The DOI matches its Crossref record; 34 of the 34 references it cites were found in a registry or an index; 4 of the 27 sources its claims cite were checked for support.
Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center
Open accessAbstract
Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and the cofactor ascorbate (vitamin C) is required to reactivate the enzyme by reducing its iron center from Fe(III) to Fe(II). Herein, we report on the discovery of the first synthetic activators of CP4H. Specifically, we find that 2,2'-bipyridine-4-carboxylate and 2,2'-bipyridine-5-carboxylate serve as ligands for the iron center in human CP4H that enhance the rate of ascorbate-dependent reactivation. This new mode of CP4H activation is available to other biheteroaryl compounds but does not necessarily extend to other prolyl 4-hydroxylases. As collagen is weakened in many indications, analogous activators of CP4H could have therapeutic benefits.
Overview
CoChat Summary
TL;DR
The paper reports the first synthetic activators of human collagen prolyl 4-hydroxylase (CP4H): 2,2′-bipyridine-4-carboxylate (bipy4C) and 2,2′-bipyridine-5-carboxylate (bipy5C). These ligands activate CP4H by enhancing ascorbate-dependent reduction of an inactive Fe(III) iron center back to the active Fe(II) state.
Key findings
- bipy4C and bipy5C activated human CP4H1 by ~3-fold under standard assay conditions; activation was dose-dependent and required vitamin C/ascorbate in a dose-dependent manner.
- Both compounds bound Fe(II), forming Fe(ligand)₃-type complexes, with Fe20-EC50 values of (33 ± 0.8) μM for bipy4C and (43 ± 2) μM for bipy5C, compared with (43 ± 2) μM for bipy.
- Structure–activity studies showed activation required both a bipyridyl chelate and a pendant carboxylate with appropriate regiochemistry: phenyl replacements, non-chelating pyridyl nitrogen, esterification, and simple pyridine carboxylates abolished activation; pythi5C activated CP4H1, whereas pythi4C and pypyr5C did not.
- Preincubation of bipy4C with Fe(II) abolished activation, and succinate (1 or 5 mM) outcompeted activation by 100 μM bipymonocarboxylates. At high α-ketoglutarate concentrations, several known inhibitors—25PDC, bipy45′DC, bipy55′DC, and pythiDC—appeared to activate CP4H1, whereas they inhibited at low α-ketoglutarate concentrations. Neither human PHD2 nor *C. reinhardtii* P4H-1 was activated by bipy4C or bipy5C.
Methods
- Human CP4H1, human PHD2, and *C. reinhardtii* P4H-1 were produced heterologously and assayed by HPLC/UPLC monitoring hydroxylation of peptide substrates; assays were performed in triplicate.
- CP4H1 activation assays used dansylGlyProProGlyOEt substrate, FeSO₄, ascorbate, DTT, α-ketoglutarate, and activators at varied concentrations; PHD2 and CrP4H-1 were assayed under comparable conditions.
- Fe(II) affinity was compared by measuring Fe20-EC50 at pH 7.0, and Fe(II) complex stoichiometry was estimated by Job’s method.
Limitations
- The paper states no explicit limitations section.
- Evident from the text: the work is entirely in vitro with purified enzymes and model peptide substrates, with no cellular or in vivo validation of CP4H activation or therapeutic benefit.
- Evident from the text: activation is context-dependent, influenced by ligand, α-ketoglutarate, succinate, and ascorbate concentrations, and the proposed mechanism is not structurally confirmed; selectivity was tested only against human PHD2 and *C. reinhardtii* P4H-1, not all prolyl 4-hydroxylases.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Verification
Claims the paper makes
23 Own result · 17 Own interpretation · 8 No full text of the sources · 7 Not checked yet · 1 Supported · 1 Supported so far · 1 Partly supported · accuracy not yet measured
The sources with a verdict support this claim; the rest weren’t checked yet, have no full text we could read, or their verdicts aren’t rated yet.
In the paper
“Whereas bipy itself does not serve as an activator of CP4H,20,22”
§ Human Collagen Prolyl 4-Hydroxylase is Activated by Ligands for its Iron Center > RESULTS AND DISCUSSION > Discovery of activators of CP4H1 and requirement for vitamin C
What the sources say
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“In subsequent investigations, we found that this activation was dose-dependent (Fig. 1A) and not outcompeted by α-ketoglutarate, which is a cosubstrate.”
§ Human Collagen Prolyl 4-Hydroxylase is Activated by Ligands for its Iron Center > RESULTS AND DISCUSSION > Discovery of activators of CP4H1 and requirement for vitamin C
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Moreover, at the lowest iron concentration tested (3 μM), we found that bipy4C appeared to inhibit the enzyme, suggesting that the preformed Fe(ligand)3 type complex could sequester iron from CP4H1 over the time-course of our assay.”
§ Human Collagen Prolyl 4-Hydroxylase is Activated by Ligands for its Iron Center > RESULTS AND DISCUSSION > Activation by Free Ligand or Fe(Ligand)3 Complex?
hedgedstates a possible cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We found that replacement of one or both of the pyridine rings with a phenyl ring (as in 4pyBA, 6Phpy3C, biphenyl4C, 3pyBA, 2Phpy4C, and biphenyl3C) abolished activation.”
§ Human Collagen Prolyl 4-Hydroxylase is Activated by Ligands for its Iron Center > RESULTS AND DISCUSSION > Determinants for the Activation of CP4H
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Likewise, repositioning of the pyridyl nitrogen in bipy4C to a non-chelating geometry (as in bipy4C*) abolished activation, as did esterification of bipy4C (as in methyl bipy4C).”
