hydA (DVU_1769) encodes the large catalytic subunit of a periplasmic [FeFe]-hydrogenase (HydAB) in Nitratidesulfovibrio vulgaris Hildenborough. The enzyme catalyzes the reversible reaction 2 H+ + 2 e- <-> H2 (EC 1.12.7.2), functioning primarily in H2 oxidation during dissimilatory sulfate reduction. The HydA subunit contains the catalytic H-cluster (a diiron center linked to a [4Fe-4S] subcluster) and two additional [4Fe-4S] ferredoxin-type clusters that mediate electron transfer. The physiological electron acceptor is Type I cytochrome c3 (TpI-c3), which shuttles electrons to membrane complexes (Hmc, Tmc, Qrc) for ultimate delivery to cytoplasmic sulfate reductases. The enzyme has high turnover with a Km for H2 of approximately 100 uM, is reversibly inhibited by CO, and can form an O2-protected inactive state likely involving sulfide ligation at the H-cluster. HydA forms a heterodimer with the small subunit HydB for full periplasmic [FeFe]-hydrogenase activity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: HydA contains multiple iron centers including three [4Fe-4S] clusters and a binuclear iron center at the H-cluster active site. X-ray crystallography at 1.6 A resolution (PMID:10368269) confirmed binding of iron ions through both the [4Fe-4S] clusters and the diiron active site. This annotation is accurate but less informative than the more specific 4Fe-4S cluster binding term. Reason: The annotation is technically correct as confirmed by structural studies (PMID:10368269). HydA binds iron both in [4Fe-4S] clusters and in the binuclear H-cluster active site. However, this is a parent term of more specific annotations already present. Supporting Evidence: PMID:10368269 The so-called H cluster of the enzyme is composed of a typical [4Fe-4S] cubane bridged to a binuclear active site Fe center file:DESVH/P07598/P07598-deep-research-falcon.md HydA contains the [FeFe]-hydrogenase H-cluster composed of a diiron center ligated to an atypical [4Fe-4S] subcluster. Two additional ferredoxin-like [4Fe-4S] clusters in HydA support intramolecular electron transfer |
| GO:0008901 ferredoxin hydrogenase activity | IEA GO_REF:0000120 | MODIFY | Summary: GO:0008901 describes catalysis of the reaction "2 reduced ferredoxin + 2 H+ = 2 oxidized ferredoxin + H2". While HydA does catalyze H2/proton interconversion, the physiological electron partner is NOT ferredoxin but rather Type I cytochrome c3 (TpI-c3). Literature consistently identifies cytochrome c3 as the native electron carrier for periplasmic [FeFe]-hydrogenase in D. vulgaris. Reason: The reaction catalyzed is correct in principle (H2 interconversion), but the specified electron partner (ferredoxin) is incorrect for this periplasmic enzyme. The physiological electron carrier is cytochrome c3, not ferredoxin. GO:0047806 (cytochrome-c3 hydrogenase activity) describes "2 H2 + ferricytochrome c3 = 4 H+ + ferrocytochrome c3" which matches the physiological function of HydAB. Proposed replacements: cytochrome-c3 hydrogenase activity Supporting Evidence: file:DESVH/P07598/P07598-deep-research-falcon.md Type I cytochrome c3 (TpI-c3) is the principal periplasmic electron carrier interacting with periplasmic hydrogenases, including the [FeFe]-hydrogenase file:DESVH/P07598/P07598-uniprot.txt Cytochrome c3 is likely to be the physiological electron carrier for the enzyme. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000043 | ACCEPT | Summary: GO:0016491 is a very high-level term for oxidoreductase activity. HydA is indeed an oxidoreductase (EC 1.12.7.2), catalyzing electron transfer between H2 and cytochrome c3. However, this term is too general and more specific hydrogenase activity terms should be used. Reason: While technically correct, this is a high-level parent term. The annotation derives from UniProtKB keyword mapping and accurately captures the oxidoreductase nature of the enzyme. More specific terms (GO:0047806 cytochrome-c3 hydrogenase activity) should also be present to provide specificity. Supporting Evidence: file:DESVH/P07598/P07598-uniprot.txt EC=1.12.7.2 |
