RSAD1 is a mitochondrial member of the radical S-adenosyl-L-methionine (SAM) superfamily and the human representative of the HemW subfamily of the anaerobic coproporphyrinogen-III oxidase (HemN-like) family. The protein carries a radical SAM core domain with the three-cysteine motif that ligates a single redox-active [4Fe-4S] cluster, which can be reduced to the [4Fe-4S]+ state, together with a C-terminal HemN_C domain and a predicted N-terminal mitochondrial transit peptide. Purified human RSAD1 binds protoporphyrin IX and its metal derivatives, including heme b, and the resulting [4Fe-4S]-heme protein binds the diatomic ligands oxygen and carbon monoxide as well as cyanide. Its bacterial orthologues (HemW of Escherichia coli and Lactococcus lactis) bind one heme per subunit, interact with heme-containing respiratory subunits and with bacterioferritins, and transfer their bound heme to a heme-depleted, inactive respiratory nitrate reductase in a manner requiring an intact iron-sulfur cluster, which is the basis for describing this protein family as heme chaperones that insert heme b into hemoproteins. Unlike the catalytic HemN proteins of the sister subfamily, HemW proteins lack oxygen-independent coproporphyrinogen III dehydrogenase (oxidase) activity, and no reaction catalysed by RSAD1 has been established: whether the human protein performs heme insertion itself, which hemoproteins it would serve, and whether it uses SAM-derived radical chemistry at all remain unknown. A single recent study reports that RSAD1 oxidizes methionine residues of picornaviral 3D RNA polymerases, destabilizing them, but this activity has not been confirmed independently. Rare variants in RSAD1 have been associated, in females only, with chronic central serous chorioretinopathy in an exome burden analysis awaiting replication.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Root-level catalytic activity from the radical SAM InterPro signatures. RSAD1 has the radical SAM architecture and a reducible [4Fe-4S] cluster (PMID:41747499), but no reaction has ever been demonstrated for it or for any HemW-subfamily protein, and the one activity its family was presumed to have (coproporphyrinogen dehydrogenase) was experimentally excluded (PMID:22142238). Reason: Per the RADICAL_SAM project's treatment rule for generic rows: where no reaction is known, GO:0003824 is already as precise as the evidence allows, so it is retained but is not a core function. It is not false - the protein belongs to a catalytic superfamily and holds the cofactor - and there is no specific descendant that the evidence supports. Supporting Evidence: file:human/RSAD1/RSAD1-uniprot.txt Binds 1 [4Fe-4S] cluster. The cluster PMID:41747499 radical S-adenosyl-l-methionine (SAM) superfamily of enzymes that utilize a |
| GO:0004109 coproporphyrinogen oxidase activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping from IPR004559 "HemW-like" to coproporphyrinogen oxidase activity. The HemW subfamily is precisely the clade shown NOT to have this activity: L. lactis HemW is devoid of coproporphyrinogen III dehydrogenase activity in vivo and in vitro (PMID:22142238), and no HemW-like protein has ever been shown to catalyse the reaction. UniProt records the same conclusion for RSAD1. Reason: A demonstrably wrong signature-to-GO mapping. The signature that triggers it (HemW-like) identifies the non-catalytic sister subfamily of HemN, and the activity was experimentally excluded for HemW proteins; the fourth cysteine of the HemN CX3CX2CXC motif is not conserved in this clade (PMID:34311096). Note also that the oxygen-independent reaction of HemN is represented by GO:0051989 coproporphyrinogen dehydrogenase activity rather than GO:0004109, so the mapped term does not even correspond to the family's ancestral chemistry. Supporting Evidence: PMID:22142238 showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly PMID:22142238 demonstrated for a HemW-like protein. We here studied HemW of L. lactis and file:human/RSAD1/RSAD1-uniprot.txt SIMILARITY: Belongs to the anaerobic coproporphyrinogen-III oxidase |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic transfer of a cytoplasmic location from bacterial family members, which are indeed cytoplasmic (and partly membrane-associated) proteins (PMID:22142238; PMID:37101575). The human protein carries a predicted mitochondrial transit peptide, is assigned to the mitochondrion by UniProt, and is a member of the MitoCoP high-confidence mitochondrial proteome (PMID:34800366). Since the mitochondrion is part of the cytoplasm, the term is true but less specific than the evidence supports. Reason: Correct but general: the node placement is sound (the family acts in the cytoplasmic compartment rather than, say, the nucleus or the extracellular space), but for the human protein the supported location is the mitochondrion, a term the gene already carries from two independent sources. Replace with GO:0005739. