Metallomics (DOI: 10.1093/mtomcs/mfaf023): https://doi.org/10.1093/mtomcs/mfaf023
What the study examined
Pseudomonas aeruginosa is a metabolically versatile, opportunistic pathogen that colonizes the lungs of people with cystic fibrosis, chronic wounds, burns, and indwelling medical devices. A defining trait is its ability to switch from aerobic respiration to anaerobic denitrification, using nitrate and nitrite as terminal electron acceptors in the low-oxygen, mucus-laden microenvironments where it forms biofilms. Both lifestyles depend heavily on iron, the most common transition-metal cofactor in biology.
Saito and McIlvin (2025), publishing in the journal Metallomics, set out to determine not just how much iron the bacterium contains but where that iron resides at the level of individual metalloproteins and protein complexes. Rather than inferring metal use from genome annotation, they measured the intact metalloproteome directly, comparing cells grown under oxic (aerobic) versus anoxic (denitrifying) conditions to see how iron allocation is remodeled with oxygen availability.
How native metalloproteomics works
The team used a native, non-denaturing two-dimensional chromatographic separation coupled to dual mass spectrometry. Cell extracts were prepared under conditions that preserve metal-protein coordination and protein-protein interactions, then fractionated first by anion-exchange chromatography and second by size-exclusion chromatography. Because the proteins are never unfolded, metals stay bound to their host proteins during separation.
Each fraction was analyzed two ways in parallel: inductively coupled plasma mass spectrometry (ICP-MS) to quantify metals such as iron, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) on trypsin-digested proteins to identify which proteins were present. Overlaying the elemental map onto the protein map lets the metal signal be assigned to specific metalloproteins, and the size dimension reveals when several iron proteins co-migrate as larger assemblies rather than isolated enzymes.
Four iron peaks with synergistic functions
The analysis resolved the iron metalloproteome into four distinct iron peaks, each a cluster of metalloproteins with related biological roles. Peak one grouped core respiratory and central-metabolism enzymes such as aconitase and catalase. Peak two concentrated oxidative-stress-response enzymes, including superoxide dismutase. Peak three collected enzymes of DNA synthesis and nitrogen assimilation, notably ribonucleotide reductases and glutamine synthetase. Peak four contained the denitrification machinery and associated copper-dependent enzymes.
Because the four peaks each contained multiple iron proteins of varying size, the authors interpret them as evidence of cytosolic super-complexes: functionally related iron enzymes that physically associate, potentially co-located with dedicated iron storage. This points to cellular organization of the metalloproteome at the protein-complex level, a logistics system for trafficking and prioritizing a scarce, reactive metal.
Ferritins localize iron storage to specific functions
Three iron-storage proteins were detected. Rather than forming a single generic reservoir, these ferritin-family proteins co-eluted with the iron peaks tied to respiration/metabolism and to DNA synthesis/nitrogen assimilation. Co-localizing storage with the enzymes that consume iron suggests a just-in-time supply arrangement, staging iron next to the pathways with the highest demand.
Under low-oxygen growth the balance among storage proteins shifted, with ferritin increasing relative to bacterioferritin. That reallocation is consistent with the broader remodeling the cell undertakes as it moves iron toward anaerobic metabolism.
Oxygen availability reshapes the iron economy
The iron peaks were larger under anoxic than oxic conditions, matching the elevated iron demand of anaerobic denitrifying growth. Most strikingly, the denitrification peak was essentially absent under oxic conditions and appeared only when the cells respired nitrogen oxides, reflecting induction of the iron- and copper-dependent nitrate, nitrite, nitric-oxide, and nitrous-oxide reductases.
Other enzyme classes tracked the switch. All three classes of ribonucleotide reductase (the oxygen-independent and oxygen-dependent forms P. aeruginosa encodes) were more prominent under anoxia, as was alkylhydroperoxide reductase C, which detoxifies organic radicals generated as a byproduct of denitrification. Conversely, an oxygen-utilizing enzyme, homogentisate 1,2-dioxygenase, was more abundant aerobically. Together these shifts show the bacterium rebuilding a substantial fraction of its iron-protein inventory in response to a single environmental variable.
