AhpF is the soluble flavoprotein reductase subunit of the E. coli AhpC/AhpF alkyl hydroperoxide reductase system. It accepts electrons from NADH through a C-terminal FAD/NADH-binding module and relays them via redox-active disulfides to its N-terminal AhpC-facing domain, regenerating oxidized AhpC for continuous hydrogen peroxide and organic hydroperoxide detoxification in the cytosol.
Definition: Catalysis of the NADH-dependent reduction of an oxidized peroxiredoxin, regenerating its peroxide-reducing cysteine residues.
Justification: AhpF is not the EC 1.11.1.26 peroxiredoxin that reduces hydroperoxide; it is the dedicated flavoprotein-disulfide reductase that accepts reducing equivalents from NADH and transfers them to the oxidized AhpC peroxiredoxin. Existing GO:0047134 captures the broader EC 1.8.1.8 chemistry but not the dedicated peroxiredoxin acceptor.
Parent term: protein-disulfide reductase [NAD(P)H] activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000302 response to reactive oxygen species | IEA GO_REF:0000002 | MODIFY | Summary: AhpF supplies reducing equivalents to AhpC during peroxide detoxification; native E. coli reconstitution directly demonstrates hydrogen peroxide turnover. Reason: AhpF performs the reductive regeneration step in the peroxide response. Refine this general response term to response to hydrogen peroxide, supported by native EcAhpF/EcAhpC assays. Proposed replacements: response to hydrogen peroxide Supporting Evidence: PMID:28270505 The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. PMID:11717276 Mutants that lack both Ahp and catalase could not scavenge H 2 O 2 . PMID:2649484 ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. |
| GO:0004791 thioredoxin-disulfide reductase (NADPH) activity | IBA GO_REF:0000033 | MODIFY | Summary: AhpF is mechanistically adjacent to thioredoxin reductases, but the E. coli enzyme reduces oxidized AhpC using NADH-derived electrons rather than functioning as a NADPH-linked thioredoxin-disulfide reductase. Reason: The PAINT node PANTHER:PTN005133404 places an NADPH/thioredoxin activity across a family that includes the distinct AhpF architecture. Native E. coli biochemical and structural work establishes an N-terminal AhpC-facing disulfide relay linked to an NADH/FAD module, rather than identifying free thioredoxin as its physiological acceptor. Generalize to the supported protein-disulfide reductase activity GO:0047134. This is an argument from target-specific donor/acceptor chemistry and architecture, not donor count or an assertion that AhpF can never reduce thioredoxin in vitro; the Salmonella homolog has very slow promiscuous thioredoxin reduction (PMID:10828978), which does not establish this specific NADPH-linked activity for E. coli. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN005133404 SUPPORTS SOURCE BUT NOT TARGET The PAINT source node supports an NADPH/thioredoxin-disulfide reductase assertion, but E. coli AhpF is an NADH-linked AhpC reductase in the same broad PTHR48105 family. Proposed replacements: protein-disulfide reductase [NAD(P)H] activity Supporting Evidence: PMID:10666639 The AhpF component (521 amino-acid residues, 56.2 kDa) belongs to the family of pyridine nucleotide-disulfide oxidoreductases and channels electrons from NAD(P)H via a series of disulfides towards the AhpC component, which finally reduces the hydro-peroxide substrates. PMID:28270505 AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. |
| GO:0005829 cytosol | HDA PMID:16858726 A complexomic study of Escherichia coli using two-dimensiona... | ACCEPT | Summary: This high-throughput complexomic study separated cytosolic and membrane E. coli protein complexes by blue-native/SDS-PAGE and identified partners by LC-MS/MS. Reason: The cached abstract does not expose the AhpF-specific result table, but the HDA cytosol call is consistent with AhpF being a soluble, cytosolic-flavoprotein component of the AhpC/AhpF peroxide-detoxifying system. Supporting Evidence: PMID:16858726 Using two-dimensional blue native/SDS-PAGE (2-D BN/SDS-PAGE) technology, the cytosolic and membrane protein complexes of Escherichia coli were separated. Then, the different partners of each protein complex were identified by LC-MS/MS. PMID:28270505 AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: AhpF is inferred by PAINT to be active in the cytosol, and the target itself has direct E. coli proteomic cytosol evidence. Reason: The IBA cytosol annotation is consistent with multiple direct proteomic cytosol rows for E. coli AhpF and with AhpF acting as a soluble electron transfer enzyme for AhpC. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN000530716 SUPPORTS TRANSFER The PAINT cytosol node is concordant with direct cytosolic proteomics evidence on E. coli AhpF. Supporting Evidence: PMID:15911532 Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. PMID:18304323 Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. |
