Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe₄S₄] Cluster Nitrosylation

Abstract

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, [(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.

Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication

“…DNA electrochemistry on Au electrode surfaces is amenable to measuring physiological potentials ranging from −200 mV to +500 mV vs. NHE (28, 50–59). However, scanning beyond this range is necessary to observe a redox signal from a protein in the absence of DNA, which has a higher (more reductive) midpoint potential due to the absence of the DNA polyanion (51, 53, 56, 60, 61). In order to measure the DNA-free and DNA-bound redox potentials of EndoIII, highly oriented pyrolytic graphite (HOPG) electrodes were used (60).…”

UvrC Coordinates an O2-Sensitive [4Fe4S] Cofactor

“…Annealing Titrations. 6 Phosphorimaging screens (GE Healthcare or Molecular Diagnostics) were exposed according to the guideline that samples with 300,000 counts require 1 hour of exposure. The exposed screens were scanned using the Figure S5.…”

Cell-Penetrating Delivery of Nitric Oxide by Biocompatible Dinitrosyl Iron Complex and Its Dermato-Physiological Implications

“…Based on the distinctive electron paramagnetic resonance (EPR) signal at g ≈ 2.03 observed in the baker’s yeast cells ( S. cerevisiae ), biosynthesis of dinitrosyl iron complexes (DNICs) was first discovered by Prof. Vanin in 1964 [ 1 , 2 , 3 , 4 ]. Later, continued investigations explored tetrahedral DNICs [(NO) 2 Fe(L) 2 ] as a natural and ubiquitous cofactor generated upon the interaction of nitric oxide (NO) with non-heme [Fe-S] proteins and cellular labile iron pools [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. Despite the identification of NO as an endothelium-derived relaxing factor (EDRF) [ 19 , 20 ], DNICs in a low-molecular-weight or protein-bound form were proposed as a “working form” of NO under physiological conditions [ 21 , 22 ].…”