Cyb5r3 encodes NADH-cytochrome b5 reductase 3, an FAD-containing flavoprotein that transfers electrons from NADH to cytochrome b5, reducing two molecules of cytochrome b5 per NADH. Alternative promoter usage and initiation give a myristoylated membrane-bound isoform anchored on the cytoplasmic face of the endoplasmic reticulum and mitochondrial outer membrane, where reduced cytochrome b5 supplies electrons to fatty acid desaturation, cholesterol biosynthesis and cytochrome P450 reactions, and a soluble erythroid isoform that drives reduction of methemoglobin in red cells. Loss-of-function variants of the human ortholog cause recessive congenital methemoglobinemia.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IBA GO_REF:0000033 | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NAD(P)H as a direct annotation (IBA, GO_REF:0000033). Reason: cytochrome-b5 reductase activity, acting on NAD(P)H directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md The rat gene symbol **Cyb5r3** corresponds to **NADH–cytochrome b5 reductase 3** (EC **1.6.2.2**), historically termed **diaphorase-1 (DIA1)** in the methemoglobin reductase literature. |
| GO:0071949 FAD binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: FAD binding is retained as contextual support for Cyb5r3, but it is not the core function (IBA, GO_REF:0000033). Reason: FAD binding records cofactor, substrate, or interaction context for Cyb5r3, but the curated core role is NADH-dependent reduction of cytochrome b5. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md **CYB5R3/Cyb5r3** encodes a **flavoprotein oxidoreductase** that uses **NADH** as electron donor to reduce various acceptors. |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: mitochondrion is retained as contextual support for Cyb5r3, but it is not the core function (IBA, GO_REF:0000033). Reason: mitochondrion records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md A 2024 review of “free radical reductases” similarly describes CYB5R3 as **membrane-tethered** on **MOM (cytosolic face), ER, and plasma membrane**, positioning it to reduce ferric heme and oxidized CoQ species. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IEA GO_REF:0000117 | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NAD(P)H as a direct annotation (IEA, GO_REF:0000117). Reason: cytochrome-b5 reductase activity, acting on NAD(P)H directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. GOA no longer carries this ARBA IEA row (retired); the same activity remains supported by IBA, ISO and IDA rows. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: cytoplasm is retained as contextual support for Cyb5r3, but it is not the core function (IEA, GO_REF:0000044). Reason: cytoplasm records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0005741 mitochondrial outer membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: mitochondrial outer membrane is retained as contextual support for Cyb5r3, but it is not the core function (IEA, GO_REF:0000044). Reason: mitochondrial outer membrane records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0005789 endoplasmic reticulum membrane | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: endoplasmic reticulum membrane is retained as contextual support for Cyb5r3, but it is not the core function (IEA, GO_REF:0000120). Reason: endoplasmic reticulum membrane records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md At the **ER**, membrane-bound Cb5R/CYB5R3 transfers electrons to pathways including **fatty-acid desaturation/elongation** and works in detoxification alongside **cytochrome b5 and cytochrome P450** systems. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: oxidoreductase activity captures part of Cyb5r3 biology, but a more specific replacement better represents the direct role (IEA, GO_REF:0000002). Reason: oxidoreductase activity is directionally related to Cyb5r3 but should be replaced by the more specific term(s): cytochrome-b5 reductase activity, acting on NADH. Proposed replacements: cytochrome-b5 reductase activity, acting on NADH Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md A recent 2024 study summarizes the core redox chemistry as **electron transfer from NADH to acceptors such as cytochrome b5 or coenzyme Q (CoQ)**, resulting in **NAD+** production. |
| GO:0090524 cytochrome-b5 reductase activity, acting on NADH | IEA GO_REF:0000120 | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NADH as a direct annotation (IEA, GO_REF:0000120). Reason: cytochrome-b5 reductase activity, acting on NADH directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md The rat gene symbol **Cyb5r3** corresponds to **NADH–cytochrome b5 reductase 3** (EC **1.6.2.2**), historically termed **diaphorase-1 (DIA1)** in the methemoglobin reductase literature. |
