Affinage

GLRX5

Glutaredoxin-related protein 5, mitochondrial · UniProt Q86SX6

Length
157 aa
Mass
16.6 kDa
Annotated
2026-04-28
26 papers in source corpus 15 papers cited in narrative 15 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

GLRX5 is a mitochondrial monothiol glutaredoxin that serves as the central hub in iron-sulfur (Fe/S) cluster biogenesis, receiving nascent [2Fe-2S] clusters from the scaffold protein ISCU (facilitated by the Hsp70 chaperone Ssq1 in a ternary complex) and distributing them to downstream acceptors required for both mitochondrial and cytoplasmic Fe/S protein maturation, heme biosynthesis, and lipoylation (PMID:11950925, PMID:23615440, PMID:40074084). The holo protein assembles as a dimer (or tetramer) with [2Fe-2S] clusters coordinated by the conserved active-site Cys67 from each protomer and two glutathione cysteinyl thiols, and it forms functionally distinct [2Fe-2S]-bridged heterodimeric complexes with BOLA1 (non-trafficking, redox role) and BOLA3 (trafficking role) (PMID:21029046, PMID:28483642, PMID:32542995). Grx5-depleted mitochondria fail to generate both intramitochondrial Fe/S clusters and the exported intermediate for cytoplasmic Fe/S assembly, positioning GLRX5 upstream of the late-acting ISA scaffold proteins (PMID:40074084). Mutations in human GLRX5 cause congenital sideroblastic anemia and variant nonketotic hyperglycinemia through impaired Fe/S cluster binding or transfer, defective ferrochelatase and ALAS2 activity, and loss of protein lipoylation (PMID:24334290, PMID:26100117, PMID:30660387).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 1999 High

    The initial identification of Grx5 as a monothiol glutaredoxin protecting against protein oxidative damage established the gene's link to redox homeostasis, but its specific molecular role was unknown.

    Evidence Genetic epistasis with other yeast glutaredoxins and protein carbonyl assays in yeast mutants

    PMID:10567543

    Open questions at the time
    • No connection to iron-sulfur cluster biogenesis established
    • No subcellular localization determined
    • Mammalian ortholog function untested
  2. 2002 High

    Demonstration that Grx5 localizes to the mitochondrial matrix and is required for Fe/S cluster assembly — not merely redox defense — repositioned the gene as an ISC machinery component, with suppression by SSQ1 and ISA2 overexpression placing it genetically within the pathway.

    Evidence Subcellular fractionation, Fe/S enzyme activity assays, iron quantification, and genetic suppressor analysis in yeast

    PMID:11950925

    Open questions at the time
    • Whether Grx5 directly handles Fe/S clusters or acts indirectly was unclear
    • Physical interaction partners within the ISC machinery not yet identified
    • Structure-function basis of the active site not resolved
  3. 2002 High

    Structure-function mutagenesis established that the active-site Cys60, adjacent Gly61, and glutathione-binding residues are essential for Grx5 function, defining the minimal chemical requirements for its biological activity.

    Evidence Site-directed mutagenesis with phenotypic assays (oxidant sensitivity, respiratory growth, iron accumulation) in yeast

    PMID:12138088

    Open questions at the time
    • No crystal structure available to rationalize residue roles
    • Mechanism of cluster coordination not yet demonstrated biochemically
  4. 2003 High

    Biochemical characterization of purified Grx5 revealed its redox chemistry — a low-pKa active-site Cys60 forms a mixed disulfide with glutathione and catalyzes deglutathionylation — establishing it as a functional glutaredoxin with distinctive kinetics compared to dithiol glutaredoxins.

    Evidence In vitro redox potential measurement, pKa determination, and thiol reductase activity assays with purified recombinant wild-type and mutant proteins

    PMID:12730244

    Open questions at the time
    • Relationship between glutaredoxin redox activity and Fe/S cluster handling not clarified
    • No structural data on cluster-bound form
  5. 2006 High

    Cross-species complementation showed that human GLRX5 and chicken cGRX5 fully rescue yeast grx5 null phenotypes, establishing evolutionary conservation of GLRX5 function in mitochondrial Fe/S cluster biogenesis and validating yeast as a model for the human protein.

