PPOX

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0004729 protoporphyrinogen oxidase activity, oxygen as acceptor	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetic (IBA) assignment of the defining catalytic activity of PPOX, transferred across the protoporphyrinogen oxidase orthology group. This is the core molecular function and is strongly supported by direct human enzyme assays. Reason: Protoporphyrinogen oxidase (EC 1.3.3.4) is the experimentally established activity of the human enzyme, purified and assayed directly, and this IBA sits at the correct level of specificity for the whole ortholog group. The current ontology label for this ID is "oxygen-dependent protoporphyrinogen oxidase activity". Supporting Evidence: PMID:7713909 Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway.
GO:0006783 heme biosynthetic process	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetic (IBA) assignment placing PPOX in the heme biosynthetic pathway. This is the core biological process for the gene: the PPOX-catalysed step is the penultimate reaction of heme synthesis. Reason: PPOX catalyses the seventh of eight steps of heme biosynthesis. The process assignment is correct and well supported; deficiency causes the heme-biosynthesis disorder variegate porphyria. Supporting Evidence: PMID:7713909 Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway.
GO:0005743 mitochondrial inner membrane	IBA GO_REF:0000033	ACCEPT	Summary: Phylogenetic (IBA) assignment of PPOX to the mitochondrial inner membrane, its established site of action. PPOX is a peripheral membrane protein on the intermembrane-space-facing (outer) surface of the inner membrane. Reason: The human enzyme is a mitochondrial inner-membrane protein; the crystallographic study explicitly describes hPPO as a mitochondrial inner membrane protein, and Reactome places it on the outer surface of the inner mitochondrial membrane. Core cellular location. Supporting Evidence: PMID:21048046 Human protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein, converts protoporphyrinogen IX to protoporphyrin IX in the heme biosynthetic pathway.
GO:0004729 protoporphyrinogen oxidase activity, oxygen as acceptor	IEA GO_REF:0000120	ACCEPT	Summary: Electronic assignment (combined IEA methods, tied to EC 1.3.3.4, RHEA:25576 and InterPro:IPR004572) of the core catalytic activity. Redundant with the experimental and IBA annotations to the same term. Reason: Correct core molecular function, independently supported by direct enzyme assays and the mapped EC number/RHEA reaction. Duplicate ID with different evidence is acceptable. Supporting Evidence: file:human/PPOX/PPOX-uniprot.txt Reaction=protoporphyrinogen IX + 3 O2 = protoporphyrin IX + 3 H2O2;
GO:0005743 mitochondrial inner membrane	IEA GO_REF:0000120	ACCEPT	Summary: Electronic (combined IEA / SubCell SL-0168) assignment of mitochondrial inner membrane localisation, matching the experimentally supported location. Reason: Consistent with the UniProt subcellular location (mitochondrion inner membrane, peripheral, intermembrane side) and with the crystallographic characterisation of the human enzyme. Core location. Supporting Evidence: file:human/PPOX/PPOX-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion inner membrane
GO:0005758 mitochondrial intermembrane space	IEA GO_REF:0000117	MARK AS OVER ANNOTATED	Summary: ARBA electronic assignment to the mitochondrial intermembrane space. PPOX faces the intermembrane space (it is a peripheral protein on the outer/IMS-facing surface of the inner membrane) but is not a soluble intermembrane-space protein. Reason: PPOX is anchored to the inner membrane with its active site oriented toward the intermembrane space; UniProt records the location as "Mitochondrion inner membrane; Peripheral membrane protein; Intermembrane side". The more specific and accurate component is mitochondrial inner membrane (GO:0005743). Assigning it as an intermembrane-space (matrix-of-the-IMS) protein overstates a free-luminal localisation, so this is an over-annotation relative to the inner-membrane term. Supporting Evidence: file:human/PPOX/PPOX-uniprot.txt {ECO:0000250\|UniProtKB:P51175}; Intermembrane side
GO:0006779 porphyrin-containing compound biosynthetic process	IEA GO_REF:0000120	ACCEPT	Summary: Electronic assignment to porphyrin-containing compound biosynthesis. This is a correct but more general parent of the specific heme biosynthetic process; PPOX produces the porphyrin protoporphyrin IX. Reason: Biologically correct: PPOX generates protoporphyrin IX, a porphyrin. The term is broader than the core heme biosynthetic process, but an IEA at this generality is acceptable and true. Supporting Evidence: PMID:21048046 converts protoporphyrinogen IX to protoporphyrin IX in the heme biosynthetic pathway