| GO:0005783 endoplasmic reticulum | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: endoplasmic reticulum is retained as contextual support for Cyb5r3, but it is not the core function (ISO, GO_REF:0000121). Reason: endoplasmic reticulum records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md A 2024 review of “free radical reductases” similarly describes CYB5R3 as **membrane-tethered** on **MOM (cytosolic face), ER, and plasma membrane**, positioning it to reduce ferric heme and oxidized CoQ species. |
| GO:0005737 cytoplasm | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: cytoplasm is retained as contextual support for Cyb5r3, but it is not the core function (ISO, GO_REF:0000121). Reason: cytoplasm records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0005737 cytoplasm | EXP PMID:1577871 A single mRNA, transcribed from an alternative, erythroid-sp... | KEEP AS NON CORE | Summary: cytoplasm is retained as contextual support for Cyb5r3, but it is not the core function (EXP, PMID:1577871). Reason: cytoplasm records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: PMID:1577871 Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. |
| GO:0090524 cytochrome-b5 reductase activity, acting on NADH | ISO GO_REF:0000121 | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NADH as a direct annotation (ISO, GO_REF:0000121). Reason: cytochrome-b5 reductase activity, acting on NADH directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0090524 cytochrome-b5 reductase activity, acting on NADH | EXP PMID:11695905 The structure and biochemistry of NADH-dependent cytochrome ... | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NADH as a direct annotation (EXP, PMID:11695905). Reason: cytochrome-b5 reductase activity, acting on NADH directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:11695905 Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. |
| GO:0090524 cytochrome-b5 reductase activity, acting on NADH | EXP PMID:8812833 High-level expression in Escherichia coli of the soluble, ca... | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NADH as a direct annotation (EXP, PMID:8812833). Reason: cytochrome-b5 reductase activity, acting on NADH directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:8812833 A T7 expression system has been produced for the high-level production of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase in Escherichia coli. |
| GO:0090524 cytochrome-b5 reductase activity, acting on NADH | EXP PMID:8978818 A role for N-myristoylation in protein targeting: NADH-cytoc... | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NADH as a direct annotation (EXP, PMID:8978818). Reason: cytochrome-b5 reductase activity, acting on NADH directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:8978818 N-myristoylation is a cotranslational modification involved in protein-protein interactions as well as in anchoring polypeptides to phospholipid bilayers; however, its role in targeting proteins to specific subcellular compartments has not been clearly defined. |
| GO:0005789 endoplasmic reticulum membrane | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: endoplasmic reticulum membrane is retained as contextual support for Cyb5r3, but it is not the core function (ISO, GO_REF:0000121). Reason: endoplasmic reticulum membrane records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | ISO GO_REF:0000121 | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NAD(P)H as a direct annotation (ISO, GO_REF:0000121). Reason: cytochrome-b5 reductase activity, acting on NAD(P)H directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IDA PMID:11695905 The structure and biochemistry of NADH-dependent cytochrome ... | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NAD(P)H as a direct annotation (IDA, PMID:11695905). Reason: cytochrome-b5 reductase activity, acting on NAD(P)H directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:11695905 Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IDA PMID:8812833 High-level expression in Escherichia coli of the soluble, ca... | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NAD(P)H as a direct annotation (IDA, PMID:8812833). Reason: cytochrome-b5 reductase activity, acting on NAD(P)H directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:8812833 A T7 expression system has been produced for the high-level production of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase in Escherichia coli. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | IDA PMID:8978818 A role for N-myristoylation in protein targeting: NADH-cytoc... | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NAD(P)H as a direct annotation (IDA, PMID:8978818). Reason: cytochrome-b5 reductase activity, acting on NAD(P)H directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:8978818 N-myristoylation is a cotranslational modification involved in protein-protein interactions as well as in anchoring polypeptides to phospholipid bilayers; however, its role in targeting proteins to specific subcellular compartments has not been clearly defined. |