| GO:0042597 periplasmic space | IEA GO_REF:0000044 | ACCEPT | Summary: HydA is unambiguously localized to the periplasmic space. This is supported by biochemical fractionation studies, the presence of an N-terminal signal peptide, and the functional requirement for interaction with periplasmic cytochrome c3. Reason: Periplasmic localization is well-established from multiple lines of evidence including classical biochemical fractionation and spheroplast complementation experiments. UniProt annotation and deep research confirm periplasmic localization. Supporting Evidence: file:DESVH/P07598/P07598-uniprot.txt SUBCELLULAR LOCATION: Periplasm. file:DESVH/P07598/P07598-deep-research-falcon.md HydAB in D. vulgaris Hildenborough is a periplasmic [FeFe]-hydrogenase |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | ACCEPT | Summary: GO:0046872 is a very general term for metal ion binding. HydA binds iron in multiple contexts (Fe-S clusters, binuclear iron center). This annotation is correct but uninformative given more specific terms are available. Reason: Technically correct as HydA binds iron ions extensively. This is a parent term of GO:0005506 (iron ion binding) which is also annotated. The annotation captures the general metal-binding property but more specific terms provide the mechanistic detail. Supporting Evidence: PMID:10368269 The so-called H cluster of the enzyme is composed of a typical [4Fe-4S] cubane bridged to a binuclear active site Fe center |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000120 | ACCEPT | Summary: HydA binds multiple iron-sulfur clusters: three [4Fe-4S] clusters (two ferredoxin-type and one as part of the H-cluster). This is well-established from X-ray crystallography and Mossbauer spectroscopy. Reason: Accurate annotation supported by high-resolution structural data. The enzyme contains three [4Fe-4S] clusters that are essential for intramolecular electron transfer. Supporting Evidence: file:DESVH/P07598/P07598-uniprot.txt Binds 3 [4Fe-4S] clusters per subunit. PMID:11456963 It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000043 | ACCEPT | Summary: HydA specifically binds three [4Fe-4S] clusters: two ferredoxin-type clusters (at domains 26-57 and 59-86) and one [4Fe-4S] subcluster as part of the H-cluster active site. X-ray crystallography at 1.6 A resolution confirmed the cluster coordination. Reason: Highly accurate and specific annotation. The [4Fe-4S] clusters are central to the electron transfer mechanism of the enzyme. Crystallographic evidence (PMID:10368269) and UniProt domain annotations confirm the presence of multiple 4Fe-4S ferredoxin-type domains. Supporting Evidence: file:DESVH/P07598/P07598-uniprot.txt Binds 3 [4Fe-4S] clusters per subunit. PMID:11456963 It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster |
| GO:0047806 cytochrome-c3 hydrogenase activity | ISS file:DESVH/P07598/P07598-deep-research-falcon.md | NEW | Summary: This is the most specific and accurate molecular function term for HydA. The enzyme catalyzes H2 oxidation with cytochrome c3 as the physiological electron acceptor, matching the reaction described by GO:0047806: "2 H2 + ferricytochrome c3 = 4 H+ + ferrocytochrome c3". Reason: This annotation is missing from the current GOA set but represents the core molecular function of the enzyme. UniProt explicitly states cytochrome c3 as the physiological electron carrier, and deep research confirms Type I cytochrome c3 (TpI-c3) as the principal electron partner. Supporting Evidence: file:DESVH/P07598/P07598-uniprot.txt Cytochrome c3 is likely to be the physiological electron carrier for the enzyme. file:DESVH/P07598/P07598-deep-research-falcon.md Type I cytochrome c3 (TpI-c3) is the principal periplasmic electron carrier interacting with periplasmic hydrogenases, including the [FeFe]-hydrogenase |
