HemF is the oxygen-dependent coproporphyrinogen III oxidase of Escherichia coli K-12 (EC 1.3.3.3), a soluble cytoplasmic homodimer. It uses molecular oxygen as electron acceptor to oxidatively decarboxylate the propionate side chains of rings A and B of coproporphyrinogen III to vinyl groups, forming protoporphyrinogen IX and hydrogen peroxide in the heme b biosynthetic pathway. Activity is lost after metal chelation and restored only by manganese, with four conserved histidines implicated in manganese coordination. E. coli also has the structurally unrelated, oxygen-independent radical SAM enzyme HemN for the same step; either suffices in unstressed aerobic cells, but hemF is induced by the OxyR regulon during hydrogen peroxide stress, when this manganese enzyme becomes the critical isozyme for sustaining heme synthesis and catalase activation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004109 coproporphyrinogen oxidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of coproporphyrinogen oxidase activity from node PTN000079415 (PANTHER PTHR10755), with E. coli HemF's own experimental annotation among the descendant evidence together with the yeast, mouse, rat and Synechocystis enzymes. Reason: GO:0004109 is defined by the oxygen-dependent reaction ("coproporphyrinogen III + 2 H+ + O2 = 2 CO2 + 2 H2O + protoporphyrinogen IX"), which is exactly the HemF chemistry. Sound node placement; the target in its own WITH/FROM marks experimental grounding. Core function. Supporting Evidence: PMID:12975365 Molecular oxygen is essential for the enzymatic reaction. file:ECOLI/hemF/hemF-uniprot.txt PANTHER; PTHR10755:SF0; OXYGEN-DEPENDENT COPROPORPHYRINOGEN-III OXIDASE, MITOCHONDRIAL; 1. |
| GO:0004109 coproporphyrinogen oxidase activity | IDA PMID:12975365 Oxygen-dependent coproporphyrinogen III oxidase (HemF) from ... | ACCEPT | Summary: Breckau et al. 2003 purified recombinant E. coli HemF and measured coproporphyrinogen III oxidase activity (Km 2.6 uM, kcat 0.17 min-1, pH optimum 6), showed that O2 is essential and acts as electron acceptor with formation of H2O2, and that the enzyme does not use protoporphyrinogen IX or coproporphyrin III. Reason: Direct enzymatic demonstration of the defining activity, with oxygen as acceptor. Core. Supporting Evidence: PMID:12975365 The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. PMID:12975365 Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. |
| GO:0004109 coproporphyrinogen oxidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: The same activity from UniProt's combined pipeline (InterPro IPR001260 aerobic coproporphyrinogen oxidase, HAMAP UniRule, RHEA:18257, EC 1.3.3.3). Reason: Unlike the HemN-family InterPro2GO mapping to this term, the aerobic HemF signatures map to the correct chemistry. Consistent with the IDA row. Supporting Evidence: file:ECOLI/hemF/hemF-uniprot.txt SIMILARITY: Belongs to the aerobic coproporphyrinogen-III oxidase family. file:ECOLI/hemF/hemF-uniprot.txt Catalyzes the aerobic oxidative decarboxylation of propionate groups of rings A and B of coproporphyrinogen-III to yield the vinyl groups in protoporphyrinogen-IX. |
| GO:0004109 coproporphyrinogen oxidase activity | IMP PMID:12975365 Oxygen-dependent coproporphyrinogen III oxidase (HemF) from ... | ACCEPT | Summary: Site-directed mutagenesis in the same study: the histidines implicated in manganese coordination and Trp274 are needed for activity, whereas a putative nucleotide-binding motif and many conserved Tyr/Trp/Cys residues are not. Reason: Mutational analysis of the purified enzyme supports the same core activity as the IDA row. Supporting Evidence: PMID:12975365 One catalytically important tryptophan residue was localized in position 274. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of cytoplasmic activity for the HemF family. The node also covers eukaryotic enzymes located in the mitochondrial intermembrane space, but bacterial HemF has no targeting signal and is a soluble cytoplasmic protein. Reason: Cytoplasm is correct and as specific as warranted for a soluble bacterial enzyme purified from the soluble fraction; it is where the activity acts. The target's own IDA is among the IBA's sources. Supporting Evidence: file:ECOLI/hemF/hemF-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. |