GO:0006785 heme B biosynthetic process	IEA GO_REF:0000117	ACCEPT	Summary: ARBA electronic assignment to heme B (protoheme) biosynthesis, the specific branch of heme synthesis to which the PPOX-generated protoporphyrin IX is committed (protoporphyrin IX + Fe2+ -> heme B). Reason: Heme b (protoheme IX) is the immediate downstream product of the protoporphyrin IX made by PPOX (via ferrochelatase). The term is a valid, more specific descendant of heme biosynthetic process. Keep as a specific process; the general heme biosynthetic process remains the core BP. Supporting Evidence: PMID:7713909 Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway.
GO:0016491 oxidoreductase activity	IEA GO_REF:0000002	MARK AS OVER ANNOTATED	Summary: InterPro2GO electronic assignment (from the flavin amine-oxidase domain IPR002937) of the broad parent activity oxidoreductase. Reason: PPOX is an oxidoreductase, so the term is not wrong, but it is far more general than the specific and experimentally established protoporphyrinogen oxidase activity (GO:0004729) that is already annotated. The generic parent adds no functional information beyond the specific child. Supporting Evidence: file:human/PPOX/PPOX-uniprot.txt Reaction=protoporphyrinogen IX + 3 O2 = protoporphyrin IX + 3 H2O2;
GO:0031966 mitochondrial membrane	IEA GO_REF:0000117	MARK AS OVER ANNOTATED	Summary: ARBA electronic assignment to the generic mitochondrial membrane. Correct but broader than the specific mitochondrial inner membrane localisation. Reason: PPOX localises specifically to the mitochondrial inner membrane (GO:0005743), which is already annotated. The generic mitochondrial membrane parent is true but redundant and less informative. Supporting Evidence: file:human/PPOX/PPOX-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion inner membrane
GO:0006783 heme biosynthetic process	IEA GO_REF:0000107	ACCEPT	Summary: Ensembl (orthology-based) electronic transfer of the core heme biosynthetic process annotation. Redundant with the IBA/ISS/IDA annotations to the same term. Reason: Correct core biological process, supported by direct experimental evidence for the human enzyme; the electronic transfer from the rat ortholog is appropriate. Supporting Evidence: PMID:7713909 Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway.
GO:0009410 response to xenobiotic stimulus	IEA GO_REF:0000107	KEEP AS NON CORE	Summary: Ensembl electronic transfer (from the rat ortholog D3ZVN7) of "response to xenobiotic stimulus". PPOX is the molecular target of diphenyl-ether herbicides (e.g. acifluorfen), which underlies this annotation, but responding to a xenobiotic is not a core evolved function of the enzyme. Reason: The annotation reflects that PPOX is inhibited by xenobiotic herbicides (acifluorfen is a specific PPO inhibitor), a pharmacologically important but peripheral, non-core aspect of the gene. It is retained as non-core rather than removed because the xenobiotic-interaction is genuine and experimentally documented for the enzyme. Supporting Evidence: PMID:7713909 this activity was inhibited by acifluorfen, a specific inhibitor of PPO
GO:0004729 protoporphyrinogen oxidase activity, oxygen as acceptor	ISS GO_REF:0000024	ACCEPT	Summary: Curator sequence-similarity (ISS) transfer of the core catalytic activity from the mouse ortholog (MGI:104968). Redundant with the direct experimental and IBA annotations. Reason: Correct core molecular function; sequence-similarity transfer is fully consistent with the directly assayed human enzyme activity. Supporting Evidence: PMID:7713909 Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway.
GO:0006783 heme biosynthetic process	ISS GO_REF:0000024	ACCEPT	Summary: Curator sequence-similarity (ISS) transfer of the core heme biosynthetic process from the mouse ortholog. Redundant with IBA/IEA/IDA annotations to the same term. Reason: Correct core biological process, consistent with all other lines of evidence. Supporting Evidence: PMID:7713909 Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway.
