Date of Award
2025
Document Type
Thesis (Ph.D.)
Department or Program
Chemistry
First Advisor
Ekaterina Pletneva
Abstract
Pseudomonas aeruginosa (Pa) is a pathogenic bacterium known for its antibiotic resistance and ability to survive in low-oxygen environments through biofilm formation. Central to its aerobic respiration under such conditions is cytochrome cbb3 oxidase, a terminal enzyme usually receiving electrons from c type cytochromes. This thesis investigates the molecular basis of electron transfer to cytochrome cbb3 oxidase, focusing on the CcoP subunit, which contains two heme c groups and is proposed to be the primary electron entry site. CcoP isoforms (CcoP1 and CcoP2) from Pa and its non-pathogenic relative Pseudomonas stutzeri (Ps) were expressed, purified, and analyzed using spectroscopic methods and binding assays. Ps CcoP1 and CcoP2 displayed high structural similarity with minor redox differences that may influence donor interactions. In Pa CcoP2, spectroscopic and structural characterizations revealed a deviation from canonical Methionine (Met) coordination in the oxidized state, consistent with a longer-than-usual distance between the C-terminal heme iron and the sulfur atom of M186 noticed in cryo-EM structure. Mutagenesis near M186 partially restored Met coordination and reduced nitric oxide (NO) binding, indicating that local structural features influence ligand stability. These results underscore the importance of axial ligand identity in electron transfer: stable Met ligation raises the heme’s reduction potential, promoting electron entry into CcoP. Conversely, unstable Met or alternative ligands may facilitate electron transfer from CcoP to downstream subunits. Furthermore, NO binding studies provide insight into Met stability and Pa’s survival under nitrosative stress. Biolayer interferometry suggested strong binding between Ps CcoP isoforms and ii i cytochrome c4 (cyt c4), with enzymatic assays further supporting cyt c4 as a physiological electron donor in Ps. However, the observed viability of Ps strains lacking cyt c4 suggested the existence of alternative redox partners, which were subsequently investigated through in-vivo pull-down assays. Recent cryo-EM analysis of the Pa cytochrome bc1–cbb3 super-complex revealed the presence of both cyt c4 and a fragment likely from cytochrome c5 (cyt c5). A truncated Pa cyt c5 was expressed, purified, and shown by NMR to bind Pa CcoP2 with higher affinity than cyt c4, suggesting its potential role in bridging electron flow to cbb₃ oxidase in Pa respiration.
Recommended Citation
Yang, Jia, "Properties and Interactions of the CcoP Subunit of Cytochrome cbb3 Oxidase" (2025). Dartmouth College Ph.D Dissertations. 342.
https://digitalcommons.dartmouth.edu/dissertations/342
