Structural and Functional Characterizations of the Two Mini-ferritins in Pseudomonas aeruginosa Revealed the Coexistence of a Classic Dps (PA0962) and a Dps-Like (PA4880) Protein

Author

Rajapaksha Pathirannahalage Nimesha Hansani Rajapaksha, Louisiana State University and Agricultural and Mechanical CollegeFollow

Degree

Doctor of Philosophy (PhD)

Department

Department of Chemistry

Document Type

Dissertation

Abstract

In the P. aeruginosa genome, gene PA0962 codes for a probable DNA-binding protein from starved cells (Pa Dps). Pa Dps is assembled from 12 monomers, each consisting of a four-helix bundle fold typical of Dps proteins. A subunit dimer of Pa Dps harbors two ferroxidase centers. Assembly of a dodecameric structure of Pa Dps is favored by the presence of divalent metal ions and at pH 6.0 in the absence or presence of divalent cations. Pa Dps is unable to form the large DNA-protein complexes typical of many Dps proteins. Instead, Pa Dps acts on plasmid DNA to form relaxed (nicked) and linearized DNA. Increasing the protein-to-DNA mole ratio results in smears and small DNA fragments in agarose gel electrophoresis. The nuclease activity of 12-mer Pa Dps is enhanced in the presence of divalent cations and low ionic strength. Pa Dps ferroxidase centers can oxidize Fe²⁺ ions using H₂O₂ as an oxidant and form a Fe³⁺ iron mineral core. The Pa Dps X-ray crystal structure revealed a unique network of 10 tyrosine residues at the subunit dimer interface, halfway between the two ferroxidase centers. Pa Dps uses this tyrosine network to trap hydroxyl radicals leaking from the ferroxidase center during Fe²⁺oxidation, thus protecting the P. aeruginosa cells from H₂O₂-mediated oxidative stress.

The gene PA4880 in the P. aeruginosa genome codes for a Dps-like protein (Pa DpsL). It is a dodecameric, nearly spherical shell-like structure that shares common structural and functional features with Pa Dps and Pa bacterioferritin. The monomer of the protein harbors a ferroxidase center, which can bind two Fe ions. The assembly of a dodecameric structure in Pa DpsL is stabilized by the presence of divalent cations. Both 6-mer and 12-mer oligomeric assemblies of Pa DpsL show nuclease activity at pH 6.0–8.5. High ionic strength favors the formation of nicked DNA. Pa DpsL ferroxidase centers utilize O₂ or H₂O₂ as the oxidant and form an Fe³⁺ iron mineral core. The formation of mineral cores was more efficient with O₂. The contribution of Pa DpsL to combat H₂O_2-mediated oxidative stress in P. aeruginosa cells was not as significant as the effect of Pa Dps under the experimental conditions employed.

Date

10-24-2023

Recommended Citation

Rajapaksha, Rajapaksha Pathirannahalage Nimesha Hansani, "Structural and Functional Characterizations of the Two Mini-ferritins in Pseudomonas aeruginosa Revealed the Coexistence of a Classic Dps (PA0962) and a Dps-Like (PA4880) Protein" (2023). LSU Doctoral Dissertations. 6303.
https://repository.lsu.edu/gradschool_dissertations/6303

Committee Chair

Rivera, Mario E.