LBA (UniProt P02234; LGBA_PHAVU, "PvLba") encodes leghemoglobin alpha / leghemoglobin component A of Phaseolus vulgaris (common/kidney bean), a member of the plant globin family (InterPro IPR000971 Globin, IPR001032 Leghaemoglobin-like, IPR019824 Leghaemoglobin_Fe_BS). It is a small (~16 kDa, 146 aa) monomeric heme protein expressed at very high abundance specifically in the cytosol of infected cells of functional legume root nodules. Its core molecular function is reversible, high-affinity binding of dioxygen at a pentacoordinated ferrous (Fe2+) heme iron. Physiologically, leghemoglobin resolves the nodule "oxygen paradox": it acts as an oxygen carrier/buffer that delivers O2 toward the symbiosomes to sustain the high respiratory flux of nitrogen-fixing bacteroids while keeping free O2 extremely low (sub-micromolar, reported <50 nM in infected cells) so that the oxygen-labile bacterial nitrogenase is not inactivated. This O2-homeostasis role is essential for symbiotic nitrogen fixation. Beyond O2 transport, PvLba also participates in nodule nitrogen/oxidative chemistry: it is nitrated in vivo on distal-pocket tyrosines (mainly Tyr-31, with Tyr-26 and Tyr-134) by a nitrite/peroxide-dependent (ferryl-Lb) mechanism, and the broader nodule hemoglobin network contributes to NO/ROS homeostasis. UniProt also notes a phosphoserine (Ser-46) that modulates the heme pocket. The protein functions in the infected-cell cytosol (a nucleus localization is asserted only by similarity/ISS and is not the site of its O2-carrier function). The gene symbol "LBA" here refers unambiguously to the bean nodule leghemoglobin and is not to be confused with unrelated "LBA" symbols (e.g. mammalian albumin) in other organisms.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Nuclear localization asserted via UniProtKB Subcellular Location keyword mapping (SL-0191). The UniProt nucleus assignment is itself only "By similarity" (ECO:0000250) to other legume globins, not experimental for PvLba. Reason: Leghemoglobin's functionally relevant location is the cytosol of infected nodule cells, where it buffers and delivers O2 to symbiosomes; the deep-research synthesis places PvLba in the infected host-cell cytosol in proximity to symbiosomes, not the nucleus. The UniProt nucleus call is inferred by similarity (ISS/By similarity) to P02240 and is not supported by gene-specific experimental evidence for the bean protein. It is retained as non-core because a globin nuclear pool, if present, is not where the O2-carrier function is executed. Supporting Evidence: file:PHAVU/LBA/LBA-deep-research-falcon.md Lb is consistently described as **nodule-localized** and functioning in the infected nodule environment |
| GO:0005829 cytosol | IEA GO_REF:0000044 | ACCEPT | Summary: Cytosolic localization assigned via UniProtKB Subcellular Location keyword mapping (SL-0091; UniProt "Cytoplasm, cytosol"). This is the functionally meaningful location of leghemoglobin. Reason: This is the correct core cellular component. Leghemoglobin is a soluble cytosolic hemeprotein present at very high concentration in the cytosol of infected nodule cells, where it buffers free O2 and ferries O2 toward the symbiosome membrane. Both UniProt ("Cytoplasm, cytosol") and the literature synthesis localize PvLba to the infected host-cell cytosol. Supporting Evidence: file:PHAVU/LBA/LBA-uniprot.txt Cytoplasm, cytosol file:PHAVU/LBA/LBA-deep-research-falcon.md Lb is described as an abundant hemeprotein (reported in the **millimolar range**) |
| GO:0019825 oxygen binding | IEA GO_REF:0000002 | ACCEPT | Summary: Oxygen-binding molecular function assigned from InterPro globin/leghemoglobin domain signatures. This is a core, correct molecular function of PvLba. Reason: Reversible O2 binding at the ferrous heme is the defining molecular activity of leghemoglobin and is directly consistent with the UniProt FUNCTION statement and the globin domain architecture. The InterPro-based assignment is at an appropriate level of specificity for a globin and is corroborated by the literature synthesis describing PvLba as an oxygen-binding heme globin. Supporting Evidence: file:PHAVU/LBA/LBA-uniprot.txt facilitates the diffusion of oxygen to the bacteroids while preventing PMID:25603991 essential role as an O2 transporter file:PHAVU/LBA/LBA-deep-research-falcon.md **only ferrous (Fe²⁺) leghemoglobin binds O₂**, so maintenance of the reduced state is integral to function |
