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Characterization of the DNA-Mediated Oxidation of Dps, A Bacterial Ferritin

Arnold, Anna R. and Zhou, Andy and Barton, Jacqueline K. (2016) Characterization of the DNA-Mediated Oxidation of Dps, A Bacterial Ferritin. Journal of the American Chemical Society, 138 (35). pp. 11290-11298. ISSN 0002-7863. PMCID PMC5014645. doi:10.1021/jacs.6b06507. https://resolver.caltech.edu/CaltechAUTHORS:20160907-083741647

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Abstract

Dps proteins are bacterial ferritins that protect DNA from oxidative stress and have been implicated in bacterial survival and virulence. In addition to direct oxidation of the Dps iron sites by diffusing oxidants, oxidation from a distance via DNA charge transport (CT), where electrons and electron holes are rapidly transported through the base-pair π-stack, could represent an efficient DNA protection mechanism utilized by Dps. Here, we spectroscopically characterize the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation using an intercalating ruthenium photooxidant and the flash-quench technique. Upon irradiation with poly(dGdC)_2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)_2 is substituted with poly(dAdT)_2, the yield of Dps oxidation is decreased significantly, consistent with guanine radical intermediates facilitating Dps oxidation. We have also explored possible protein electron transfer (ET) intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the iron-binding ferroxidase site (W52 in E. coli Dps). In EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, a W52A Dps mutant was significantly deficient compared to WT Dps in forming the characteristic EPR signal at g = 4.3, consistent with W52 acting as an ET hopping intermediate. This effect is mirrored in vivo in E. coli survival in response to hydrogen peroxide, where mutation of W52 leads to decreased survival under oxidative stress.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/jacs.6b06507DOIArticle
http://pubs.acs.org/doi/abs/10.1021/jacs.6b06507PublisherArticle
http://pubs.acs.org/doi/suppl/10.1021/jacs.6b06507Related ItemSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014645PubMed CentralArticle
ORCID:
AuthorORCID
Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 2016 American Chemical Society. Received: June 23, 2016. Published: August 29, 2016. Publication Date (Web): August 29, 2016. We are grateful for the financial support of the National Institutes of Health and the Moore Foundation. A.R.A. was supported by the National Institute on Aging of the NIH on a predoctoral NRSA (F31AG040954), and A.Z. through an NSF fellowship. We thank Dr. Angelo Di Bilio for his assistance with EPR instrumentation. We also thank Stephanie Gu for her contributions to the hydrogen peroxide survival assay during her Caltech SURF experience. The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
NIH Predoctoral FellowshipF31AG040954
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Summer Undergraduate Research Fellowship (SURF)UNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
Issue or Number:35
PubMed Central ID:PMC5014645
DOI:10.1021/jacs.6b06507
Record Number:CaltechAUTHORS:20160907-083741647
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160907-083741647
Official Citation:Characterization of the DNA-Mediated Oxidation of Dps, A Bacterial Ferritin Anna R. Arnold, Andy Zhou, and Jacqueline K. Barton Journal of the American Chemical Society 2016 138 (35), 11290-11298 DOI: 10.1021/jacs.6b06507
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:70184
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:07 Sep 2016 16:57
Last Modified:11 Nov 2021 04:25

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