Functional Annotation Report: hemN (PP_4264, UniProt Q88F35) in Pseudomonas putida KT2440

Summary

The gene hemN (ordered locus PP_4264; UniProt accession Q88F35) of Pseudomonas putida KT2440 encodes the oxygen-independent (anaerobic) coproporphyrinogen-III oxidase (EC 1.3.98.3), a soluble cytoplasmic enzyme of the heme (tetrapyrrole) biosynthetic pathway. Its primary and defining function is to catalyze the oxidative decarboxylation of the two ring-A/ring-B propionate side chains of coproporphyrinogen III to the corresponding vinyl groups of protoporphyrinogen IX, executing the antepenultimate step of protoporphyrin-IX biosynthesis without requiring molecular oxygen. This assignment is supported by direct biochemical and structural characterization of the Escherichia coli ortholog, by phenotypic/regulatory studies of orthologs in closely related Pseudomonas and Alcaligenes species, and by sequence-level confirmation that the P. putida protein carries every diagnostic catalytic motif at conserved positions.

Mechanistically, HemN is the founding structurally-characterized member of the radical S-adenosyl-L-methionine (radical SAM) superfamily. It harbors a [4Fe-4S] cluster ligated by three cysteines of a conserved CX₃CX₂C motif (the CANICYYC sequence at residues 63–70 in Q88F35) and binds two molecules of SAM in adjacent sites. Reductive cleavage of one SAM generates a highly reactive 5′-deoxyadenosyl radical that abstracts a hydrogen from a substrate propionate; the second SAM acts as a hydrogen relay. The two propionate decarboxylations proceed sequentially through a mono-decarboxylated intermediate, harderoporphyrinogen, consuming two SAM and releasing two CO₂, two 5′-deoxyadenosine, and two L-methionine per turnover (Rhea:15425).

Biologically, HemN provides the O₂-independent route to protoporphyrinogen IX, complementing the oxygen-dependent enzyme HemF (PP_0073) and enabling heme synthesis under anaerobic/microaerobic conditions. In Pseudomonas, hemN transcription is controlled by the Fnr-type redox regulators Anr and Dnr, and loss of HemN function causes accumulation and excretion of coproporphyrin III, the oxidized form of the blocked substrate. The P. putida genome additionally encodes a second HemN-family paralog (PP_3781), giving a redundant, redox-responsive coproporphyrinogen-III oxidase system that parallels the HemF/HemN/HemZ arrangement documented in P. aeruginosa and Bacillus subtilis. The enzyme carries out its function in the cytoplasm as a soluble monomer.

Identity verification

The gene symbol "hemN" corresponds precisely to the UniProt protein description, the organism is correct, and the protein family/domains align with the characterized literature. No gene-symbol ambiguity was encountered — "hemN" is used uniformly across bacteria for this radical-SAM enzyme.

Key Findings

Finding 1 — HemN is the oxygen-independent coproporphyrinogen-III oxidase of heme biosynthesis

The core function of HemN is the O₂-independent conversion of coproporphyrinogen III to protoporphyrinogen IX (EC 1.3.98.3): the oxidative decarboxylation of the two propionate side chains on pyrrole rings A and B of coproporphyrinogen III to vinyl groups. In vitro anaerobic enzyme assays of recombinant E. coli HemN directly demonstrated coproporphyrinogen-III oxidase activity (PMID: 12114526), described explicitly as: "In bacteria the oxygen-independent coproporphyrinogen-III oxidase catalyzes the oxygen-independent conversion of coproporphyrinogen-III to protoporphyrinogen-IX." The structural study reinforced the physiological essentiality: "HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis" (PMID: 14633981).

The physiological substrate is coproporphyrinogen III specifically (the type-III isomer). HemN acts on the two propionates of rings A and B, retaining the other two propionates (rings C and D) in protoporphyrinogen IX — the same regiochemical outcome as the unrelated O₂-dependent HemF but achieved by radical chemistry. The P. putida HemN (hemN/PP_4264) is orthologous to the characterized enzymes and belongs to the anaerobic coproporphyrinogen-III oxidase family (InterPro IPR004558), so the reaction assignment transfers with high confidence given the conserved sequence signatures (Finding 5).

