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Published September 7, 2016 | public
Journal Article Open

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


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.

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.

Attached Files

Supplemental Material - ja6b06507_si_001.pdf

Accepted Version - nihms812219.pdf


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August 20, 2023
August 20, 2023