    Evidence Heterologous expression of human/chicken GLRX5 in yeast grx5Δ with phenotypic rescue assays

    PMID:16566929

    Open questions at the time
    • Direct biochemical activity of human GLRX5 not yet characterized
    • Disease relevance in humans not yet demonstrated
  6. 2010 High

    In vivo interaction of Grx5 with the late-acting ISA scaffold proteins Isa1/Isa2 in fission yeast, combined with suppressor genetics, clarified that Grx5 acts at the interface between early and late Fe/S assembly steps and also supports mitochondrial DNA integrity.

    Evidence Bimolecular fluorescence complementation (BiFC) in S. pombe, multicopy suppressor screens, Fe/S enzyme activity assays

    PMID:20085751

    Open questions at the time
    • Direction of cluster flow between Grx5 and Isa proteins not determined
    • Mechanism of mitochondrial DNA maintenance effect unclear
  7. 2011 High

    The crystal structure of human GLRX5 revealed that the holo form is a tetramer with two [2Fe-2S] clusters each coordinated by Cys67 from two protomers and two glutathione molecules, providing the first atomic-level picture of how a monothiol glutaredoxin binds Fe/S clusters.

    Evidence X-ray crystallography, analytical ultracentrifugation, mass spectrometry, and in vitro activity assays

    PMID:21029046

    Open questions at the time
    • Mechanism of cluster release to downstream acceptors not structurally resolved
    • Role of oligomeric state transitions in cluster transfer unknown
  8. 2013 High

    Two advances established the mechanistic role of GLRX5 in human disease and in cluster transfer: (1) the Hsp70 chaperone Ssq1 forms a ternary complex with Isu1 and Grx5 to facilitate rapid Fe/S cluster transfer from scaffold to Grx5, and (2) GLRX5 mutations were shown to cause variant nonketotic hyperglycinemia through deficient protein lipoylation, with transfection rescue confirming causality.

    Evidence In vitro and in vivo interaction assays with ATPase stimulation and cluster transfer kinetics (yeast); patient cell biochemical assays with transfection rescue (human)

    PMID:23615440 PMID:24334290

    Open questions at the time
    • Structural basis of the Ssq1-Grx5 interaction not resolved
    • Precise mechanism linking Fe/S cluster deficiency to lipoylation failure not delineated
  9. 2016 High

    Separation-of-function mutations in human GLRX5 (K101Q preventing cluster binding vs. L148S impairing cluster transfer) demonstrated that cluster acquisition and cluster donation are mechanistically distinct steps, revealing GLRX5 as a multifunctional node rather than a simple carrier.

    Evidence GLRX5 knockout K562 cells complemented with site-directed mutants, with Fe/S enzyme activity assays for multiple downstream targets

    PMID:26100117

    Open questions at the time
    • Structural basis for why L148S selectively impairs transfer but not binding is unknown
    • Whether distinct acceptor proteins require different GLRX5 surfaces is untested
  10. 2017 High

    Spectroscopic characterization of GLRX5 heterodimeric complexes with BOLA1 and BOLA3 revealed they coordinate distinct [2Fe-2S] cluster types — reduced Rieske-type for BOLA1-GLRX5 and oxidized ferredoxin-like for BOLA3-GLRX5 — suggesting bifurcation of Fe/S cluster function into redox sensing and trafficking branches.

    Evidence UV/vis, CD, EPR, and NMR spectroscopy on reconstituted complexes; glutathione coordination analysis; cluster transfer kinetics

    PMID:28483642 PMID:29264659

    Open questions at the time
    • In vivo relevance of BOLA1-GLRX5 vs. BOLA3-GLRX5 partitioning not demonstrated
    • Downstream acceptors for the BOLA3-GLRX5 complex not identified
  11. 2020 High

    Systematic donor/acceptor specificity testing confirmed that the BOLA1-GLRX5 complex accepts clusters from ISCU but cannot donate them to acceptor proteins, supporting a non-trafficking redox role, while distinguishing it functionally from BOLA1 homodimers that show facile cluster exchange.