Finding 2 — HemN is a radical SAM enzyme with a [4Fe-4S] cluster and two bound SAM molecules

HemN was the first radical SAM enzyme to have its crystal structure solved (2.07 Å, E. coli), defining the structural template for the entire superfamily. The structure shows a [4Fe-4S] cluster ligated by three cysteines of the conserved CXXXCXXC motif, with a SAM molecule coordinating the fourth, unique iron: "HemN binds a 4Fe-4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen" (PMID: 14633981). The SAM sulfonium sits ~3.5 Å from the cluster, positioned for single-electron transfer and reductive cleavage to a 5′-deoxyadenosyl radical.

A striking, functionally important feature is that HemN binds a second SAM immediately adjacent to the first: "HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations" (PMID: 14633981). This provides the structural basis for the two sequential decarboxylation events.

Biochemical work confirmed the cluster's identity and the essentiality of its ligands: the enzyme contains an O₂-sensitive Fe-S cluster, and "Cysteine residues Cys(62), Cys(66), and Cys(69), which are part of the conserved CXXXCXXC motif found in all HemN proteins, are essential for iron-sulfur cluster formation and enzyme function" (PMID: 12114526). The oxygen sensitivity of this cluster is the molecular basis for HemN serving as the anaerobic branch of coproporphyrinogen oxidation.

Finding 3 — Sequential radical decarboxylation via a harderoporphyrinogen intermediate, requiring SAM and NAD(P)H

The catalytic cycle proceeds by radical chemistry and requires SAM, NAD(P)H, and additional cytoplasmic components (PMID: 12114526): "The enzyme requires S-adenosyl-l-methionine (SAM), NAD(P)H, and additional cytoplasmatic components for catalysis." Mechanistic re-investigation established that the two propionate groups are decarboxylated one at a time, passing through the mono-decarboxylated intermediate harderoporphyrinogen (PMID: 30884058). The revised mechanism assigns distinct roles to the two SAM molecules: "a SAM serves as a hydrogen relay which mediates a radical-based hydrogen transfer from the propionate to the 5'-deoxyadenosyl (dAdo) radical generated from another SAM in the active site." One SAM is the source of the initiating 5′-deoxyadenosyl radical, while the second SAM mediates hydrogen transfer during radical-based decarboxylation.

Finding 4 — Cytoplasmic soluble monomer; Anr/Dnr redox regulation; loss-of-function causes coproporphyrin III accumulation

Anaerobically purified HemN is a monomeric, soluble cytoplasmic protein of native mass ~52 kDa (PMID: 12114526): "Anaerobically purified HemN is a monomeric protein with a native M(r) = 52,000 +/- 5,000." This establishes both the oligomeric state (monomer) and subcellular localization (cytoplasm). HemN has no signal peptide or membrane-spanning region, and tetrapyrrole biosynthesis occurs in the bacterial cytoplasm.

In P. aeruginosa, hemN is strongly induced under anaerobic conditions through two Fnr-type redox regulators: "An approximately 10-fold anaerobic induction of hemN gene expression was mediated by the dual action of Anr and a second Fnr-type regulator, Dnr" (PMID: 9767567). The physiological importance is shown by the loss-of-function phenotype: "An anr mutant did not contain detectable amounts of hemN mRNA and accumulated coproporphyrin III both aerobically and anaerobically, indicating the importance of HemN for aerobic and anaerobic haem formation" (PMID: 9767567). Accumulation of coproporphyrin III (the oxidized, aromatized form of unprocessed coproporphyrinogen III) is the diagnostic metabolic signature of a blocked coproporphyrinogen-III oxidase step.

The orthologous Alcaligenes eutrophus hemN shows the same logic: Fnr-regulated, O₂-repressed, required for anaerobic growth on nitrate/nitrite, with coproporphyrin III excretion upon inactivation (PMID: 9396835). Because P. putida KT2440 encodes the Anr regulator and hemN (PP_4264), the same redox-responsive control is expected in the target organism (inferred by orthology).

Finding 5 — Sequence-level confirmation of catalytic motifs in Q88F35

Direct inspection of the 460-aa, 52.6 kDa P. putida KT2440 HemN sequence (Q88F35) confirms the two essential functional motifs at conserved positions:

• The radical-SAM [4Fe-4S]-binding motif CANICYYC (a CxxxCxxC motif) at residues 63–70, positionally equivalent to the essential Cys62/Cys66/Cys69 of E. coli HemN. Their essentiality was established experimentally: "Cysteine residues Cys(62), Cys(66), and Cys(69) ... are essential for iron-sulfur cluster formation and enzyme function" (PMID: 12114526).
• The SAM-binding GGGTP motif at residues 113–117, matching the E. coli Gly111/Gly113 whose mutation abolished activity: "Mutation of Gly(111) and Gly(113), which are part of the potential GGGTP S-adenosyl-l-methionine binding motif, completely abolished enzymatic function" (PMID: 12114526).

