CAREER: Bacterial cytochrome c biogenesis synthase function, specificity and evolution

About This Grant

Microbes inhabit nearly every environment on Earth, acting as key components of ecosystems, drivers of biogeochemical cycles and significantly impacting human health. The ability to thrive in diverse environments is in part due to microbes’ ability to convert diverse energy sources into cellular energy, often via multiple branched electron transport chains. Cytochromes c (cyt c) are a key component of most electron transport chains and much effort has been devoted to understanding the diverse roles of individual cyt c isoforms. Over one hundred different cyt c have been identified, yet all are made in the same way, a process called cyt c biogenesis. Despite the large number and diversity in function of cyt c, only three pathways exist to make it: System I (prokaryotes), System II (prokaryotes), and System III (eukaryotes). Understanding how these three pathways function is a fundamental biological question that is still not well understood. This project will obtain a detailed, mechanistic understanding of the bacterial System II cyt c biogenesis pathway through structure-function analysis. The team integrates undergraduate researchers into the project via direct participation in the research objectives. Additional educational opportunities from this project include classroom experiences to expand undergraduate knowledge, both of research and of STEM careers. These activities will increase STEM identity as a way to improve retention in STEM, leading to a more multitalented STEM workforce. Cyt c biogenesis requires the covalent attachment of heme to a conserved motif on cyt c. This project focuses on the bacterial System II cyt c biogenesis pathway composed of two proteins, CcsB/A, proposed to be a bi-functional enzyme for transmembrane heme transport and attachment to cyt c. Comparison of System II proteins across bacteria has determined that CcsB/A’s have low sequence identity, can be encoded with different genetic arrangements and exhibit variability in protein size and predicted structure. Therefore, a comparative study of System II pathways will be undertaken to 1) determine if holocytochrome c synthase function is conserved despite protein variability and 2) elucidate the System II/cytochrome c interaction domain to determine specificity of heme attachment. Additionally, the distribution of bacterial cyt c pathways, System I and II, across bacteria will be determined bioinformatically to define the evolution of these pathways. These in-depth structure-function studies will provide a mechanistic understanding of System II, provide tools that can be expanded for the study of the other cytochrome c biogenesis pathways and lay the foundation for future studies to probe the regulation and impact of cyt c biogenesis in the context of bacterial metabolism and bioenergetics. This project is jointly funded by the Cellular Dynamics and Function program, the Division of Molecular and Cellular Bioscience, and the Established Program to Stimulate Competitive Research (EPSCoR). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.