| GO:0019420 dissimilatory sulfate reduction | ISS file:DESVH/P07598/P07598-deep-research-falcon.md | NEW | Summary: HydA functions as an entry point for electrons into the dissimilatory sulfate reduction pathway. By oxidizing H2 in the periplasm and transferring electrons via cytochrome c3 to membrane complexes (Hmc, Tmc, Qrc), HydA supports the reduction of sulfate to H2S as the terminal electron acceptor. Reason: No biological process annotation currently exists for HydA. The enzyme's role in dissimilatory sulfate reduction is well-documented and represents its primary physiological function in sulfate-respiring conditions. Supporting Evidence: file:DESVH/P07598/P07598-uniprot.txt May be involved in hydrogen uptake for the reduction of sulfate to hydrogen sulfide in an electron transport chain. file:DESVH/P07598/P07598-deep-research-falcon.md H2 diffuses to the periplasm, where HydAB (HydA/HydB) and other periplasmic hydrogenases oxidize H2, delivering electrons to TpI-c3 and then across the membrane (via Hmc/Tmc/Qrc) to the cytoplasmic sulfate-reduction pathway |
| GO:1902421 hydrogen metabolic process | ISS file:DESVH/P07598/P07598-deep-research-falcon.md | NEW | Summary: HydA catalyzes the reversible interconversion of H2 and protons, directly participating in hydrogen metabolism. While the enzyme can catalyze both H2 uptake and evolution, the primary in vivo function is H2 oxidation during sulfate respiration. Reason: This biological process term captures the core metabolic role of the enzyme in H2 cycling. The hydrogen cycling model in Desulfovibrio is well-established. Supporting Evidence: file:DESVH/P07598/P07598-deep-research-falcon.md Core reaction: reversible interconversion of molecular hydrogen and protons/electrons, 2 H+ + 2 e |
| GO:0019645 anaerobic electron transport chain | ISS file:DESVH/P07598/P07598-deep-research-falcon.md | NEW | Summary: HydA is a component of the anaerobic electron transport chain in sulfate-reducing bacteria. It oxidizes H2 and transfers electrons to cytochrome c3, which then delivers electrons to membrane complexes for ultimate transfer to cytoplasmic sulfate reductases. Reason: HydA functions within an anaerobic electron transport chain where sulfate (not oxygen) serves as the terminal electron acceptor. The enzyme is a key entry point for electrons from H2 into this chain. Supporting Evidence: file:DESVH/P07598/P07598-deep-research-falcon.md Electrons from periplasmic carriers are shuttled to cytoplasmic sulfate-reduction enzymes via multiheme transmembrane complexes (Hmc, Tmc) and the quinone-interfacing Qrc complex |
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Download this section (compressed HTML)Q: What is the precise stoichiometry of HydA:HydB in the active holoenzyme complex?
Q: Are there conditions under which the periplasmic [FeFe]-hydrogenase operates in the H2-evolution direction in vivo?
Q: What are the specific regulatory mechanisms controlling hydA expression in response to H2 availability and sulfate levels?
Experiment: Deletion mutant studies to quantify the contribution of HydAB specifically (versus other hydrogenases) to sulfate reduction with different electron donors.
Hypothesis: HydAB is the primary periplasmic hydrogenase responsible for H2 oxidation during sulfate respiration.
Type: Genetic deletion/complementation
Experiment: In vivo crosslinking studies to map the interaction interface between HydA and cytochrome c3.
Hypothesis: HydA interacts directly with cytochrome c3 via a specific protein-protein interface.
Type: Crosslinking mass spectrometry
Experiment: Time-resolved spectroscopy to characterize electron transfer kinetics between HydA and membrane complexes (Hmc, Tmc, Qrc).
Hypothesis: Electron transfer from HydA to membrane complexes occurs via cytochrome c3 as an obligate intermediate.
Type: Time-resolved spectroscopy
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