Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe₄S₄] Cluster Nitrosylation
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)_2Fe(NO)_2]−, and Roussin's red ester, [(μ-Cys)_2Fe_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 ^(14)N hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin's red ester. Whole-protein mass spectrometry of EndoIII nitrosylated with ^(14)NO and ^(15)NO support the assignment of a protein-bound [(μ-Cys)_2Fe_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.
© 2018 American Chemical Society. Received: July 12, 2018; Published: August 26, 2018. This work was supported by GM126904 from the National Institutes of Health (to J.K.B.). The Caltech EPR facility is supported by the National Science Foundation (NSF-1531940) and the Dow Next Generation Educator Fund. The Proteome Exploration Laboratory is supported by the Beckman Institute and the National Institutes of Health (1S10OD02001301). This research benefited from the use of the Autoflex MALDI TOF mass spectrometer in the Caltech CCE Multiuser Mass Spectrometry Laboratory, acquired with funds from the Dow Corporation. The authors thank Andy Zhou for assistance with protein overexpression and purification. The authors declare no competing financial interest.
Accepted Version - nihms-989219.pdf
Supplemental Material - ja8b07362_si_001.pdf