| GO:0020037 heme binding | IEA GO_REF:0000002 | ACCEPT | Summary: Heme-binding molecular function assigned from InterPro globin/leghemoglobin domain signatures. Correct and core: leghemoglobin is a b-type heme protein. Reason: Heme binding is essential to leghemoglobin function: O2 is bound at a pentacoordinated ferrous heme b, and UniProt annotates heme b binding residues (e.g. His-93 proximal, and contacts at 46 and 96) on the bean protein. The deep-research synthesis explicitly describes PvLba as a heme-binding globin. This is the more informative and specific molecular-function counterpart to the broad SPKW "metal ion binding" term and is the preferred replacement target for it. Supporting Evidence: file:PHAVU/LBA/LBA-uniprot.txt Leghemoglobin that reversibly binds oxygen O(2) through a file:PHAVU/LBA/LBA-deep-research-falcon.md **Leghemoglobins (Lbs)** are **heme-containing globins** |
| GO:0005634 nucleus | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Nuclear localization transferred by sequence-similarity (ISS) from UniProtKB:P02240 (soybean leghemoglobin a). Duplicates the IEA nucleus annotation with a different evidence path. Reason: This ISS annotation rests on similarity to soybean P02240 rather than on gene-specific experimental data for PvLba, and the nucleus is not where leghemoglobin carries out its oxygen-carrier function (the infected-cell cytosol is). As with the IEA nucleus annotation, it is retained but marked non-core: any nuclear globin pool is not the locus of the protein's core O2-buffering/transport activity. Supporting Evidence: file:PHAVU/LBA/LBA-deep-research-falcon.md functioning in the infected nodule environment where symbiosomes and nitrogenase activity occur |
| GO:0005344 oxygen carrier activity | IEA GO_REF:0000043 | ACCEPT | Summary: SPKW (GO_REF:0000043) annotation derived from the UniProt keyword "Oxygen transport"; snapshot-only, removed in the current GOA release. Oxygen carrier activity is the defining molecular function of leghemoglobin. Reason: GOA's removal of this annotation was NOT justified - it caused collateral damage by dropping a correct, CORE molecular-function term. Leghemoglobin is, by definition, an oxygen carrier: it reversibly binds O2 at a ferrous heme and shuttles/buffers it in nodule cells. The deep-research synthesis describes PvLba as an oxygen-binding heme globin that facilitates O2 diffusion/transport and buffers O2 in nodules, occurring at high (millimolar) concentration in the infected host-cell cytosol as a "high-abundance oxygen buffer/transport system". UniProt's FUNCTION statement states the protein "facilitates the diffusion of oxygen to the bacteroids". This is the most precise and most informative MF for leghemoglobin and should be retained (it complements the InterPro "oxygen binding" term). Re-add and ACCEPT. Supporting Evidence: PMID:25603991 essential role as an O2 transporter file:PHAVU/LBA/LBA-deep-research-falcon.md Lb is described as an abundant hemeprotein (reported in the **millimolar range**) file:PHAVU/LBA/LBA-uniprot.txt facilitates the diffusion of oxygen to the bacteroids while preventing |
| GO:0015671 oxygen transport | IEA GO_REF:0000043 | ACCEPT | Summary: SPKW (GO_REF:0000043) annotation derived from the UniProt keyword "Oxygen transport"; snapshot-only, removed in the current GOA release. This is the biological-process counterpart of leghemoglobin's oxygen-carrier molecular function. Reason: GOA's removal of this annotation was NOT justified - it is collateral damage. Oxygen transport is exactly the process leghemoglobin performs: it delivers O2 toward the symbiosomes to support bacteroid respiration while buffering free O2. The deep-research synthesis states leghemoglobins "function as O2 transporters delivering O2 to symbiosomes" and that PvLba "facilitates O2 diffusion/transport and buffers O2 in nodules", and UniProt records that the protein facilitates the diffusion of oxygen to the bacteroids. The term is accurate and core; re-add and ACCEPT. (It is more informative than the bare "nodulation" process term that was also keyword-derived.) Supporting Evidence: PMID:25603991 essential role as an O2 transporter file:PHAVU/LBA/LBA-deep-research-falcon.md PvLba supports this by **delivering O₂ at low free concentrations** (oxygen buffering/transport) to infected nodule cells and symbiosomes file:PHAVU/LBA/LBA-uniprot.txt facilitates the diffusion of oxygen to the bacteroids while preventing |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | MODIFY | Summary: SPKW (GO_REF:0000043) annotation derived from the UniProt keywords "Metal-binding" and "Iron"; snapshot-only, removed in the current GOA release. Leghemoglobin binds the iron atom of its heme prosthetic group, so the term is correct but very broad. Reason: