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)₂Fe₂(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 ¹⁴N hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin’s red ester. Whole-protein mass spectrometry of EndoIII nitrosylated with ¹⁴NO and ¹⁵NO support the assignment of a protein-bound [(μ-Cys)₂Fe₂(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.