§ Human Collagen Prolyl 4-Hydroxylase is Activated by Ligands for its Iron Center > RESULTS AND DISCUSSION > Determinants for the Activation of CP4H
plainstates a cause- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
32 more claims
Create a free account to see all 58 claims and to verify the 8 references still open.
- Probably not a substantial claim. 8
- It may state two separate claims as one. 15
Citations
Cites
Molecular Mechanisms of Stress-Responsive Changes in Collagen and Elastin Networks in Skin
Aziz, J., Shezali, H., Radzi, Z., Yahya, N. A., Abu Kassim, N. H., Czernuszka, J., & Rahman, M. T. (2016) · Skin Pharmacology and Physiology · doi:10.1159/000447017 · Cited by 7,323
Collagen Structure and Stability
Shoulders, M. D., & Raines, R. T. (2009) · Annual Review of Biochemistry · doi:10.1146/annurev.biochem.77.032207.120833 · Cited by 3,923
Self-citationDioxygen Activation at Mononuclear Nonheme Iron Active Sites: Enzymes, Models, and Intermediates
Costas, M., Mehn, M. P., Jensen, M. P., & QUE, L. J. (2004) · Chemical Reviews · doi:10.1021/cr020628n · Cited by 2,511
Fe(II)/α-Ketoglutarate-Dependent Hydroxylases and Related Enzymes
Hausinger, R. P. (2004) · Critical Reviews in Biochemistry and Molecular Biology · doi:10.1080/10409230490440541 · Cited by 937
Enzymatic Assembly of Overlapping DNA Fragments
Gibson, D. G. (2011) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/b978-0-12-385120-8.00015-2 · Cited by 763
Gorres, K. L., & Raines, R. T. (2010) · Critical Reviews in Biochemistry and Molecular Biology · doi:10.3109/10409231003627991 · Cited by 591
Self-citationBerg, R. A., & Prockop, D. J. (1973) · Biochemical and Biophysical Research Communications · doi:10.1016/0006-291x(73)90961-3 · Cited by 582
[27] Determination of binding stoichiometry by the continuous variation method: The job plot
Huang, C. Y. (1982) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/s0076-6879(82)87029-8 · Cited by 544
Sakakibara, S., Inouye, K., Shudo, K., Kishida, Y., Kobayashi, Y., & Prockop, D. J. (1973) · Biochimica et Biophysica Acta (BBA) - Protein Structure · doi:10.1016/0005-2795(73)90164-5 · Cited by 275
Myllyharju, J. (2008) · Annals of Medicine · doi:10.1080/07853890801986594 · Cited by 269
Research works printed but not linked: 1
- Job P., 1928, Annali di Chimica Applicata, 9, 113
Cited by
Boo, Y. C. (2022) · Antioxidants · doi:10.3390/antiox11091663 · Cited by 230
Iron bioavailability from food fortification to precision nutrition. A review
Blanco-Rojo, R., & Vaquero, M. P. (2018) · Innovative Food Science & Emerging Technologies · doi:10.1016/j.ifset.2018.04.015 · Cited by 190
Kontoghiorghes, G. J., Kolnagou, A., Kontoghiorghe, C., Mourouzidis, L., Timoshnikov, V. A., & Polyakov, N. E. (2020) · Medicines · doi:10.3390/medicines7080045 · Cited by 117
Timmins, A., Saint-André, M., & de Visser, S. P. (2017) · Journal of the American Chemical Society · doi:10.1021/jacs.7b02839 · Cited by 98
Collagen Prolyl 4-Hydroxylase as a Therapeutic Target: Miniperspective
Vasta, J. D., & Raines, R. T. (2018) · Journal of Medicinal Chemistry · doi:10.1021/acs.jmedchem.8b00822 · Cited by 82
Self-citation
Metadata
- Authors
- Vasta, J. D., & Raines, R. T.
- Affiliations
- University of Wisconsin–Madison (US)
- Corresponding author
- Raines, Ronald T.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2016-05-31
- Volume
- 55
- Issue
- 23
- Pages
- 3224-3233
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- PMC
- PMC5141248
- OpenAlex
- W2403625569
- Cited by
- 36
- References
- 34
- Citations by year
- 2026: 5 · 2025: 5 · 2024: 4 · 2023: 4 · 2022: 5 · 2021: 1 · 2020: 3 · 2019: 3 · 2018: 3 · 2017: 2 · 2016: 1
- FWCI
- 1.11
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR044276)
- Topics
- Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
- Physical Sciences › Materials Science › Biomaterials › Collagen: Extraction and Characterization
- Physical Sciences › Chemistry › Inorganic Chemistry › Vanadium and Halogenation Chemistry
- Keywords
- collagen prolyl 4-hydroxylase, proline hydroxylation, iron centers, enzyme activation, oxidative inactivation, scurvy, alpha-ketoglutarate-dependent dioxygenase
- MeSH terms
- Ascorbic Acid, Catalysis, Humans, Hydroxylation, Iron, Kinetics, Ligands, Proline, 2,2'-Dipyridyl, Catalytic Domain, Prolyl Hydroxylases
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
A language model drew the claims from the paper on 2026-11-11, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).
A second model labelled each claim on 2026-11-11 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).
Cited sources are checked with a language model’s help (gpt-6-sol).
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
How to cite
Cite CoChat for the checks on this page, and the paper for its own findings.
- This page
- CoChat. (2026). Citation check: Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center. https://app.cochat.ai/papers/10.1021/acs.biochem.6b00251
- The paper
- Vasta, J. D., & Raines, R. T. (2016). Human Collagen Prolyl 4-Hydroxylase Is Activated by Ligands for Its Iron Center. Biochemistry. https://doi.org/10.1021/acs.biochem.6b00251
Data sources
Bibliographic data: Semantic Scholar, OpenAlex, Crossref.
Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.