GOA's removal of this generic keyword-derived term lost little, because the biology is better captured by more specific terms. PvLba does bind a metal ion - the heme iron - and only the ferrous (Fe2+) form binds O2 efficiently, consistent with canonical globin chemistry. However, "metal ion binding" (the broad parent) is uninformative for a globin: the precise, gene-relevant terms are "heme binding" (GO:0020037, already present and ACCEPTed in current GOA, capturing binding of the heme b cofactor) and "iron ion binding" (GO:0005506, capturing the coordinated heme iron). The annotation should therefore be re-added but MODIFIED to these specific terms rather than retained at the broad metal-ion level. Tier B (broad/over-annotated but not wrong). Proposed replacements: heme binding iron ion binding Supporting Evidence: file:PHAVU/LBA/LBA-uniprot.txt Leghemoglobin that reversibly binds oxygen O(2) through a file:PHAVU/LBA/LBA-uniprot.txt pentacoordinated heme iron file:PHAVU/LBA/LBA-deep-research-falcon.md **only ferrous (Fe²⁺) leghemoglobin binds O₂**, so maintenance of the reduced state is integral to function |
| GO:0009877 nodulation | IEA GO_REF:0000043 | MARK AS OVER ANNOTATED | Summary: SPKW (GO_REF:0000043) annotation derived from the UniProt keyword "Nodulation"; snapshot-only, removed in the current GOA release. The keyword reflects that leghemoglobin is a hallmark protein OF the nodule, not that the protein drives the nodulation/organogenesis program. Reason: GOA's removal of this annotation was JUSTIFIED. "Nodulation" denotes the developmental process of nodule formation/organogenesis triggered by rhizobial symbiosis. PvLba is not part of that developmental program; it is a highly abundant nodule-EXPRESSED oxygen carrier whose function is O2 binding/transport and buffering within the already-formed nodule. The keyword captures the nodule expression/localization context ("localized in host cells associated with the symbiosis"; "specialization for infected nodule cells") rather than a developmental role - expression and location are not the same as participation in the organogenesis process. The protein's genuine biological contribution (nodule O2 homeostasis enabling symbiotic nitrogen fixation) is already and better captured by "oxygen transport" (GO:0015671, re-added above) and is more precisely described as symbiotic nitrogen fixation support. The bare "nodulation" term is an over-broad/contextual over-annotation; its removal is appropriate. Tier A (removal justified). If a process term beyond oxygen transport were desired, "nitrogen fixation" (GO:0009399) would be the biologically apt downstream context. Proposed replacements: oxygen transport Supporting Evidence: file:PHAVU/LBA/LBA-deep-research-falcon.md Lb is a **marker of functional (pink) nodules** file:PHAVU/LBA/LBA-deep-research-falcon.md accumulate to very high levels in legume root nodules PMID:25603991 essential role as an O2 transporter |
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Download this section (compressed HTML)Q: Are the millimolar levels of leghemoglobin in bean nodules rate-limiting for symbiotic nitrogen fixation, and does natural genotype variation in PvLba abundance correlate with fixation performance?
Suggested experts: Manuel Becana, Michael Udvardi
Q: What is the in vivo functional consequence of distal-pocket tyrosine nitration (Tyr-31/Tyr-26/Tyr-134) and Ser-46 phosphorylation for the O2 affinity and oxygen-carrier activity of PvLba during nodule development and senescence?
Suggested experts: Manuel Becana
Experiment: Generate PvLba loss-of-function (RNAi or CRISPR) bean nodules and measure free O2 concentration, bacteroid respiration, nitrogenase activity and nitro-oxidative stress markers, to test whether bean leghemoglobin is required for nodule O2 homeostasis as shown for Lotus leghemoglobins.
Hypothesis: Loss of PvLba elevates free O2 and accumulates ROS/NO in infected cells, impairing nitrogenase activity and symbiotic nitrogen fixation.
Type: reverse-genetics phenotyping of symbiotic nitrogen fixation
Experiment: Measure O2 association/dissociation kinetics and equilibrium affinity of recombinant wild-type PvLba versus Tyr31-nitrated and Ser46-phosphomimic variants, and determine whether these PTMs shift the protein between O2-buffering and O2-delivery modes.
Hypothesis: Distal-pocket nitration and Ser-46 phosphorylation modulate heme-pocket chemistry and thereby tune leghemoglobin between buffering and delivering O2 to symbiosomes.
Type: in vitro ligand-binding kinetics on modified protein
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