument is integral to the proposed research. Further, the applied structural techniques rely on diverse tools from computation, mathematics, chemistry, biochemistry, and microbiology, providing a range of potential research projects with a common focus on answering biological questions. At the same time, active-learning modules developed specifically for a first year Introduction to Biology class at Florida State University will reinforce and enhance classroom lessons on biophysics, biochemistry, molecular biology and cellular biology. SiR is an engaging, effective foundation for classroom exercises because it is a novel model to explain basic life science concepts not explored in most lecture courses or textbooks. These classroom modules developed with, and for, students aspiring to teach K-12 science and technology further broadens the long-term impact of this proposal on improving general science education in the United States.