    Evidence CD spectroscopy-monitored cluster transfer kinetics with reconstituted holo complexes and multiple donor/acceptor combinations in vitro

    PMID:32542995

    Open questions at the time
    • Physiological function of the BOLA1-GLRX5 redox complex in mitochondria remains uncharacterized
    • Whether BOLA3-GLRX5 serves as a direct cluster donor to specific targets is untested in vitro
  12. 2025 High

    Reconstitution in isolated mitochondria definitively positioned Grx5 upstream of Isa1/Isa2, showing that Grx5 depletion abolishes both intramitochondrial Fe/S cluster synthesis and generation of the exported cytoplasmic Fe/S assembly intermediate, while Isa protein depletion only blocks [4Fe-4S] biogenesis.

    Evidence Fe/S cluster synthesis assays in isolated yeast mitochondria, import of purified Grx5 precursor for rescue, comparison with Isa1/Isa2 depletion

    PMID:40074084

    Open questions at the time
    • Identity of the exported (Fe-S)int intermediate remains unknown
    • Whether human GLRX5 occupies the identical epistatic position in isolated human mitochondria is untested
    • Direct structural characterization of the ISCU-to-Grx5 cluster transfer intermediate is lacking

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the identity of the exported Fe/S intermediate that Grx5 generates for cytoplasmic assembly, the structural basis for selective cluster donation to distinct acceptor proteins, and the in vivo physiological role of the BOLA1-GLRX5 redox complex.
  • No structural model of the GLRX5-acceptor transfer complex exists
  • The exported (Fe-S)int species has not been chemically identified
  • In vivo phenotypic consequences of selectively disrupting BOLA1-GLRX5 vs. BOLA3-GLRX5 are unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140104 molecular carrier activity 4 GO:0016491 oxidoreductase activity 3
Localization
GO:0005739 mitochondrion 5
Pathway
R-HSA-1430728 Metabolism 5 R-HSA-1643685 Disease 2
Partners
BOLA1BOLA3FDX1HSPA9ISCA1ISCA2ISCU
Complex memberships
BOLA1-GLRX5 heterodimerBOLA3-GLRX5 heterodimerSsq1-Isu1-Grx5 ternary complex