GO:0004729 protoporphyrinogen oxidase activity, oxygen as acceptor	EXP PMID:23467411 Quantitative structural insight into human variegate porphyr...	ACCEPT	Summary: Experimental (EXP) annotation of protoporphyrinogen oxidase activity from the quantitative structural/kinetic study of wild-type and VP-mutant human PPO, which assayed kcat/Km of the enzyme and characterised the FAD- and O2-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. Reason: Direct experimental determination of the core catalytic activity of the human enzyme, including quantitative kinetics; the defining function of the gene. Supporting Evidence: PMID:23467411 catalyzes the oxidation of protoporphyrinogen IX (protogen) to protoporphyrin IX (porphyrin) in the presence of cofactor FAD and molecular oxygen
GO:0005743 mitochondrial inner membrane	ISS GO_REF:0000024	ACCEPT	Summary: Curator sequence-similarity (ISS) transfer of mitochondrial inner membrane localisation from the mouse ortholog (P51175). Consistent with the experimentally supported location. Reason: Correct core location; matches the UniProt subcellular location and the crystallographic description of the human enzyme. Supporting Evidence: PMID:21048046 Human protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein
GO:0005743 mitochondrial inner membrane	ISS GO_REF:0000024	ACCEPT	Summary: Duplicate GOA line of the curator sequence-similarity (ISS) transfer of mitochondrial inner membrane localisation from the mouse ortholog (P51175); differs only in annotation date/source. Same conclusion as the other ISS localisation annotation. Reason: Correct core location; matches the UniProt subcellular location and the crystallographic description of the human enzyme. Duplicate of the equivalent GO:0005743 ISS annotation. Supporting Evidence: PMID:21048046 Human protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein
GO:0004729 protoporphyrinogen oxidase activity, oxygen as acceptor	IDA PMID:21048046 Structural insight into human variegate porphyria disease.	ACCEPT	Summary: Direct assay (IDA) of protoporphyrinogen oxidase activity from the 1.9 Angstrom crystal structure study of human PPO in complex with FAD and the inhibitor acifluorfen, which biochemically characterised the wild-type enzyme and 47 VP-causing mutants. Reason: Direct experimental evidence for the core catalytic function of the human enzyme, including structural and kinetic characterisation. Defining molecular function. Supporting Evidence: PMID:21048046 we report the crystal structure of hPPO in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of 1.9
GO:0006785 heme B biosynthetic process	IDA PMID:7713909 Cloning of a human cDNA for protoporphyrinogen oxidase by co...	ACCEPT	Summary: Direct assay (IDA) placing PPOX in heme (heme B / protoheme) biosynthesis, from the cDNA cloning study that complemented an E. coli hemG (PPO-deficient) mutant and demonstrated acifluorfen-inhibitable PPO activity in transfected cells. Reason: Functional complementation of a heme-pathway (hemG) mutant plus demonstration of PPO activity directly establishes PPOX's role in producing the protoporphyrin IX committed to heme b synthesis. Keep as a specific heme-branch process alongside the general core heme biosynthetic process. Supporting Evidence: PMID:7713909 a hemG mutant strain of Escherichia coli deficient in PPO is defective in aerobic respiration
GO:0005739 mitochondrion	HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an...	ACCEPT	Summary: High-throughput (HTP) proteomic assignment of PPOX to the mitochondrion, from a quantitative high-confidence human mitochondrial proteome (MitoCoP) study. Broader than, but consistent with, the specific inner-membrane localisation. Reason: Correct organellar localisation supported by a large-scale, high-confidence mitochondrial proteome. The term is more general than mitochondrial inner membrane but is accurate; retained as a valid (if less specific) location annotation. Supporting Evidence: PMID:34800366 defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP)
GO:0005758 mitochondrial intermembrane space	TAS Reactome:R-HSA-189423	MARK AS OVER ANNOTATED	Summary: Reactome traceable-author-statement placing PPO on the outer surface of the inner mitochondrial membrane (facing the intermembrane space). As with the IEA IMS annotation, the intermembrane-space term overstates a soluble-luminal localisation. Reason: Reactome explicitly states the protein resides on the outer surface of the inner mitochondrial membrane, i.e. it is inner-membrane-associated with its active site facing the intermembrane space, not a free intermembrane-space protein. The specific, accurate component is mitochondrial inner membrane (GO:0005743); the IMS assignment is an over-annotation. Supporting Evidence: Reactome:R-HSA-189423 The protein resides on the outer surface of the inner mitochondrial membrane.
