Pseudomonas aeruginosa Dps (PA0962) Functions in H2O2 Mediated Oxidative Stress Defense and Exhibits In Vitro DNA Cleaving Activity

Abstract

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps.

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

“…The chromatogram ( Figure 1A , red trace) shows only partial reassembly of the 12-mer. This observation is different from that made with 12-mer Pa Dps, which disassembles in the absence of MgSO 4 , but can be fully reassembled into 12-mer when MgSO 4 is added ( Rajapaksha et al, 2023 ). To investigate the nature of the irreversible disassembly of Pa DpsL we resorted to SDS PAGE and mass spectrometry.…”

“…A similar plasmid DNA nicking and cleaving activity was recently reported for Pa Dps ( Rajapaksha et al, 2023 ). Consequently, we decided to compare the activity of both proteins under identical conditions.…”

“…This issue was investigated by titrating solutions of the 12-mer Pa DpsL in 50 mM Tris (pH 7.5) containing 1 mM MgSO 4 with aliquots delivering 50 Fe 2+ /12-mer to a total of 300 Fe 2+ /12-mer. These experiments were conducted in an anaerobic glove box using H 2 O 2 as oxidant, or in air, with O 2 as the electron acceptor, in a manner similar to that reported for Pa Dps ( Rajapaksha et al, 2023 ).…”

“…Consequently, in addition to accumulating iron at concentrations much higher than those enabled by the poor solubility of Fe 3+ near neutral pH, ferritin and ferritin-like molecules minimize the toxic effects expected from unregulated Fe 2+ /Fe 3+ redox cycling ( Bradley et al, 2016 ; Rivera, 2017 ; Rivera, 2023 ). The P. aeruginosa PAO1 genome contains four genes encoding iron storage proteins: ftnA (PA4235) encodes a bacterial ferritin (FtnA) ( Yao et al, 2011 ), bfrB (PA3531) codes for a bacterioferritin (BfrB) ( Weeratunga et al, 2009 ; Weeratunga et al, 2010 ), gene PA0962 codes a Dps ( Rajapaksha et al, 2023 ), and gene PA4880, the subject of this report, is annotated to encode a probable bacterioferritin. Understanding the function of all these proteins is an important step toward gaining a more comprehensive understanding of the fate of iron in bacterial cells, and how storage molecules contribute to managing the benefits and risks associated with the chemistry of essential iron.…”

“…Much less is known about the function of the other two proteins thought to participate in iron storage, the products of genes PA0962, and PA4880. A recent investigation has demonstrated that the product of gene PA0962 is a Dps (DNA binding protein under starvation) ( Rajapaksha et al, 2023 ), but nothing is known about the product of gene PA4880, which is annotated as a putative bacterioferritin. Hence, the objective of this investigation was to characterize biochemically and structurally the product of gene PA4880.…”