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1999 Yeast Grx5 is a monothiol glutaredoxin that protects against protein oxidative damage; loss of Grx5 causes constitutive protein carbonylation (including transketolase oxidation), and synthetic lethality with grx2 and grx3/grx4 double mutations establishes a complex functional relationship among yeast glutaredoxins. Genetic epistasis (double mutants), protein carbonyl assays, oxidant sensitivity assays Molecular and cellular biology High 10567543
2002 Yeast Grx5 localizes to the mitochondrial matrix (mature form lacks first 29 aa signal sequence) and is required for Fe/S cluster assembly; grx5 null mutants accumulate iron and show inactivation of Fe/S enzymes; overexpression of SSQ1 or ISA2 suppresses grx5 phenotypes, placing Grx5 in the mitochondrial ISC machinery. Subcellular fractionation, genetic epistasis (suppressor overexpression), Fe/S enzyme activity assays, iron quantification Molecular biology of the cell High 11950925
2002 Structure-function analysis of yeast Grx5 using site-directed mutagenesis shows Cys60 and Gly61 are essential for biological function (Fe/S assembly, oxidant resistance, respiratory growth); Gly115 and Gly116 are important for glutathione-cleft formation; Phe50 mutation disrupts beta-sheet of the thioredoxin fold and inhibits Grx5 function; Cys117 is dispensable for function. Site-directed mutagenesis, phenotypic assays (oxidant sensitivity, respiratory growth, iron accumulation), 3D structural modeling The Journal of biological chemistry High 12138088
2003 Purified yeast Grx5 has a redox potential of -175 mV; the conserved Cys60 (pKa 5.0) forms a transient mixed disulfide with glutathione upon GSSG treatment; this promotes lowering of Cys117 pKa (8.2→lower), triggering intramolecular disulfide bond formation between Cys60 and Cys117; the intramolecular disulfide is reduced by GSH ~20-fold slower than E. coli Grx1; wild-type Grx5 efficiently reduces glutathionylated substrate proteins. In vitro biochemical assays: redox potential measurement, iodoacetamide titration, GSSG incubation with purified recombinant proteins (WT, C60S, C117S mutants), thiol reductase activity assay The Journal of biological chemistry High 12730244
2006 Human GLRX5 and chicken cGRX5 contain mitochondrial targeting sequences and, when expressed in yeast grx5 null mutants, rescue all defects (oxidant sensitivity, amino acid auxotrophy, iron accumulation), demonstrating functional conservation of GLRX5 in mitochondrial Fe/S cluster biogenesis. Heterologous complementation in yeast, mitochondrial targeting experiments, phenotypic rescue assays FEBS letters High 16566929
2010 In fission yeast, Grx5 resides in mitochondria, is required for Fe/S enzyme activity in both mitochondria and cytoplasm, and physically interacts with A-type Fe/S scaffold proteins Isa1 and Isa2 in vivo; overexpression of isa1+ or isa2+ (but not isu1+) suppresses grx5 deletion; Grx5 also supports mitochondrial DNA integrity. Bimolecular fluorescence complementation (BiFC) for in vivo protein interaction, multicopy suppressor screen, Fe/S enzyme activity assays, mitochondrial DNA quantification Biochemical and biophysical research communications High 20085751
2011 Crystal structure of human GLRX5 reveals that the holo protein assembles as a tetramer (dimer of dimers) with two [2Fe-2S] clusters buried at the dimer interface, each coordinated by the active-site Cys67 from two protomers and two glutathione cysteinyl thiols; the apo protein is monomeric; apo-GLRX5 reduces glutathione mixed disulfides and acts as an electron donor for mammalian ribonucleotide reductase (100-fold less active than GLRX2); cysteine residues are glutathionylated in the absence of cluster, protecting them from further oxidation. X-ray crystallography, gel-filtration chromatography, analytical ultracentrifugation, mass spectrometry, in vitro glutaredoxin activity assays The Biochemical journal High 21029046
2013 The yeast mitochondrial Hsp70 chaperone Ssq1 interacts with Grx5 at a binding site distinct from its Isu1-binding site; Grx5 binding is strongest for ADP-bound Ssq1; the Ssq1–Isu1–Grx5 ternary proximity facilitates rapid Fe/S cluster transfer from Isu1 to Grx5; Grx5 and its bound Fe/S cluster are required for maturation of all cellular Fe/S proteins regardless of cluster type or subcellular location. In vivo and in vitro interaction assays (co-purification, pulldown), ATPase stimulation assays, Fe/S cluster transfer assays, genetic complementation Molecular biology of the cell High 23615440