| GO:0005829 cytosol | IDA PMID:15911532 Localization, annotation, and comparison of the Escherichia ... | ACCEPT | Summary: Lopez-Campistrous et al. assigned E. coli proteins, including this EcoCyc-curated AhpF hit, to cytosol, periplasm, inner membrane, or outer membrane fractions by 2D gel electrophoresis and tandem mass spectrometry. Reason: This direct localization row is credible for AhpF: the original work used biochemical fractionation before mass spectrometry, and independent cytosolic proteomics and AhpF mechanism papers agree with a soluble cytosolic enzyme. Supporting Evidence: PMID:15911532 Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. |
| GO:0005829 cytosol | IDA PMID:17309111 Comparison of SDS- and methanol-assisted protein solubilizat... | ACCEPT | Summary: Zhang et al. compared SDS and methanol solubilization for LC-MS/MS of an E. coli K-12 inner-membrane fraction; EcoCyc used this proteomics dataset for an AhpF cytosol call. Reason: The cached abstract does not show the AhpF-specific supplemental row, but cytosolic localization is independently supported for this soluble, AhpC-reducing flavoprotein, supporting the experimental cytosol assignment despite the limited cached text. Supporting Evidence: PMID:17309111 In this work, two methods of protein solubilization, namely using 60% methanol or 1% SDS, to dissolve and analyze the inner membrane fraction of an Escherichia coli K12 cell lysate were compared. PMID:18304323 Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. |
| GO:0005829 cytosol | IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol. | ACCEPT | Summary: Ishihama et al. profiled proteins in the E. coli cytosolic fraction by LC-MS/MS, providing direct mass-spectrometry evidence for cytosolic AhpF. Reason: AhpF is a soluble, cytosolic AhpC reductase, and this IDA row comes from the most directly relevant E. coli cytosol proteomic dataset among the localization references. Supporting Evidence: PMID:18304323 Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. |
| GO:0005829 cytosol | IEA GO_REF:0000117 | ACCEPT | Summary: The ARBA cytosol prediction agrees with several direct E. coli proteomic cytosol annotations and with the soluble AhpC reductase mechanism of AhpF. Reason: This electronic row is a broad localization prediction, but it is independently corroborated by HDA/IDA cytosol rows and by biochemical work on soluble EcAhpF. Supporting Evidence: PMID:18304323 Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. |
| GO:0006979 response to oxidative stress | NAS PMID:28270505 Essential role of the flexible linker on the conformational ... | MODIFY | Summary: AhpF supplies reducing equivalents to AhpC during peroxide detoxification; native E. coli reconstitution directly demonstrates hydrogen peroxide turnover. Reason: AhpF performs the reductive regeneration step in the peroxide response. Native EcAhpF/EcAhpC assays specifically support response to hydrogen peroxide. The oxidative-stress parent also covers the independently supported response to alkyl hydroperoxide, supporting both specific replacements. Proposed replacements: response to hydrogen peroxide response to alkyl hydroperoxide Supporting Evidence: PMID:28270505 The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. PMID:11717276 Mutants that lack both Ahp and catalase could not scavenge H 2 O 2 . PMID:2649484 ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. |
| GO:0009321 alkyl hydroperoxide reductase complex | IDA PMID:25372677 Structure, mechanism and ensemble formation of the alkylhydr... | ACCEPT | Summary: Dip et al. solved E. coli AhpF and AhpC structures and demonstrated AhpF binding to AhpC, supporting AhpF as a component of the alkyl hydroperoxide reductase complex. Reason: The specific GO:0009321 complex term is appropriate for the E. coli AhpC/AhpF enzyme system because EcAhpF physically binds the AhpC ring to form the transient ensemble that accelerates hydroperoxide scavenging. Supporting Evidence: PMID:25372677 Solution X-ray scattering analysis of EcAhpF unravels the flexibility of its N-terminal domain, and its binding to EcAhpC was demonstrated by isothermal titration calorimetry. These studies suggest a novel overall mechanistic model of AhpR as a hydroperoxide scavenger, in which the dimeric, extended AhpF prefers complex formation with the AhpC ring to accelerate the catalytic activity and thus to increase the chance of rescuing the cell from ROS. |