GO:0004729 protoporphyrinogen oxidase activity, oxygen as acceptor	IDA PMID:7713909 Cloning of a human cDNA for protoporphyrinogen oxidase by co...	ACCEPT	Summary: Direct assay (IDA) of protoporphyrinogen oxidase activity from the original human cDNA cloning: COS-1 cells transfected with the cDNA showed markedly higher, acifluorfen- inhibitable PPO activity than controls. Reason: Direct experimental demonstration of the core catalytic activity of the human gene product. Defining molecular function. Supporting Evidence: PMID:7713909 A homogenate of the monkey kidney COS-1 cells that had been transfected with the cDNA had much higher PPO activity than an extract of control cells, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO.
GO:0006779 porphyrin-containing compound biosynthetic process	IDA PMID:7713909 Cloning of a human cDNA for protoporphyrinogen oxidase by co...	ACCEPT	Summary: Direct assay (IDA) placing PPOX in porphyrin-containing compound biosynthesis, from the cloning/complementation study. PPOX generates the porphyrin protoporphyrin IX. Reason: Correct but general process; PPOX produces protoporphyrin IX, a porphyrin. The specific heme biosynthetic process is the core BP, with this as a valid broader annotation. Supporting Evidence: PMID:7713909 Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway.
GO:0006783 heme biosynthetic process	IDA PMID:7713909 Cloning of a human cDNA for protoporphyrinogen oxidase by co...	ACCEPT	Summary: Direct assay (IDA) of PPOX's role in heme biosynthesis, from functional complementation of an E. coli hemG (PPO-deficient) mutant with the human cDNA. This is the core biological process for the gene. Reason: Functional complementation of a heme-pathway mutant and restoration of PPO activity directly establishes the role of PPOX in heme biosynthesis. Core BP. Supporting Evidence: PMID:7713909 Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway.
GO:0031966 mitochondrial membrane	IMP PMID:7713909 Cloning of a human cDNA for protoporphyrinogen oxidase by co...	MARK AS OVER ANNOTATED	Summary: Mutant-phenotype-based (IMP) localisation to the mitochondrial membrane, from the cloning study showing the in vitro-translated protein associates with isolated mitochondria without size change, indicating no cleaved targeting presequence. Reason: The evidence supports mitochondrial (membrane) localisation, but the generic mitochondrial membrane term is broader than the experimentally and structurally supported mitochondrial inner membrane (GO:0005743), which is already annotated. Kept but flagged as an over-general component. (Deferring to the curator, this is not a removal — the localisation is genuine.) Supporting Evidence: PMID:7713909 after incubation with isolated mitochondria the protein was found to be located in the mitochondria, having just the same size as before, an indication that PPO is a mitochondrial enzyme and has no apparent transport-specific leader sequence.
GO:0050660 flavin adenine dinucleotide binding	TAS PMID:7713909 Cloning of a human cDNA for protoporphyrinogen oxidase by co...	ACCEPT	Summary: Traceable author statement that PPOX binds FAD. The N-terminal sequence contains a classic dinucleotide-binding motif, and the crystal structure resolves one FAD bound per subunit; FAD is the essential redox cofactor for the oxidation reaction. Reason: PPOX is a flavoprotein that binds one FAD per subunit as its catalytic cofactor, confirmed both by sequence motif and by crystal structures. This is a genuine, though secondary/enabling, molecular function supporting the core oxidase activity. Supporting Evidence: PMID:7713909 The NH2-terminal amino acid sequence of the deduced PPO contains a conserved amino acid sequence that forms the dinucleotide-binding site in many flavin-containing proteins. file:human/PPOX/PPOX-uniprot.txt Note=Binds 1 FAD per subunit.

Gene Description

Existing Annotations Review

Core Functions

FAD- and oxygen-dependent six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX, the seventh (penultimate) step of heme biosynthesis, catalysed at the mitochondrial inner membrane.

Binds one non-covalent FAD cofactor per subunit, the flavin redox centre required for the oxidase activity.

References

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