| GO:0005737 cytoplasm | IDA PMID:12975365 Oxygen-dependent coproporphyrinogen III oxidase (HemF) from ... | ACCEPT | Summary: EcoCyc IDA for cytoplasm from Breckau et al. 2003. The cached record is the abstract, which describes purification of the soluble overexpressed enzyme but does not itself state a localization experiment. Reason: The location is right for this soluble enzyme and consistent with the IBA and IEA rows; deferring to the curator, who read the full text, for the specific evidence. Supporting Evidence: PMID:12975365 Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Cytoplasm from UniProt's subcellular-location mapping and HAMAP rule. Reason: Consistent with the IBA and IDA rows for this term. Supporting Evidence: file:ECOLI/hemF/hemF-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. |
| GO:0006779 porphyrin-containing compound biosynthetic process | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the aerobic coproporphyrinogen oxidase signatures. Reason: Correct but general. HemF's product protoporphyrinogen IX is used only for protoheme (heme b) and the hemes derived from it, so the endpoint is GO:0006785 heme B biosynthetic process (an is_a descendant via heme biosynthetic process), as for ECOLI/hemN and PSEPK/hemF. Proposed replacements: heme B biosynthetic process Supporting Evidence: PMID:12975365 During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. |
| GO:0006783 heme biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of heme biosynthesis for the HemF family. Reason: HemF catalyses a step of the shared trunk of heme synthesis; its product feeds heme b and the hemes o and d made from it, so the term is accurate at this level. The heme B endpoint is recorded in core_functions. Supporting Evidence: PMID:12975365 During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. |
| GO:0006783 heme biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Heme biosynthesis from UniProt's combined pipeline (HAMAP UniRule, ARBA). Reason: Same claim as the IBA row; supported by the curated function statement. Supporting Evidence: file:ECOLI/hemF/hemF-uniprot.txt FUNCTION: Involved in the heme biosynthesis. |
| GO:0006783 heme biosynthetic process | IMP PMID:8300522 Isolation of the hemF operon containing the gene for the Esc... | ACCEPT | Summary: Troup et al. 1994 cloned E. coli hemF by functional complementation of a Saccharomyces cerevisiae HEM13 (coproporphyrinogen oxidase) mutant. Reason: Rescue of a heme-deficient yeast coproporphyrinogen oxidase mutant shows that the E. coli gene product performs this heme-biosynthetic step. Consistent with the IBA and IEA rows. Supporting Evidence: PMID:8300522 Here we report the cloning of the hemF gene, encoding the aerobic coproporphyrinogen III oxidase from Escherichia coli, by functional complementation of a Saccharomyces cerevisiae HEM13 mutant. |
| GO:0030145 manganese ion binding | IDA PMID:12975365 Oxygen-dependent coproporphyrinogen III oxidase (HemF) from ... | ACCEPT | Summary: Metal chelator treatment inactivated HemF and only manganese restored full activity; His96, His106, His145 and His175 were implicated in manganese coordination. Reason: Direct biochemical evidence that the E. coli enzyme binds manganese as an activating cofactor, independently supported by the 2015 finding that HemF behaves as a manganoprotein in vivo (PMID:25664592). Manganese dependence is not shared by all HemF-family members, but it is established for this protein. Supporting Evidence: PMID:12975365 Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. |
| GO:0030145 manganese ion binding | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule (HAMAP MF_00333) transfer of manganese ion binding. Reason: Consistent with the IDA row for this term. Supporting Evidence: file:ECOLI/hemF/hemF-uniprot.txt Name=Mn(2+); Xref=ChEBI:CHEBI:29035; Evidence={ECO:0000255|HAMAP-Rule:MF_00333, ECO:0000269|PubMed:12975365}; |