Relevance to the metal-microbiome-disease axis
This is a study of essential-metal biology in a pathogen, and its clearest disease connection runs through nutritional immunity: the host defends itself by withholding iron (via lactoferrin and transferrin) and other metals (via calprotectin), while pathogens deploy siderophores and metalloregulatory circuits to fight back. By showing exactly which iron enzymes P. aeruginosa relies on, and that anaerobic denitrification is especially iron-hungry, the work identifies the metabolic pressure points that iron restriction targets in infected, low-oxygen tissue such as cystic-fibrosis mucus and biofilms.
The findings also frame how perturbations to metal availability can ripple into disease. Within the metal-microbiome-disease axis, the governing idea is that shifts in metal supply reshape which microbes and metabolic modes thrive. Iron availability tips the balance between P. aeruginosa lifestyles, and heavy-metal exposures that disturb host and microbial metal homeostasis, through competition for uptake, mismetallation of enzymes, or disruption of metalloregulation, could plausibly alter that balance. Those downstream links are mechanistic hypotheses rather than direct findings of this paper, which stops at defining the iron metalloproteome itself; establishing an exposure-to-disease chain would require studies that manipulate metal exposure and measure microbiome and host outcomes.
Key findings
- Native two-dimensional metalloproteomics resolved the iron content of Pseudomonas aeruginosa into four distinct iron peaks, each a cluster of functionally related metalloproteins.
- The four peaks map to (1) respiration and central metabolism, (2) oxidative-stress response, (3) DNA synthesis and nitrogen assimilation, and (4) denitrification plus associated copper enzymes.
- Three ferritin-family storage proteins co-eluted with the respiration and DNA-synthesis peaks, co-locating iron storage with the enzymes that consume it.
- Iron peaks were larger under anoxic conditions, and the denitrification peak was present only under anoxia, showing anaerobic denitrifying growth demands more iron than aerobic respiration.
- Anoxia increased all three classes of ribonucleotide reductase and alkylhydroperoxide reductase C, while ferritin rose relative to bacterioferritin; the oxygen-using enzyme homogentisate 1,2-dioxygenase was higher under oxic growth.
- The co-migration of multiple iron proteins as larger assemblies suggests cytosolic super-complexes, implying the metalloproteome is organized at the protein-complex level for metal trafficking and prioritization.
Frequently asked questions
What is a metalloproteome, and how was the iron metalloproteome of P. aeruginosa measured?
The metalloproteome is the full set of an organism's metal-binding proteins together with the metals bound to them. Saito and McIlvin measured it natively: cell extracts were fractionated by anion-exchange then size-exclusion chromatography without denaturing the proteins, and each fraction was analyzed by ICP-MS for iron and by LC-MS/MS for protein identity, letting iron signals be assigned to specific proteins.
Why does Pseudomonas aeruginosa need more iron when growing without oxygen?
Without oxygen, P. aeruginosa switches to denitrification, respiring nitrate and nitrite through a suite of iron- and copper-dependent reductases that are not made during aerobic growth. It also upregulates iron-dependent enzymes such as ribonucleotide reductases. The study found the iron peaks were larger under anoxic conditions and that the denitrification iron peak appeared only under anoxia.
What role do ferritins play in the P. aeruginosa iron metalloproteome?
Ferritins and bacterioferritins store iron in a safe, non-reactive form. The study detected three storage proteins that co-eluted with the iron peaks for respiration/metabolism and for DNA synthesis/nitrogen assimilation, suggesting iron is stored next to the pathways that need it most. Under low oxygen, ferritin increased relative to bacterioferritin.
How does this study relate to infection and the metal-microbiome-disease axis?
P. aeruginosa is a major opportunistic pathogen, and mapping its iron enzymes reveals the metabolic targets of host nutritional immunity, which withholds iron to starve invaders. It connects to the metal-microbiome-disease axis by showing how metal availability governs which microbial lifestyles thrive; links from heavy-metal exposure to disease through this pathway remain mechanistic hypotheses beyond the scope of this metalloproteome mapping.