| GO:0005741 mitochondrial outer membrane | IDA PMID:8978818 A role for N-myristoylation in protein targeting: NADH-cytoc... | KEEP AS NON CORE | Summary: mitochondrial outer membrane is retained as contextual support for Cyb5r3, but it is not the core function (IDA, PMID:8978818). Reason: mitochondrial outer membrane records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: PMID:8978818 N-myristoylation is a cotranslational modification involved in protein-protein interactions as well as in anchoring polypeptides to phospholipid bilayers; however, its role in targeting proteins to specific subcellular compartments has not been clearly defined. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md Membrane targeting includes regulation by **N-terminal myristoylation**, which influences targeting (e.g., MOM vs ER). |
| GO:0005789 endoplasmic reticulum membrane | IDA PMID:8978818 A role for N-myristoylation in protein targeting: NADH-cytoc... | KEEP AS NON CORE | Summary: endoplasmic reticulum membrane is retained as contextual support for Cyb5r3, but it is not the core function (IDA, PMID:8978818). Reason: endoplasmic reticulum membrane records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: PMID:8978818 N-myristoylation is a cotranslational modification involved in protein-protein interactions as well as in anchoring polypeptides to phospholipid bilayers; however, its role in targeting proteins to specific subcellular compartments has not been clearly defined. |
| GO:0005829 cytosol | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: cytosol is retained as contextual support for Cyb5r3, but it is not the core function (ISO, GO_REF:0000121). Falcon notes a soluble, N-terminally truncated erythrocyte isoform consistent with a cytosolic/soluble pool. Reason: cytosol records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md **Soluble erythrocyte isoform** (N-terminally truncated), critical for methemoglobin reduction. |
| GO:0031966 mitochondrial membrane | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: mitochondrial membrane is retained as contextual support for Cyb5r3, but it is not the core function (ISO, GO_REF:0000121). Reason: mitochondrial membrane records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0071949 FAD binding | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: FAD binding is retained as contextual support for Cyb5r3, but it is not the core function (ISO, GO_REF:0000121). Reason: FAD binding records cofactor, substrate, or interaction context for Cyb5r3, but the curated core role is NADH-dependent reduction of cytochrome b5. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0071949 FAD binding | IDA PMID:11695905 The structure and biochemistry of NADH-dependent cytochrome ... | KEEP AS NON CORE | Summary: FAD binding is retained as contextual support for Cyb5r3, but it is not the core function (IDA, PMID:11695905). Reason: FAD binding records cofactor, substrate, or interaction context for Cyb5r3, but the curated core role is NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:11695905 Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. |
| GO:0071949 FAD binding | IDA PMID:8812833 High-level expression in Escherichia coli of the soluble, ca... | KEEP AS NON CORE | Summary: FAD binding is retained as contextual support for Cyb5r3, but it is not the core function (IDA, PMID:8812833). Reason: FAD binding records cofactor, substrate, or interaction context for Cyb5r3, but the curated core role is NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:8812833 A T7 expression system has been produced for the high-level production of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase in Escherichia coli. |
| GO:0006809 nitric oxide biosynthetic process | ISO GO_REF:0000121 | MARK AS OVER ANNOTATED | Summary: nitric oxide biosynthetic process (GO:0006809) is the chemical formation of NO. Falcon deep research clarifies that CYB5R3's connection to NO biology is via maintaining soluble guanylate cyclase (sGC) heme in the reduced NO-responsive state (NO signaling/transduction), NOT direct catalysis of NO synthesis. The protein does not chemically form NO, so this term overshoots the evidence and is an over-annotation. Reason: CYB5R3 does not directly catalyze the NO-forming reaction; its connection to NO biology is heme-redox control of sGC (sustaining NO/cGMP/PKG signaling), per falcon. The orthology-derived GO:0006809 (NO biosynthesis) overshoots the actual evidence, which supports NO signaling rather than NO biosynthesis. No rat-specific NO-forming assay supports this term. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: ROLE CONFLATION Sources checked: UniProtKB:P00387 · human CYB5R3 SUPPORTS SOURCE BUT NOT TARGET The human CYB5R3 evidence concerns heme-redox support of NO/sGC signaling rather than catalysis of NO formation; the biosynthetic-process term over-scopes what transfers to rat Cyb5r3. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md In a 2023 pulmonary fibrosis model, CYB5R3 is described as maintaining **sGC heme iron redox state**, enabling NO responsiveness and downstream **cGMP/PKG** signaling. |
