Iron Metalloproteome of Pseudomonas Aeruginosa: A Review

Overview

This paper characterizes how Pseudomonas aeruginosa organizes and reallocates iron across metalloproteins when grown with and without oxygen, with implications for denitrifying infections and redox-flexible metabolism in host and environmental matrices.

What was studied and how?

Aim: to define the Pseudomonas aeruginosa iron metalloproteome under oxic versus anoxic (nitrate-respiring) growth and relate iron allocation to functional protein groupings. The primary matrix was native, detergent-free cell extracts; aliquots from oxic and anoxic cultures in marine broth (anoxic medium contained 0.88 mM nitrate) were separated by two-dimensional native chromatography—anion exchange (AE) followed by size exclusion (SE)—and analyzed by dual mass spectrometry: LC–MS/MS for proteins and ICP-MS for metals (Fe monitored as ^56Fe in “ppb”; each SE fraction = 0.5 mL). Global (denaturing) proteomics validated oxygen acclimation. Microbial system: P. aeruginosa isolates (strain 2-54; PAO1 genome used for annotation). Metals/metalloproteins emphasized Fe (heme, Fe-S, ferritins), with contextual Cu in denitrification (e.g., azurin); explicit chemical oxidation states were not resolved by XAS but inferred by prosthetic groups (heme, Fe–S). Analytical specifics include AE (50 mM Tris, pH 8.8; NaCl gradient), SE (TSKgel G3000SWxl), ICP-MS (KED-He mode; isotopes included ^56Fe, ^63Cu, etc.), and Orbitrap-based proteomics of fractionated digests.

Most important findings

Critical point	Details
Four Fe peaks define functional “modules”	Fe Peaks 1–4 mapped to respiration/carbon catabolism (Peak 1), oxidative stress (Peak 2), DNA synthesis & nitrogen assimilation (Peak 3), and denitrification (Peak 4). Fe peak maxima (ppb, per 0.5 mL fraction): Peak 1 oxic 40.5 vs anoxic 158.96; Peak 2: 25.7 vs 46.2; Peak 3: 11.6 vs 57.8; Peak 4: 4.1 vs 28.7.
Oxygen drives greater Fe demand anoxically	Peaks 3–4 enlarged markedly in anoxia despite similar protein loading, consistent with nitrate respiration; Peak 4 nearly absent under oxic growth.
Peak 1 composition & shifts	Aconitase (PA1787), HmgA, HdhA, catalase, and ferritins (BfrB/FtnA) co-eluted; HmgA abundant in oxic; HdhA increased in anoxia; ferritins co-localized with Fe maximum.
Antioxidant module (Peak 2)	Fe-SodB aligned with Fe; quinone oxidoreductase present; two AhpC peroxiredoxins co-eluted and increased under anoxia.
DNA synthesis module (Peak 3)	RNRs (NrdA/NrdB/NrdD) increased under anoxia; NrdB aligned with Fe; GS, Fe–S biogenesis proteins, and a third ferritin (PA4880/Dps-like) co-occurred.
Denitrification module (Peak 4)	NirS, NapA, NirN, multiple c-type cytochromes, IscU, PhuT observed; Cu-azurin co-eluted with Fe peak.
Iron storage architecture	Three ferritins detected: BfrB (PA3531), FtnA (PA4235) at Peak 1; PA4880 (Dps-like) at Peak 3; proportion of FtnA increased at low O₂; PA4880 dominated ferritin spectral counts.
Protein assemblies suggested	Multiple proteins eluted earlier than monomeric size predictions, consistent with multi-protein complexes that may coordinate Fe use.

Strengths

The study employs native two-dimensional metalloproteomics with orthogonal AE–SE separation and dual MS readouts, enabling direct alignment of Fe peaks with protein IDs at high resolution. The authors quantify Fe peak magnitudes (ppb basis per fraction) and map them to coherent metabolic functions, capturing oxygen-dependent re-allocation from oxic respiration toward denitrification and dNTP synthesis. Parallel global proteomics verifies physiological acclimation (e.g., denitrification apparatus; RNR classes) and supports metalloproteomic assignments. The explicit tracking of ferritin variants across AE/SE space provides organism-level context for iron storage during redox shifts. Evidence from elution behavior and column calibration strengthens the inference of higher-order assemblies relevant to Fe trafficking.

Any Limitations

Membrane-associated denitrification proteins may be under-recovered in the non-detergent workflow, potentially underestimating Peak 4 iron. The isolate was likely a ship/handling contaminant rather than open-ocean origin; results rely on PAO1 annotation mapping. Oxidation states were inferred from prosthetic groups rather than determined by dedicated speciation methods, and absolute cellular Fe burdens beyond peak ppb were not reported.

Future Perspectives

For infections characterized by hypoxia and nitrate availability, the anoxic expansion of Fe-intensive modules (RNRs; denitrification proteins) highlights candidate targets and diagnostics focused on Fe-dependent activity, ferritin dynamics, and Cu-partner enzymes like azurin. The native 2D approach also surfaces putative protein assemblies that may govern Fe prioritization; extending this with targeted proteomics and structural interrogation could refine therapeutic strategies that disrupt Fe storage/use (e.g., ferritin composition) or oxidative-stress defenses (e.g., AhpC) during denitrifying growth. Applying this framework to clinical isolates and sputum/biofilm matrices would enhance translational relevance.

Conclusion

This metalloproteomic analysis shows that P. aeruginosa reorganizes iron across four functional modules and increases total Fe allocation during anoxic, nitrate-respiring growth. Key iron users include aconitase and ferritins (Peak 1), SodB and quinone oxidoreductase with AhpC peroxiredoxins (Peak 2), RNRs and nitrogen metabolism proteins with Dps-like storage (Peak 3), and denitrification enzymes plus c-type cytochromes with Cu-azurin (Peak 4). These findings connect oxygen tension to iron demand, storage composition, and respiratory strategy, informing pathogenesis, diagnostics, and Fe-targeted interventions.

Citation

Saito MA, McIlvin MR. The iron metalloproteome of Pseudomonas aeruginosa under oxic and anoxic conditions. Metallomics. 2025;17:mfaf023. doi:10.1093/mtomcs/mfaf023