Cyb5r3 encodes NADH-cytochrome b5 reductase 3, a flavoprotein that transfers electrons from NADH to cytochrome b5. The review accepts the cytochrome-b5 reductase activities as direct functions, modifies generic oxidoreductase activity, keeps cofactor/nucleotide binding and membrane/localization terms as context, and removes the uninformative ND biological_process annotation.
| 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. 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 | REMOVE | Summary: biological_process is removed because it is unsupported or uninformative for Cyb5r3's reviewed role (ND, GO_REF:0000015). Reason: biological_process is not supported as a direct or useful contextual annotation for Cyb5r3 after review against 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: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. |
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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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