| GO:0050421 nitrite reductase (NO-forming) activity | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: nitrite reductase (NO-forming) activity is retained as contextual support for Cyb5r3, but it is not the core function (ISO, GO_REF:0000121). Reason: nitrite reductase (NO-forming) activity is kept only as non-core orthology-derived context; the cached rat literature primarily supports NADH-cytochrome b5 reductase activity, not a dedicated rat NO-forming assay. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:1903958 nitric-oxide synthase complex | ISO GO_REF:0000121 | MARK AS OVER ANNOTATED | Summary: nitric-oxide synthase complex (GO:1903958) is a complex capable of nitric-oxide synthase activity. Neither the cached rat literature nor falcon provides evidence for CYB5R3 physical association with the NOS complex; falcon shows CYB5R3 maintains soluble guanylate cyclase (sGC) heme (the NO receptor), not NOS. This orthology-derived complex annotation lacks supporting evidence and is an over-annotation. Reason: No rat-specific or falcon evidence supports CYB5R3 membership in the nitric-oxide synthase complex; CYB5R3's documented NO-related role is sGC heme maintenance, not NOS association. Consistent with the over-annotation call on GO:0006809. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: UniProtKB:P00387 · human CYB5R3 SUPPORTS SOURCE BUT NOT TARGET A nitric-oxide synthase complex component term does not follow from the human donor's redox role; rat Cyb5r3's NO-related activity is sGC heme maintenance, not NOS complex membership. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0005811 lipid droplet | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: lipid droplet is retained as contextual support for Cyb5r3, but it is not the core function (ISO, GO_REF:0000121). Falcon corroborates that membrane CYB5R3 participates in lipid/cholesterol metabolism, consistent with lipid-associated localization. Reason: lipid droplet records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. file:rat/Cyb5r3/Cyb5r3-deep-research-falcon.md A 2024 cancer-focused study reiterates participation of membrane CYB5R3 in lipid/cholesterol metabolism and drug metabolism, while emphasizing its broad metabolic involvement. |
| GO:0008150 biological_process | ND GO_REF:0000015 | ACCEPT | Summary: Root biological process ND annotation: when it was made, no biological process had been curated for this gene product from literature. Reason: ACCEPT under the ND acceptance rule (projects/NO_DATA.md). The evidence this review cites for the biological process is inferred (from orthologs, family membership or sequence features) or comes from other species; none of it is literature on this gene product in this species. This was judged from the review's own content during the NO_DATA re-action pass, not from a new literature search. Earlier action: REMOVE. Possibly stale: the 2026-11-06 ND citation pass found literature on this gene in this species that may identify a biological process; see Cyb5r3-notes.md, section "ND literature-search citations (2026-11-06)". A curator should check it before this ACCEPT stands. |
| GO:0016208 AMP binding | IDA PMID:14609324 The structure of the S127P mutant of cytochrome b5 reductase... | MARK AS OVER ANNOTATED | Summary: AMP binding (GO:0016208) is binding to free AMP as a ligand. PMID:14609324 instead shows that in the S127P methemoglobinemia mutant the AMP/ADP moiety of the bound FAD cofactor is displaced into the NADH substrate pocket, acting as a substrate inhibitor. This is a crystallographic observation about cofactor geometry in a disease mutant, not demonstration of free-AMP binding. The annotation is an over-annotation derived from misinterpreting the structural data. Reason: PMID:14609324 describes the FAD prosthetic group's own AMP/ADP moiety occupying the NADH substrate site in the S127P mutant, not binding of free AMP as a ligand. This is a structural artifact, so AMP binding overshoots the evidence. Supporting Evidence: PMID:14609324 the adenosine diphosphate (ADP) moiety of the FAD prosthetic group is displaced into the corresponding ADP binding site of the physiological substrate, NADH, thus acting as a substrate inhibitor which is consistent with both the spectroscopic and kinetic data. |