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000358281 SUPPORTS TRANSFER The ancestral node's cytoplasmic location is not wrong for the human protein, since the mitochondrion is part of the cytoplasm; the transfer simply cannot express the eukaryote-specific relocation of this family member into the organelle, which is why the row is refined rather than removed. Proposed replacements: mitochondrion Supporting Evidence: file:human/RSAD1/RSAD1-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion PMID:41747499 discovered mitochondrial protein that plays an important yet not fully |
| GO:0005737 cytoplasm | IEA GO_REF:0000002 | MODIFY | Summary: Cytoplasm from the HemW-like InterPro signature, reflecting the cytoplasmic location of the bacterial orthologues. For the human protein the specific compartment is the mitochondrion (UniProt; MitoCoP, PMID:34800366), which is part of the cytoplasm. Reason: Same judgement as the IBA row for this term: true but less specific than the evidence supports, so MODIFY to the supported descendant GO:0005739 mitochondrion rather than removing a correct broad location. Proposed replacements: mitochondrion Supporting Evidence: file:human/RSAD1/RSAD1-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: RSAD1 is a member of MitoCoP, a stringently filtered high-confidence human mitochondrial proteome built from subtractive proteomics, spatial proteomics, importomics and curation (PMID:34800366). This agrees with the predicted N-terminal mitochondrial transit peptide and with UniProt's assignment, and with the description of the human protein as mitochondrial in dedicated biochemistry (PMID:41747499). Reason: Mitochondrion is the supported site of action and the most specific compartment the evidence establishes (no sub-mitochondrial localization has been determined). Core location for the gene's heme-binding function. Supporting Evidence: PMID:34800366 We defined a human mitochondrial high-confidence proteome (MitoCoP) of 1,134 protein-coding genes file:human/RSAD1/RSAD1-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Mitochondrion from UniProt's subcellular-location mapping, based on the predicted transit peptide and sequence-similarity reasoning; independently supported experimentally by the MitoCoP proteome (PMID:34800366). Reason: Consistent with the HTP row for the same term and with UniProt's curated location. Accepted as the core location. Supporting Evidence: file:human/RSAD1/RSAD1-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion |
| GO:0006779 porphyrin-containing compound biosynthetic process | IBA GO_REF:0000033 | REMOVE | Summary: Phylogenetic inference that RSAD1 participates in tetrapyrrole biosynthesis, propagated from an ancestral node whose descendant evidence comes from HemN-type bacterial and cyanobacterial proteins that catalyse the coproporphyrinogen III step of heme biosynthesis. RSAD1 belongs to the HemW subfamily, which does not perform that step (PMID:22142238; PMID:29282292), and whose characterized role - binding heme and delivering it to hemoproteins - lies downstream of porphyrin biosynthesis. Reason: This is an argument with the node placement, not with the number of donors. The biosynthetic function was placed at a node that subsumes both the catalytic HemN clade and the HemW clade, but HemW-like proteins form a distinct clade in which the coproporphyrinogen dehydrogenase chemistry is absent - demonstrated by direct in vivo and in vitro assay in L. lactis (PMID:22142238) and reflected in the loss of the fourth motif cysteine (PMID:34311096). RSAD1 is therefore outside the clade that inherited the biosynthetic step. Its own characterized property, heme binding and handover, acts on the finished tetrapyrrole and so does not place it in porphyrin biosynthesis either: the gene does none of the work of making a porphyrin. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000358281 SUPPORTS SOURCE BUT NOT TARGET The node's descendant evidence is HemN-type coproporphyrinogen dehydrogenase activity, which is genuinely biosynthetic in those donors; the node is placed too deep, because the HemW/RSAD1 subfamily descending from it was experimentally shown to lack that chemistry and acts on finished heme instead. Supporting Evidence: PMID:22142238 showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly PMID:22142238 HemW-like proteins form a distinct phylogenetic clade that has not previously PMID:29282292 indicate that the radical SAM protein family HemW/RSAD1 is a heme chaperone |