2013 Mutations in human GLRX5 cause variant nonketotic hyperglycinemia with deficiency of lipoylation of mitochondrial proteins and deficient glycine cleavage enzyme activity; transfection with native GLRX5 corrects the biochemical deficiency in patient cells, establishing GLRX5 as required for lipoylation/Fe-S-dependent cofactor biosynthesis. Patient cell biochemical assays, transfection rescue experiments, enzyme activity measurements Brain : a journal of neurology High 24334290
2016 In human K562 cells, the GLRX5 missense mutation K101Q prevents [Fe-S] cluster binding to GLRX5, while L148S allows cluster binding but impairs transfer to downstream Fe/S protein acceptors (IRP1, mitochondrial aconitase, ferrochelatase); different conserved residues affect distinct downstream Fe/S biosynthesis steps, demonstrating GLRX5 is multifunctional. GLRX5 knockout K562 cells, site-directed mutagenesis, functional complementation, Fe/S enzyme activity assays (succinate dehydrogenase, aconitase, IRP1, ferrochelatase), lipoylation assays Journal of cellular biochemistry High 26100117
2017 Human GLRX5 forms [2Fe-2S] cluster-bridged heterodimeric complexes with mitochondrial BOLA1 and BOLA3; BOLA1-GRX5 complex coordinates a reduced Rieske-type [2Fe-2S]1+ cluster, while BOLA3-GRX5 coordinates an oxidized ferredoxin-like [2Fe-2S]2+ cluster; BOLA1-GRX5 has higher cluster binding affinity and is preferentially formed; the two complexes have distinct redox properties suggesting different molecular functions. UV/vis, CD, EPR, NMR spectroscopy, computational protein-protein docking, experimentally-driven structural modeling Biochimica et biophysica acta. General subjects High 28483642
2017 Human Grx5 binds [2Fe-2S] clusters with glutathione providing cysteinyl coordination to iron; disruption of glutathione-protein hydrogen bonding/ionic contacts (by natural and non-natural amino acid substitutions) alters cluster chirality (CD spectra) but does not abolish cluster transfer to apo-ferredoxin 1; Grx5 can be reconstituted with non-glutathione thiol ligands (DTT, L-cysteine) with comparable cluster transfer rates. In vitro chemical reconstitution with mutant proteins and glutathione analogs, CD spectroscopy, cluster transfer kinetics to apo-ferredoxin 1 Journal of biological inorganic chemistry High 29264659
2019 GLRX5 mutations in a congenital sideroblastic anemia patient (Cys67Tyr and Met128Lys) impair ferrochelatase activity (without porphyrin accumulation) and ALAS2 activity; structural analysis confirms Cys67 coordinates the [2Fe-2S] cluster and suggests Met128 mediates partner interactions; GLRX5 deficiency also causes oxidative stress with decreased aconitase activity and mitochondrial respiratory chain dysfunction. Patient-derived lymphoblastoid and CD34+ cells, enzyme activity assays (ferrochelatase, ALAS2, aconitase, complex I/IV), 3D structural analysis, glutathione measurement Molecular genetics and metabolism Medium 30660387
2020 [2Fe-2S]-bridged BOLA1-GLRX5 heterodimer accepts clusters from ISCU or [2Fe-2S](GS)4 but not from ISCA1 or ISCA2, and is incapable of donating its cluster to apo protein acceptors, supporting a non-trafficking (likely redox) role for this complex; the BOLA1 homodimer, in contrast, shows facile cluster exchange. CD spectroscopy-monitored cluster transfer kinetics, in vitro reconstitution of holo complexes, donor/acceptor specificity assays The FEBS journal High 32542995
2025 Using isolated yeast mitochondria, Grx5-depleted mitochondria fail to synthesize Fe/S clusters for intramitochondrial proteins and also fail to generate the (Fe-S)int intermediate exported to the cytoplasm for cytoplasmic Fe/S assembly; import of purified Grx5 precursor into Grx5-depleted mitochondria rescues both defects; by contrast, Isa1- or Isa2-depleted mitochondria can still synthesize [2Fe-2S] but not [4Fe-4S] clusters and can still support cytoplasmic Fe/S assembly, positioning Grx5 upstream of Isa1/Isa2 as a central hub for Fe/S cluster intermediate trafficking. Isolated mitochondria reconstitution assays, Fe/S cluster synthesis assays in isolated organelles, protein import into isolated mitochondria, genetic depletion strains The Journal of biological chemistry High 40074084