| GO:0043531 ADP binding | IDA PMID:14609324 The structure of the S127P mutant of cytochrome b5 reductase... | MARK AS OVER ANNOTATED | Summary: ADP binding (GO:0043531) is binding to free ADP as a ligand. PMID:14609324 instead shows that in the S127P methemoglobinemia mutant the ADP moiety of the bound FAD cofactor is displaced into the corresponding ADP binding site of the NADH substrate, acting as a substrate inhibitor. This is a crystallographic observation about cofactor geometry in a disease mutant, not demonstration of free-ADP binding. The annotation is an over-annotation derived from misinterpreting the structural data. Reason: PMID:14609324 describes the FAD prosthetic group's own ADP moiety occupying the NADH substrate site in the S127P mutant, not binding of free ADP as a ligand. This is a structural artifact, so ADP binding overshoots the evidence. Supporting Evidence: PMID:14609324 the adenosine diphosphate (ADP) moiety of the FAD prosthetic group is displaced into the corresponding ADP binding site of the physiological substrate, NADH, thus acting as a substrate inhibitor which is consistent with both the spectroscopic and kinetic data. |
| GO:0050660 flavin adenine dinucleotide binding | IDA PMID:14609324 The structure of the S127P mutant of cytochrome b5 reductase... | KEEP AS NON CORE | Summary: flavin adenine dinucleotide binding is retained as contextual support for Cyb5r3, but it is not the core function (IDA, PMID:14609324). Reason: flavin adenine dinucleotide binding records cofactor, substrate, or interaction context for Cyb5r3, but the curated core role is NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:14609324 Methemoglobinemia, the first hereditary disease to be identified that involved an enzyme deficiency, has been ascribed to mutations in the enzyme cytochrome b(5) reductase. |
| GO:0051287 NAD binding | IDA PMID:14609324 The structure of the S127P mutant of cytochrome b5 reductase... | KEEP AS NON CORE | Summary: NAD binding is retained as contextual support for Cyb5r3, but it is not the core function (IDA, PMID:14609324). Reason: NAD binding records cofactor, substrate, or interaction context for Cyb5r3, but the curated core role is NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:14609324 Methemoglobinemia, the first hereditary disease to be identified that involved an enzyme deficiency, has been ascribed to mutations in the enzyme cytochrome b(5) reductase. |
| GO:0005789 endoplasmic reticulum membrane | IDA PMID:14561759 Proteomic analysis of rat liver peroxisome: presence of pero... | KEEP AS NON CORE | Summary: endoplasmic reticulum membrane is retained as contextual support for Cyb5r3, but it is not the core function (IDA, PMID:14561759). Reason: endoplasmic reticulum membrane records localization or complex context for Cyb5r3, not the defining molecular function. Supporting Evidence: PMID:14561759 Subcellular proteomics, which includes isolation of subcellular components prior to a proteomic analysis, is advantageous not only in characterizing large macro-molecular complexes such as organelles but also in elucidating mechanisms of protein transport and organelle biosynthesis. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | TAS PMID:2391344 Molecular cloning of a cDNA encoding rat NADH-cytochrome b5 ... | ACCEPT | Summary: Cyb5r3's NADH-dependent reduction of cytochrome b5 supports retaining cytochrome-b5 reductase activity, acting on NAD(P)H as a direct annotation (TAS, PMID:2391344). Reason: cytochrome-b5 reductase activity, acting on NAD(P)H directly matches Cyb5r3's documented role in NADH-dependent reduction of cytochrome b5. Supporting Evidence: PMID:2391344 Rat cDNA encoding NADH-cytochrome b5 reductase (b5R) was isolated from a rat liver cDNA library using a human b5R cDNA as a probe. |
| GO:0050660 flavin adenine dinucleotide binding | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: flavin adenine dinucleotide binding is retained as contextual support for Cyb5r3, but it is not the core function (ISO, GO_REF:0000121). Reason: flavin adenine dinucleotide binding records cofactor, substrate, or interaction context for Cyb5r3, but the curated core role is NADH-dependent reduction of cytochrome b5. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | ISO GO_REF:0000121 | ACCEPT | Summary: ISO transfer of cytochrome-b5 reductase activity, acting on NAD(P)H from pig CYB5R3 (RGD:14151460); donor-split companion of the accepted dog-donor ISO and rat IDA rows for the same term. Reason: CYB5R3 orthologs share the conserved FAD/NADH-binding reductase fold and NADH-dependent cytochrome b5 reduction; the activity is also shown directly for rat Cyb5r3 (IDA/EXP rows from PMID:11695905, PMID:8812833, PMID:8978818). This is the core catalytic function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0004128 cytochrome-b5 reductase activity, acting on NAD(P)H | ISO GO_REF:0000121 | ACCEPT | Summary: ISO transfer of cytochrome-b5 reductase activity, acting on NAD(P)H from human CYB5R3 (UniProtKB:P00387); donor-split companion of the accepted dog-donor ISO and rat IDA rows for the same term. Reason: CYB5R3 orthologs share the conserved FAD/NADH-binding reductase fold and NADH-dependent cytochrome b5 reduction; the activity is also shown directly for rat Cyb5r3 (IDA/EXP rows from PMID:11695905, PMID:8812833, PMID:8978818). This is the core catalytic function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0005789 endoplasmic reticulum membrane | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: ISO transfer of located_in endoplasmic reticulum membrane from human CYB5R3 (UniProtKB:P00387); companion of the is_active_in ER membrane ISO row from the same donor and of the rat IDA row (PMID:8978818). Reason: The myristoylated membrane-bound isoform is anchored on the cytoplasmic face of the ER membrane, as shown for rat Cyb5r3, so the location is correct; it is kept as non-core localization context. Supporting Evidence: UniProtKB:P20070 SUBCELLULAR LOCATION: [Isoform 1]: Endoplasmic reticulum membrane |
| GO:0071949 FAD binding | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: ISO transfer of FAD binding from pig CYB5R3 (RGD:14151460); donor-split companion of the FAD binding ISO (dog donor) and IDA rows, kept as non-core cofactor context. Reason: FAD is the bound prosthetic group of cytochrome b5 reductase and is required for catalysis, so the term is correct, but cofactor binding is a component of the reductase activity rather than a separate core function. Supporting Evidence: UniProtKB:P20070 COFACTOR: Name=FAD; |
| GO:0071949 FAD binding | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: ISO transfer of FAD binding from human CYB5R3 (UniProtKB:P00387); donor-split companion of the FAD binding ISO (dog donor) and IDA rows, kept as non-core cofactor context. Reason: FAD is the bound prosthetic group of cytochrome b5 reductase and is required for catalysis, so the term is correct, but cofactor binding is a component of the reductase activity rather than a separate core function. Supporting Evidence: UniProtKB:P20070 COFACTOR: Name=FAD; |
| GO:0071949 FAD binding | ISO GO_REF:0000121 | KEEP AS NON CORE | Summary: ISO transfer of FAD binding from human CYB5R3 (UniProtKB:P00387, including the soluble isoform P00387-2); donor-split companion of the FAD binding ISO (dog donor) and IDA rows, kept as non-core cofactor context. Reason: FAD is the bound prosthetic group of cytochrome b5 reductase and is required for catalysis, so the term is correct, but cofactor binding is a component of the reductase activity rather than a separate core function. Supporting Evidence: UniProtKB:P20070 COFACTOR: Name=FAD; |
| GO:0090524 cytochrome-b5 reductase activity, acting on NADH | ISO GO_REF:0000121 | ACCEPT | Summary: ISO transfer of cytochrome-b5 reductase activity, acting on NADH from pig CYB5R3 (RGD:14151460); donor-split companion of the accepted dog-donor ISO and rat EXP rows for the same term. Reason: CYB5R3 orthologs share the conserved FAD/NADH-binding reductase fold and NADH-dependent cytochrome b5 reduction; the activity is also shown directly for rat Cyb5r3 (IDA/EXP rows from PMID:11695905, PMID:8812833, PMID:8978818). This is the core catalytic function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
| GO:0090524 cytochrome-b5 reductase activity, acting on NADH | ISO GO_REF:0000121 | ACCEPT | Summary: ISO transfer of cytochrome-b5 reductase activity, acting on NADH from human CYB5R3 (UniProtKB:P00387); donor-split companion of the accepted dog-donor ISO and rat EXP rows for the same term. Reason: CYB5R3 orthologs share the conserved FAD/NADH-binding reductase fold and NADH-dependent cytochrome b5 reduction; the activity is also shown directly for rat Cyb5r3 (IDA/EXP rows from PMID:11695905, PMID:8812833, PMID:8978818). This is the core catalytic function. Supporting Evidence: UniProtKB:P20070 FUNCTION: Catalyzes the reduction of two molecules of cytochrome b5 using NADH as the electron donor. |
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Download this section (compressed HTML)Q: Falcon deep research frames CYB5R3 as a prominent one-electron coenzyme Q (ubiquinone) reductase (contrasted with NQO1's two-electron mechanism). Should rat Cyb5r3 carry a curated MF annotation for one-electron CoQ/ubiquinone reductase activity, and is there direct rat experimental evidence for this acceptor in vivo?
Q: Falcon (citing Gutierrez-Merino et al. 2021) reports NADH:ascorbate-radical reductase activity for the cytochrome b5 reductase family. Is there sufficient rat-specific evidence to annotate Cyb5r3 with a monodehydroascorbate/ascorbate-radical reductase molecular function?
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