Detection of Iron Protein Supercomplexes in Pseudomonas aeruginosa by Native Metalloproteomics

Pseudomonas aeruginosa is a major contributor to infections in humans and is widely distributed in the environment. It is capable of aerobic and anaerobic growth, providing adaptability to environmental changes and in confronting immune responses. We applied high-throughput native 2-dimensional metalloproteomics to P. aeruginosa under oxic and anoxic conditions. Clear changes were observed in response to low O ₂ , including inducing the denitrification pathway and a variety of metal responsive proteins. The iron metalloproteome revealed four major iron peaks comprised of proteins with similar or synergistic functions: 1) respiratory and metabolic enzymes, 2) oxidative stress response enzymes, 3) DNA synthesis and nitrogen assimilation enzymes, and 4) denitrification enzymes and related copper enzymes. Three ferritins were observed in the metalloproteome and were coordinating with iron peaks 1 and 3. A number of metalloenzymes were more abundant at low oxygen, including: alkylhydroperoxide reductase C to deactivate organic radicals caused by denitrification, all three classes of ribonucleotide reductases, ferritin (increasing in ratio relative to bacterioferritin), and numerous denitrification enzymes. Superoxide dismutase and homogentisate 1,2-dioxygenase were more abundant at high oxygen. The four Fe peaks contained multiple iron metalloproteins of varying size, implying the presence cytosolic super protein complexes with related functions and dedicated iron storage. This also implies a cellular organization at the protein complex level optimized for metal trafficking and priotization. Together these results provide insight into the cytosol metallome and its response to oxygen and demonstrate the emerging capabilities of metalloproteomic methods in understanding metal use and protein-protein interactions in biological systems.