| GO:0009321 alkyl hydroperoxide reductase complex | IPI PMID:25372677 Structure, mechanism and ensemble formation of the alkylhydr... | ACCEPT | Summary: The physical AhpF-AhpC interaction observed by Dip et al. supports the IPI assertion that AhpF is part of the alkyl hydroperoxide reductase complex. Reason: This GO:0009321 row has distinct interaction evidence from the same structural/biophysical study. The paper directly reports EcAhpF binding to EcAhpC and models a catalytic AhpR ensemble. Supporting Evidence: PMID:25372677 Solution X-ray scattering analysis of EcAhpF unravels the flexibility of its N-terminal domain, and its binding to EcAhpC was demonstrated by isothermal titration calorimetry. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: AhpF is an oxidoreductase, but GO:0016491 is only a broad parent term for its AhpC reductase activity. Reason: The InterPro signatures correctly recognize a pyridine nucleotide/FAD redox enzyme, but AhpF is more specifically a protein-disulfide reductase that transfers electrons from NADH through its own redox centers to oxidized AhpC. Proposed replacements: protein-disulfide reductase [NAD(P)H] activity Supporting Evidence: PMID:28270505 AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. |
| GO:0032991 protein-containing complex | IEA GO_REF:0000117 | MODIFY | Summary: AhpF is part of a protein-containing complex, but the generic ARBA term is less informative than the experimentally supported alkyl hydroperoxide reductase complex term already present for AhpF. Reason: GO:0032991 is true but too general for AhpF. Dip et al. directly demonstrated binding between EcAhpF and EcAhpC and support replacing this generic complex assertion with GO:0009321 alkyl hydroperoxide reductase complex. Proposed replacements: alkyl hydroperoxide reductase complex Supporting Evidence: PMID:25372677 Solution X-ray scattering analysis of EcAhpF unravels the flexibility of its N-terminal domain, and its binding to EcAhpC was demonstrated by isothermal titration calorimetry. |
| GO:0042744 hydrogen peroxide catabolic process | IDA PMID:28270505 Essential role of the flexible linker on the conformational ... | ACCEPT | Summary: AhpF participates in hydrogen peroxide catabolism by regenerating reduced AhpC with NADH-derived electrons, enabling continued AhpC peroxidase turnover. Reason: AhpC performs the peroxide attack, but AhpF does part of the work of this two-subunit process: it accepts electrons from NADH and transfers them through its redox centers to oxidized AhpC, and AhpF linker mutants reduce H2O2 less effectively in the AhpF-AhpC ensemble. Supporting Evidence: PMID:28270505 The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. We propose that a concerted movement involving the NTD, C-terminal NADH, and FAD domains, and the flexible linker between them is essential for optimal intra-domain cross-talk and for efficient electron transfer to the redox partner AhpC required for peroxidation. |
| GO:0045454 cell redox homeostasis | IBA GO_REF:0000033 | ACCEPT | Summary: AhpF's AhpC reductase reaction is an antioxidant redox function that supports cell redox homeostasis. Reason: AhpF directly regenerates oxidized AhpC for repeated removal of metabolically generated peroxide, thereby maintaining the cellular redox environment. Native E. coli operon genetics and enzyme reconstitution independently support this core homeostatic role. It is not classified as peripheral merely because it describes the overall redox outcome. Propagation Review Root cause: NO FAILURE NON CORE Sources checked: PANTHER:PTN005133404 SUPPORTS TRANSFER The source node's molecular-function specificity should not be transferred to AhpF, but the node is still compatible with the broad antioxidant redox-homeostasis process. Supporting Evidence: PMID:28270505 Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. PMID:11717276 These data demonstrate that in wild-type cells Ahp rather than catalase is the primary scavenger of an endogenous inducer of the OxyR regulon. |
