Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 140, Issue 37, Pages 11800-11810Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.8b07362
Keywords
-
Categories
Funding
- National Institutes of Health [GM126904, 1S100D02001301]
- National Science Foundation [NSF-1531940]
- Dow Next Generation Educator Fund
- Beckman Institute
- Dow Corporation
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [1531940] Funding Source: National Science Foundation
Ask authors/readers for more resources
Here we characterize the [Fe4S4] cluster nitrosylation of a DNA repair enzyme, endonuclease III (EndoIII), using DNA-modified gold electrochemistry and protein film voltammetry, electrophoretic mobility shift assays, mass spectrometry of whole and trypsin-digested protein, and a variety of spectroscopies. Exposure of EndoIII to nitric oxide under anaerobic conditions transforms the [Fe4S4] cluster into a dinitrosyl iron complex, [(Cys)(2)Fe(NO)(2)](-), and Roussin's red ester, [(mu-Cys)(2)Fe-2(NO)(4)], in a 1:1 ratio with an average retention of 3.05 +/- 0.01 Fe per nitrosylated cluster. The formation of the dinitrosyl iron complex is consistent with previous reports, but the Roussin's red ester is an unreported product of EndoIII nitrosylation. Hyperfine sublevel correlation (HYSCORE) pulse EPR spectroscopy detects two distinct classes of NO with N-14 hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin's red ester. Whole-protein mass spectrometry of EndoIII nitrosylated with (NO)-N-14 and (NO)-N-15 support the assignment of a protein-bound [(mu-Cys)(2)Fe-2(NO)(4)] Roussin's red ester. The [Fe4S4](2+/3+) redox couple of DNA-bound EndoIII is observable using DNA-modified gold electrochemistry, but nitrosylated EndoIII does not display observable redox activity using DNA electrochemistry on gold despite having a similar DNA-binding affinity as the native protein. However, direct electrochemistry of protein films on graphite reveals the reduction potential of native and nitrosylated EndoIII to be 127 +/- 6 and -674 +/- 8 mV vs NHE, respectively, corresponding to a shift of approximately -800 mV with cluster nitrosylation. Collectively, these data demonstrate that DNA-bound redox activity, and by extension DNA-mediated charge transport, is modulated by [Fe4S4] cluster nitrosylation.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available