| GO:0033194 response to hydroperoxide | IEP PMID:25664592 The induction of two biosynthetic enzymes helps Escherichia ... | MODIFY | Summary: Mancini and Imlay 2015 found that hemF is induced about 6-fold by OxyR during protracted low-level H2O2 stress. Reason: The stimulus is hydrogen peroxide itself, but GO:0033194 is defined for hydroperoxides, "monosubstitution products of hydrogen peroxide, HOOH", and GO:0042542/GO:0070301 are not its descendants. The sound claim is a lateral correction to GO:0070301 cellular response to hydrogen peroxide. Peripheral to the catalytic function, so not core. Proposed replacements: cellular response to hydrogen peroxide Supporting Evidence: PMID:25664592 OxyR induced synthesis of two members of the nine-step heme biosynthetic pathway: ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF). |
| GO:0033194 response to hydroperoxide | IMP PMID:25664592 The induction of two biosynthetic enzymes helps Escherichia ... | MODIFY | Summary: In the same study hemF deletion mutants accumulated porphyrins, had low catalase and cytochrome oxidase activities and could not cope with exogenous H2O2, whereas hemN mutants were unaffected. Reason: Same correction as the IEP row: the stimulus is H2O2, not an organic hydroperoxide, so the term is GO:0070301 cellular response to hydrogen peroxide. HemF contributes by sustaining heme synthesis for catalase; non-core. Proposed replacements: cellular response to hydrogen peroxide Supporting Evidence: PMID:25664592 Indeed, in wild-type katG+ backgrounds ΞhemF mutants were unable to cope with exogenous H2O2 PMID:25664592 These data all indicate that during H2O2 stress the induced mangano-enzyme HemF becomes the critical coproporphyrinogen III oxidase. |
| GO:0042803 protein homodimerization activity | IDA PMID:13129604 Oxygen-dependent coproporphyrinogen-III oxidase from Escheri... | KEEP AS NON CORE | Summary: Macieira et al. 2003 purified His-tagged E. coli HemF, which behaved as a compact dimer of 35 kDa subunits. Reason: Homodimerization is directly observed and correct, but it is a structural property ancillary to the oxidase activity; matches the treatment in PSEPK/hemF. Supporting Evidence: PMID:13129604 had a subunit molecular mass of 35 kDa and behaved as a compact shaped dimer |
| GO:0042803 protein homodimerization activity | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule transfer of homodimerization. Reason: Same non-core structural claim as the IDA row. Supporting Evidence: file:ECOLI/hemF/hemF-uniprot.txt SUBUNIT: Homodimer. |
| GO:0042803 protein homodimerization activity | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Manual transfer of homodimerization from yeast HEM13 (P36551). Reason: Same non-core claim, already directly supported by the IDA row for E. coli HemF. Supporting Evidence: PMID:13129604 had a subunit molecular mass of 35 kDa and behaved as a compact shaped dimer |
| GO:0046906 tetrapyrrole binding | IMP PMID:12975365 Oxygen-dependent coproporphyrinogen III oxidase (HemF) from ... | KEEP AS NON CORE | Summary: EcoCyc IMP for tetrapyrrole binding from the Breckau et al. study, in which HemF acts on coproporphyrinogen III, discriminates against protoporphyrinogen IX and coproporphyrin III, and is inhibited by protoporphyrin IX. Reason: HemF necessarily binds its tetrapyrrole substrate and is inhibited by the tetrapyrrole protoporphyrin IX, so the claim is true; it is substrate and inhibitor binding entailed by the oxidase activity rather than an independent function. Supporting Evidence: PMID:12975365 HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. |
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Download this section (compressed HTML)Q: Is manganese a catalytic cofactor of E. coli HemF or a stimulatory/structural metal, given that the paper proposes both metal-dependent and metal-independent routes?
Experiment: Determine the metal content (ICP-MS) and activity of HemF purified from wild-type and OxyR-induced cells grown with defined Mn and Fe, and of His-to-Ala variants.
Hypothesis: Mn(2+) bound by His96/His106/His145/His175 is present in HemF isolated from peroxide-stressed cells and is required for turnover.
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