| GO:0006779 porphyrin-containing compound biosynthetic process | IEA GO_REF:0000002 | REMOVE | Summary: The same biosynthetic process claim, this time mapped from the HemW-like InterPro signature. The signature identifies the subfamily shown not to catalyse the coproporphyrinogen step (PMID:22142238), and heme binding and delivery is downstream of porphyrin biosynthesis. Reason: Consistent with the IBA row for the same term. The InterPro2GO mapping inherits the historical mis-annotation of HemW-like proteins as coproporphyrinogen dehydrogenases (PMID:34311096); RSAD1 executes no step of porphyrin biosynthesis. Supporting Evidence: PMID:22142238 (coproporphyrinogen III dehydrogenases). However, such a function has never been file:human/RSAD1/RSAD1-uniprot.txt family. HemW subfamily. |
| GO:0020037 heme binding | IDA PMID:29282292 The radical SAM protein HemW is a heme chaperone. | ACCEPT | Summary: Direct assay: the human HemW orthologue RSAD1 stably bound heme (PMID:29282292), in the study that characterized bacterial HemW as a heme chaperone binding one heme per subunit and transferring it to a heme-depleted nitrate reductase. Independently confirmed on purified human protein, which binds protoporphyrin IX and its metal derivatives including Fe-PPIX with an association constant of about 1.6e6 per molar, the heme complex also binding O2, CO and cyanide (PMID:41747499). UniProt records the same conclusion. Reason: Experimentally established, specific, and the single best-supported molecular function of this gene product; core. Two independent laboratories demonstrate heme/porphyrin binding by the purified human protein. Supporting Evidence: PMID:29282292 domain-containing 1 (RSAD1) stably bound heme. In conclusion, our findings PMID:41747499 reconstituted hRSAD1-[4Fe-4S] protein. The association constant (KA) for file:human/RSAD1/RSAD1-uniprot.txt FUNCTION: May be a heme chaperone, appears to bind heme. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: Generic iron-sulfur cluster binding from the radical SAM signatures. The cluster RSAD1 binds is specifically a [4Fe-4S] cluster: UniProt records that it binds 1 [4Fe-4S] cluster coordinated by three cysteines and SAM, and reconstituted human protein shows a [4Fe-4S] cluster cycling between the 2+ and 1+ states by UV-visible and EPR spectroscopy (PMID:41747499). Reason: Correct but general. GO:0051539 4 iron, 4 sulfur cluster binding is the descendant the radical SAM cluster always satisfies, and the gene already carries it; per the RADICAL_SAM project's treatment rule for generic rows, replace rather than keep the parent. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:human/RSAD1/RSAD1-uniprot.txt Binds 1 [4Fe-4S] cluster. The cluster PMID:41747499 a [4Fe-4S]2+ cluster that could be reduced to the [4Fe-4S]+ state, and was |
| GO:0051539 4 iron, 4 sulfur cluster binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference of [4Fe-4S] cluster binding across the HemN/HemW family. Sound node placement - the cluster-binding cysteine motif and the cofactor are conserved in the HemW clade - and directly confirmed for the human protein by cluster reconstitution with UV-visible and EPR characterization (PMID:41747499) and for bacterial HemW, where an intact cluster is required for dimerization and heme transfer though not for heme binding (PMID:29282292). Reason: True and as precise as the evidence allows. Per the RADICAL_SAM project's treatment rule, GO:0051539 is core only where catalysis is established; no reaction is known for RSAD1, so the cluster cannot be called the catalytic cofactor of a demonstrated activity. It is retained as a well-supported non-core cofactor-binding function. Supporting Evidence: PMID:41747499 a [4Fe-4S]2+ cluster that could be reduced to the [4Fe-4S]+ state, and was PMID:29282292 intact iron-sulfur cluster was required for HemW dimerization and HemW-catalyzed |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: The same [4Fe-4S] cluster binding claim from the HemW-like InterPro signature, reinforcing the IBA row and the direct spectroscopic characterization of the reconstituted human protein (PMID:41747499). Reason: Consistent with the IBA row for this term: correct and appropriately specific, but not core while no catalytic reaction of RSAD1 is established. Supporting Evidence: file:human/RSAD1/RSAD1-uniprot.txt Binds 1 [4Fe-4S] cluster. The cluster |
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Download this section (compressed HTML)Q: Does human RSAD1 transfer its bound heme into a specific mitochondrial apo-hemoprotein, as bacterial HemW does with respiratory nitrate reductase, and which protein is the acceptor?
