Investigation of the pyruvate: ferredoxin oxidoreductase and its redox partner, ferredoxin
Embargo Date
2027-02-07
OA Version
Citation
Abstract
On a global scale, Fe–S clusters (among other essential redox cofactors, such as hemes, flavins, or amino acid residues) drive the core metabolic reactions of life by transporting electrons through a suite of redox-active enzymes—the oxidoreductase superfamily (Fuchs, 2011). On a per-active-site basis, these enzymes are the most efficient catalysts for several chemical reactions crucial for renewable energy (storage and usage); thus, understanding their inner workings is paramount for developing alternative, green technologies (Reda et al., 2008; Wang et al., 2014). Therefore, this dissertation examines the mechanistic principles of one class of enzyme catalyst bearing Fe–S clusters, the 2-oxoacid:ferredoxin oxidoreductase (OFOR) enzyme superfamily. Although OFORs are considered reversible enzymes, they appear to have an inherent bias toward either the reductive or oxidative chemistry, often believed to reflect the native function of the enzyme (Li et al., 2016). However, revealing the factors that influence an enzyme’s directionality has been difficult. Therefore, by examining a series of unique OFOR enzymes and mutants, this dissertation addresses the following questions: Given the diversity of OFOR enzymes (i.e., cofactor content, number of subunits, and domain modularity), what factors control catalytic bias of CO2 fixation or evolution? What role does the OFOR’s partner protein, ferredoxin, play in biasing reaction directionality?
The electrochemical study of the structurally unique PFOR enzymes from C. tepidum, M. marinus, and M. acetivorans will provide critical information regarding the significance of the domain and structure composition of an OFOR enzyme. It will further reveal whether the native function of the enzyme influences the resting-state reduction potentials of the [4Fe–4S] clusters in the ET chain. Detailed site-directed mutagenesis studies of the Ct PFOR will provide a foundation for understanding the relationship between cluster potentials and ET/catalytic rates of the OFOR family and give insight into the role the protein matrix plays in tuning cluster potentials.
Furthermore, electrocatalytic studies of the Ct PFOR/Fd system will provide an example of how the identity of a partner protein could direct or support enzyme catalysis and elucidate factors that contribute to successful intermolecular-ET. Understanding how Fd characteristics influence catalysis applies to many other biological systems, including the chemistry of hydrogenases or nitrogenases. Finally, the study of the Fe proteins from the nitrogenase provides insight into the thermodynamic driving force that initiates nitrogen fixation in the catalytic component of the nitrogenase, further improving our understanding of how the nitrogenase accomplishes its chemistry.
Description
2024