UniProt (rule-based, PIRNR000167) assigns the exact catalytic reaction (Rhea:15425): coproporphyrinogen III + 2 SAM → protoporphyrinogen IX + 2 5′-deoxyadenosine + 2 L-methionine + 2 CO₂ (EC 1.3.98.3); cofactor: one [4Fe-4S] cluster coordinated by 3 cysteines plus an exchangeable SAM; pathway: protoporphyrin-IX biosynthesis (protoporphyrinogen-IX from coproporphyrinogen-III, AdoMet route, step 1/1); subcellular location: cytoplasm.

Finding 6 — A redundant coproporphyrinogen-III oxidase system in P. putida KT2440

P. putida KT2440 encodes three coproporphyrinogen-III oxidase-related genes, giving redundancy across oxygen regimes:

This mirrors the HemF + HemN (+ HemZ) arrangement documented in P. aeruginosa (PMID: 9767567) and B. subtilis (PMID: 10498703). In P. aeruginosa, "Mutation of hemN and hemF did not abolish aerobic or anaerobic growth, indicating the existence of an additional HemN-type enzyme, which was termed HemZ" (PMID: 9767567) — paralleling the three P. putida genes. Caveat: some HemN-family proteins (HemW-type) are radical-SAM heme chaperones rather than coproporphyrinogen oxidases, so PP_3781's exact activity should not be assumed from family membership alone. This redundancy explains why heme synthesis is robust across the aerobic–anaerobic spectrum that P. putida encounters in soil and rhizosphere niches, and why loss of a single enzyme is typically non-lethal yet causes coproporphyrin III accumulation when the alternate route cannot compensate.

Mechanistic Model / Interpretation

Position in the heme biosynthetic pathway

  ...  →  Coproporphyrinogen III
     │
     │  [Oxidative decarboxylation of 2 propionate → 2 vinyl groups]
     │
┌────────────┴─────────────┐
│                          │
   O2-DEPENDENT               O2-INDEPENDENT
   HemF (PP_0073)             HemN (PP_4264)  ← TARGET
   EC 1.3.3.3                 EC 1.3.98.3      (+ paralog PP_3781)
   uses O2                    radical-SAM; no O2 required
│                          │
└────────────┬─────────────┘
     ▼
   Protoporphyrinogen IX
     │
     ▼  (HemG/HemY protoporphyrinogen oxidase)
     Protoporphyrin IX
     │
     ▼  (HemH ferrochelatase)
   Heme b

Catalytic cycle of HemN (radical-SAM chemistry)

   [4Fe-4S]1+  +  SAM(1)  ──►  [4Fe-4S]2+  +  L-Met  +  5'-deoxyadenosyl radical (dAdo•)
                                              │
                                              ▼
        dAdo•  abstracts H from propionate β-carbon of substrate
                                              │
                                              ▼
      Substrate radical  →  decarboxylation (–CO2)  →  vinyl group formed
                                              │
        SAM(2) acts as H-relay in radical-based H transfer
                                              │
                                              ▼
        Repeat on 2nd propionate (via harderoporphyrinogen intermediate)
                                              │
                                              ▼
   Net: Coproporphyrinogen III + 2 SAM  →  Protoporphyrinogen IX
              + 2 CO2 + 2 5'-deoxyadenosine + 2 L-Met     (Rhea:15425)

The coherent picture is of a cytoplasmic, soluble, monomeric radical-SAM metalloenzyme that uses an oxygen-sensitive [4Fe-4S] cluster and two SAM cofactors to perform demanding C–C bond chemistry (decarboxylation of unactivated propionate side chains) without molecular oxygen. This capability is precisely why the enzyme exists as a distinct paralog to the oxygen-dependent HemF: it allows the cell to complete heme biosynthesis when oxygen is scarce or absent. In Pseudomonas, this need arises under low-oxygen/denitrifying conditions, when the Anr/Dnr regulators induce hemN. The metabolic redundancy — HemF for aerobic conditions, HemN and PP_3781 for anaerobic/microaerobic conditions — insulates the essential supply of heme (for cytochromes, catalases, and other hemoproteins) from the oxygen fluctuations that an environmentally versatile soil/rhizosphere organism like P. putida KT2440 routinely experiences.