Suggested experts: Dieter Jahn, Martina Jahn, Gunhild Layer
Q: Is the [4Fe-4S] cluster of RSAD1 used for radical SAM chemistry, or only for the dimerization and heme-handover function it serves in bacterial HemW?
Suggested experts: Joan B Broderick, William E Broderick, Squire J Booker
Q: Is the reported methionine-oxidation activity of RSAD1 on picornaviral 3D polymerases reproducible with a cluster-loaded human enzyme, and does it act on endogenous mitochondrial substrates?
Suggested experts: Joan B Broderick, Squire J Booker
Q: Does RSAD1 have a role in heme trafficking between the mitochondrion and the rest of the cell, given that its bacterial counterpart cycles between a heme-free cytosolic and a heme-loaded membrane-associated form?
Suggested experts: Dieter Jahn, Marc Solioz
Experiment: Map the RSAD1 interactome in human mitochondria by proximity labelling and by immunoprecipitation from cells grown under heme-limiting and heme-replete conditions, then test cluster-dependent heme transfer in vitro from heme-loaded, reconstituted RSAD1 to each candidate apo-hemoprotein, reading out restored holoprotein spectrum and enzymatic activity as was done for bacterial HemW and nitrate reductase.
Hypothesis: RSAD1 is a mitochondrial heme chaperone that delivers heme b to one or more apo-subunits of the respiratory chain.
Type: interactomics plus in vitro heme transfer assay
Experiment: With anaerobically reconstituted human RSAD1, quantify 5'-deoxyadenosine and methionine formation under reducing conditions in the apo-, heme-loaded and partner-bound states, comparing wild type with cluster-ligand cysteine mutants, to establish whether SAM is cleaved at all and whether cleavage is coupled to heme handover.
Hypothesis: RSAD1 performs reductive SAM cleavage and therefore has a genuine radical SAM activity.
Type: anaerobic enzymology / SAM cleavage assay
Experiment: Generate RSAD1 knockout human cell lines and assay respiratory complex assembly and activity, cytochrome spectra, total and labile heme pools, and heme-dependent enzyme activities, with rescue by wild type versus cluster-binding and heme-binding mutants to establish which molecular property is required.
Hypothesis: Loss of RSAD1 impairs maturation of a mitochondrial hemoprotein.
Type: cell-based loss-of-function with biochemical phenotyping
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: No reaction catalysed by RSAD1 is known: it is unknown whether the protein performs radical SAM chemistry at all, and if so what its substrate and chemistry are.
OPEN BIOLOGY MF_DARK
What is known: Established: the radical SAM core domain, three-cysteine cluster motif and SAM-binding residues are present and a [4Fe-4S] cluster can be reconstituted and reduced; the one activity the family was presumed to have, oxygen-independent coproporphyrinogen III dehydrogenase, was experimentally excluded for HemW-subfamily proteins.
Significance: RSAD1 is one of the few human radical SAM proteins whose chemistry is still unassigned; settling it would also decide whether the heme chaperone role is catalytic or purely a binding and handover function.
What would resolve it: Reductive SAM-cleavage assays (5'-deoxyadenosine formation) with the reconstituted human protein under candidate substrate conditions, including the heme-loaded state, and unbiased metabolomic search for turnover products.
Provenance (the field's own admissions):
Gap: The physiological consequence of loss of RSAD1 in human cells and tissues is unknown: no human disease has been firmly linked to it and no mitochondrial phenotype of RSAD1 loss has been characterized.
OPEN BIOLOGY BP_DARK
What is known: Established: RSAD1 is a high-confidence mitochondrial protein expressed broadly; a female-specific rare-variant burden association with chronic central serous chorioretinopathy has been reported but explicitly requires replication, and the only organismal phenotype reported for the mouse gene is increased susceptibility to picornavirus challenge.
Significance: Without a loss-of-function phenotype the pathway context of this heme-binding protein cannot be fixed, which is what keeps its biological process aspect dark.
What would resolve it: Characterize RSAD1 knockout human cells for respiratory chain assembly, hemoprotein content and heme distribution, and replicate the chorioretinopathy association in independent cohorts.
Provenance (the field's own admissions):
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