| GO:0050660 flavin adenine dinucleotide binding | IEA GO_REF:0000120 | MODIFY | Summary: AhpF contains a C-terminal FAD-binding domain, and FAD binding is required for its intramolecular electron-transfer cycle. Reason: The flavin-binding site is directly established by E. coli structural work. Refine the parent covering either flavin oxidation state to the supported descendant GO:0071949 FAD binding, which the gene already carries. This retains the same cofactor-binding claim without treating the electronic evidence code differently. Proposed replacements: FAD binding Supporting Evidence: PMID:28270505 AhpF is a homodimeric protein encompassed by an N-terminal domain (NTD) and a C-terminal domain (CTD), which includes an FAD- and NADH-binding site as well as the redox-active center |
| GO:0051287 NAD binding | IEA GO_REF:0000002 | MODIFY | Summary: EcAhpF binds the reduced substrate NADH and oxidized product NAD+ during its electron-transfer cycle. Reason: The full mechanistic study describes both substrate-NADH-bound and product-NAD+-bound crystal structures. Refine the parent NAD-binding term to both supported oxidation-state-specific descendants, rather than choosing only one state. Proposed replacements: NADH binding NAD+ binding Supporting Evidence: PMID:28270505 the substrate (NADH)- and product (NAD + )-bound crystal structures revealed that the NADH domain has to rotate back-and-forth by about 65° |
| GO:0071949 FAD binding | IDA PMID:10666639 Crystallization and preliminary X-ray analysis of the cataly... | ACCEPT | Summary: FAD binding is a genuine molecular feature of AhpF's C-terminal catalytic core. Reason: The original EcoCyc IDA cites an abstract-only crystallization paper on the E. coli AhpF catalytic core, and later structural/mechanistic work states directly that the EcAhpF C-terminal domain includes an FAD- and NADH-binding site. Supporting Evidence: PMID:10666639 Crystals of the proteolytically truncated AhpF component (residues Asn208-Ala521) of the alkyl hydroperoxide reductase from Escherichia coli were grown under oxidizing conditions. PMID:28270505 AhpF is a homodimeric protein encompassed by an N-terminal domain (NTD) and a C-terminal domain (CTD), which includes an FAD- and NADH-binding site as well as the redox-active center |
| GO:0098869 cellular oxidant detoxification | IEA GO_REF:0000120 | ACCEPT | Summary: AhpF is part of the AhpC/AhpF antioxidant system and directly supplies NADH-derived electrons for detoxification of hydrogen peroxide and other oxidants. Reason: The UniProt2GO process assertion follows from the current AhpF disulfide-reductase reaction and is supported by biochemical evidence that AhpF accepts NADH reducing equivalents and transfers them to AhpC for peroxide detoxification. Supporting Evidence: PMID:28270505 In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. |
| GO:0102039 NADH-dependent peroxiredoxin activity | IEA GO_REF:0000002 | ACCEPT | Summary: GO:0102039 captures the NADH-dependent AhpC/AhpF peroxidase system, but AhpF itself carries out the disulfide-reductase half of that reaction. Reason: Native E. coli genetics and purified EcAhpC/EcAhpF reconstitution support the coupled NADH-dependent hydroperoxide-reduction reaction. AhpC performs peroxide reduction while AhpF oxidizes NADH and regenerates AhpC; this annotation is accepted for the experimentally established enzyme component without asserting that the isolated subunit performs both half-reactions. AhpF is the NADH-oxidizing component that regenerates reduced AhpC. Supporting Evidence: PMID:10666639 The AhpF component (521 amino-acid residues, 56.2 kDa) belongs to the family of pyridine nucleotide-disulfide oxidoreductases and channels electrons from NAD(P)H via a series of disulfides towards the AhpC component, which finally reduces the hydro-peroxide substrates. PMID:28270505 In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. |
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Download this section (compressed HTML)Q: Under which oxidative-stress regimes does E. coli AhpC/AhpF primarily detoxify hydrogen peroxide versus organic hydroperoxides or peroxynitrite in vivo?
Q: How much does the E. coli AhpF cycle use NADPH in vivo, if at all, compared with its biochemically characterized NADH-linked AhpC reductase activity?
Experiment: Compare peroxide clearance and AhpC redox-state recovery in wild-type E. coli and separation-of-function ahpF mutants with impaired NADH binding, FAD reduction, linker motion, or N-terminal AhpC reduction after defined hydrogen peroxide and organic hydroperoxide challenges.
Hypothesis: Mutations that trap AhpF in a CTD-facing state will specifically delay AhpC reduction and sensitize cells to peroxides even when AhpC itself is present.
Type: Redox proteomics and targeted complementation
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