Detection of Iron Protein Supercomplexes in Pseudomonas aeruginosa by Native Metalloproteomics

Abstract

Pseudomonas aeruginosa is a major contributor to human infections and is widely distributed in the environment. Its ability for growth under aerobic and anaerobic conditions provides adaptability to environmental changes and in confronting immune responses. We applied native 2-dimensional metalloproteomics to P. aeruginosa to examine how use of iron within the metallome responds to oxic and anoxic conditions. Analyses revealed four iron peaks comprised of metalloproteins with synergistic functions, including: 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. Fe peaks were larger under anoxic conditions, consistent with increased iron demand due to anaerobic metabolism and with the denitrification peak absent under oxic conditions. Three ferritins co-eluted with the first and third iron peaks, localizing iron storage with these functions. Several enzymes were more abundant at low oxygen, including alkylhydroperoxide reductase C that deactivates organic radicals produced by denitrification, all three classes of ribonucleotide reductases (including monomers and oligomer forms), ferritin (increasing in ratio relative to bacterioferritin), and denitrification enzymes. Superoxide dismutase and homogentisate 1,2-dioxygenase were more abundant at high oxygen. Several Fe peaks contained iron metalloproteins that co-eluted earlier than their predicted size, implying additional protein-protein interactions and suggestive of cellular organization that contributes to iron prioritization in Pseudomonas with its large genome and flexible metabolism. This study characterized the iron metalloproteome of one of the more complex prokaryotic microorganisms, attributing enhanced iron use under anaerobic denitrifying metabolism to its specific metalloprotein constituents. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/633287v3_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@112b9a5org.highwire.dtl.DTLVardef@5a39a2org.highwire.dtl.DTLVardef@17dbce9org.highwire.dtl.DTLVardef@1fd858f_HPS_FORMAT_FIGEXP M_FIG C_FIG The iron metalloproteome of Pseudomonas aeruginosa was examined using native (non-denaturing) 2-dimensional chromatographic separation coupled to elemental and proteomic mass spectrometries. (A) Four major iron peaks were observed that corresponded to multi-protein complexes associated with respiratory, (B) antioxidant, DNA production, and denitrification functions, and associated iron storage and supply. The results suggest the presence of protein assemblies with potential roles in iron homeostasis and trafficking.