Source papers

Stage 0 corpus · 26 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2002 Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes. Molecular biology of the cell 379 11950925
1999 Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae. Molecular and cellular biology 246 10567543
2013 Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5. Brain : a journal of neurology 170 24334290
2003 Biochemical characterization of yeast mitochondrial Grx5 monothiol glutaredoxin. The Journal of biological chemistry 108 12730244
2013 The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation. Molecular biology of the cell 105 23615440
2011 The crystal structure of human GLRX5: iron-sulfur cluster co-ordination, tetrameric assembly and monomer activity. The Biochemical journal 101 21029046
2006 Prokaryotic and eukaryotic monothiol glutaredoxins are able to perform the functions of Grx5 in the biogenesis of Fe/S clusters in yeast mitochondria. FEBS letters 63 16566929
2002 Structure-function analysis of yeast Grx5 monothiol glutaredoxin defines essential amino acids for the function of the protein. The Journal of biological chemistry 63 12138088
2010 Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast. Biochemical and biophysical research communications 53 20085751
2017 Structural insights into the molecular function of human [2Fe-2S] BOLA1-GRX5 and [2Fe-2S] BOLA3-GRX5 complexes. Biochimica et biophysica acta. General subjects 44 28483642
2008 AtGRX4, an Arabidopsis chloroplastic monothiol glutaredoxin, is able to suppress yeast grx5 mutant phenotypes and respond to oxidative stress. FEBS letters 38 18275854
2016 Functional Analysis of GLRX5 Mutants Reveals Distinct Functionalities of GLRX5 Protein. Journal of cellular biochemistry 35 26100117
2004 Predictive reconstruction of the mitochondrial iron-sulfur cluster assembly metabolism. II. Role of glutaredoxin Grx5. Proteins 25 15382238
2019 GLRX5 mutations impair heme biosynthetic enzymes ALA synthase 2 and ferrochelatase in Human congenital sideroblastic anemia. Molecular genetics and metabolism 24 30660387
2020 A novel lncRNA BADLNCR1 inhibits bovine adipogenesis by repressing GLRX5 expression. Journal of cellular and molecular medicine 19 32449295
2024 Salvia miltiorrhiza Bge. processed with porcine cardiac blood inhibited GLRX5-mediated ferroptosis alleviating cerebral ischemia-reperfusion injury. Phytomedicine : international journal of phytotherapy and phytopharmacology 12 38677272
2023 Exosomes from hypoxic pretreated ADSCs attenuate ultraviolet light-induced skin injury via GLRX5 delivery and ferroptosis inhibition. Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology 11 38100056
2021 Case Report: A Variant Non-ketotic Hyperglycinemia With GLRX5 Mutations: Manifestation of Deficiency of Activities of the Respiratory Chain Enzymes. Frontiers in genetics 8 34054912
2025 Mitochondrial glutaredoxin Grx5 functions as a central hub for cellular iron-sulfur cluster assembly. The Journal of biological chemistry 7 40074084
2021 HACE1, GLRX5, and ELP2 gene variant cause spastic paraplegies. Acta neurologica Belgica 7 33813722
2017 Investigation of glutathione-derived electrostatic and hydrogen-bonding interactions and their role in defining Grx5 [2Fe-2S] cluster optical spectra and transfer chemistry. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 7 29264659
2020 Cluster exchange reactivity of [2Fe-2S]-bridged heterodimeric BOLA1-GLRX5. The FEBS journal 6 32542995
2021 GLRX5-associated [Fe-S] cluster biogenesis disorder: further characterisation of the neurological phenotype and long-term outcome. Orphanet journal of rare diseases 5 34732213
2024 Case report: Unveiling genetic and phenotypic variability in Nonketotic hyperglycinemia: an atypical early onset case associated with a novel GLRX5 variant. Frontiers in genetics 1 39323869
2009 Cloning, overproduction, purification, crystallization and preliminary X-ray diffraction analysis of yeast glutaredoxin Grx5. Acta crystallographica. Section F, Structural biology and crystallization communications 1 19478456
2025 Infant With a Severe Form of GLRX5-Related Atypical Hyperglycinemia Exhibiting Novel Cardiac and Neurologic Disease Manifestations at Autopsy. Pediatric and developmental pathology : the official journal of the Society for Pediatric Pathology and the Paediatric Pathology Society 0 40415601