Evidence Base

All directly cited snippets were verified against the source abstracts. The primary biochemical/structural/mechanistic evidence (PMIDs 12114526, 14633981, 30884058) derives from E. coli HemN, and the primary regulatory/phenotypic evidence (PMIDs 9767567, 9396835) from P. aeruginosa and A. eutrophus orthologs; the transfer to P. putida PP_4264 rests on orthology plus direct sequence-motif conservation (Finding 5).

Supported and Refuted Hypotheses

Limitations and Knowledge Gaps

1. No direct experimental characterization of the P. putida KT2440 protein. All enzymatic, structural, and mechanistic data come from orthologs (chiefly E. coli HemN). The functional assignment to Q88F35 is by orthology and conserved catalytic motifs, not by direct assay of the P. putida enzyme. Confidence is nonetheless high given the family-defining conserved motifs (CXXXCXXC, GGGTP) and unambiguous domain architecture.

2. Regulation in P. putida is inferred, not measured. Anr/Dnr control of hemN is demonstrated in P. aeruginosa and Fnr control in A. eutrophus; the presence of Anr and hemN in KT2440 makes the same control likely, but the specific promoter architecture (Anr-box position) and induction magnitude in KT2440 have not been experimentally confirmed. Note also that, unlike P. aeruginosa, P. putida KT2440 cannot grow by denitrification, so the HemN role likely relates to microaerobic rather than fully anaerobic physiology.

3. Functional status of the paralog PP_3781 is unverified. It is annotated as an oxygen-independent coproporphyrinogen-III oxidase family protein, but whether it is catalytically active, whether it is a true HemN/HemZ or a HemW-type radical-SAM heme chaperone, and its physiological contribution in KT2440 are unknown.

4. Substrate-specificity fine detail. While the reaction and the ring-A/B propionate targets are established for the family, the precise regiochemical order of the two decarboxylations and any P. putida-specific kinetic parameters (K_m, k_cat, redox-partner identity) are undefined.

5. Physiological reductant in vivo. In vitro assays used NAD(P)H plus additional cytoplasmic components; the actual in vivo electron donor that reduces the [4Fe-4S] cluster in P. putida is not established.

Proposed Follow-up Experiments / Actions

1. Heterologous expression and anaerobic enzyme assay of recombinant P. putida HemN (Q88F35) to directly confirm coproporphyrinogen-III oxidase activity, measure kinetic parameters, and verify harderoporphyrinogen-intermediate formation, closing the orthology-inference gap.

2. Targeted gene deletion of PP_4264 (and combinatorial deletions with PP_0073/hemF and PP_3781) in KT2440, followed by growth phenotyping across aerobic, microaerobic, and anaerobic conditions, with HPLC/fluorescence monitoring for coproporphyrin III accumulation as the diagnostic readout.

3. Test the redundancy hypothesis directly: determine whether a hemN single mutant is heme-prototrophic under microaerobiosis (predicting compensation by PP_3781/HemF), and whether a hemN/PP_3781 double mutant becomes conditionally heme-auxotrophic.

4. Map and validate the Anr/Dnr regulation: identify Anr/Dnr binding site(s) upstream of PP_4264 and quantify anaerobic/microaerobic induction by RT-qPCR or reporter fusion in wild-type vs. an anr mutant of KT2440.

5. Biochemically characterize PP_3781 to determine whether it is an active anaerobic copro-III oxidase (HemN/HemZ-type) or a HemW-type protein with a different in vivo role.

6. EPR/Mössbauer or structural analysis (or a high-confidence AlphaFold model plus cofactor docking) of P. putida HemN to confirm the [4Fe-4S] cluster geometry and the two-SAM arrangement predicted from the E. coli structure.

Conclusion

hemN (PP_4264; UniProt Q88F35) encodes the oxygen-independent, cytoplasmic, radical-SAM coproporphyrinogen-III oxidase of Pseudomonas putida KT2440. It catalyzes the oxidative decarboxylation of the two ring-A/B propionate side chains of coproporphyrinogen III to yield protoporphyrinogen IX in heme biosynthesis, using a [4Fe-4S] cluster and two SAM molecules to drive sequential radical decarboxylations through a harderoporphyrinogen intermediate. It is the anaerobic counterpart of the oxygen-dependent HemF within a redundant, redox-regulated (Anr/Dnr) system, ensuring heme supply irrespective of oxygen availability. The identity is unambiguous and all diagnostic catalytic motifs are conserved in the target sequence.

Artifacts

• OpenScientist final report
• OpenScientist final report

Citations

1. PMID:12114526
2. PMID:14633981
3. PMID:30884058
4. PMID:9767567
5. PMID:9396835
6. PMID:10498703