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. 2018 Sep 19;140(37):11800-11810.
doi: 10.1021/jacs.8b07362. Epub 2018 Sep 6.

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

Levi A Ekanger, Paul H Oyala, Annie Moradian, Michael J Sweredoski, Jacqueline K Barton

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

Levi A Ekanger et al. J Am Chem Soc. .

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)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.

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Figures

Figure 1.
Figure 1.
Structure of a protein-bound [Fe4S4]2+ cluster, dinitrosyl iron complex, Roussin’s red ester, and the Roussin’s red salt anion.
Figure 2.
Figure 2.
Spectroscopic characterization of EndoIII [Fe4S4] cluster nitrosylation. (Top) Representation of the conversion of native EndoIII into nitrosylated EndoIII by a 10 min exposure to NO (50 equiv). (Bottom left) UV−vis spectra of native (black) and nitrosylated (red) EndoIII revealing a 46 nm hypsochromic shift (inset). (Bottom middle) Representative results of the ferene colorimetric assay for native EndoIII (black), nitrosylated EndoIII (red), and a buffer blank (dotted line). Native EndoIII contains 4.00 ± 0.09 Fe per cluster, and this value decreased to 3.05 ±0.01 Fe for nitrosylated EndoIII. (Bottom right) CW EPR spectra of nitrosylated EndoIII before (red) and after (blue) a 30 min treatment with sodium dithionite (25 equiv) recorded at 60 K, microwave frequency of 9.3701 GHz, and microwave power of 0.204 mW. All experiments were performed in the absence of DNA and buffer was pH 7.5 and contained 20 mM phosphates, 150 mM NaCl, 0.5 mM EDTA, and 10% v/v glycerol.
Figure 3.
Figure 3.
X-band HYSCORE spectra and simulations of 14N hyperfine couplings. (Top left) X-band HYSCORE spectrum of nitrosylated EndoIII acquired at 341.8 mT (g = 2.036) corresponding to the [(Cys)2Fe(NO)2]− dinitrosyl iron complex. (Bottom left) nitrosylated EndoIII after treatment with dithionite acquired at 348.5 mT (g = 1.998) corresponding to the [(μ-Cys)2Fe2(NO)4]− reduced Roussin’s red ester. Left panels show the experimental spectra, with intensities indicated by colors ranging from blue to red in order of increasing intensity. The rightmost panels depict the experimental data in gray and overlay simulations from a relatively strongly coupled 14N nucleus (blue, denoted N1) and a relatively weakly coupled 14N nucleus (red, denoted N2). Specific simulation parameters are detailed in Table 1. Acquisition parameters: temperature = 20 K; microwave frequency = 9.740 GHz (a), 9.744 GHz (b); MW pulse lengths π/2, π = 8, 16 ns; τ = 138 ns (a), 138 ns (b); t1 = t2 = 100 ns; Δt1 = Δt2 = 16 ns; shot repetition time (srt) = 1 ms.
Figure 4.
Figure 4.
Deconvoluted mass spectra of (left) native (black) and nitrosylated (red) EndoIII and (right) nitrosylated EndoIII prepared using 14NO (solid) and 15NO (dotted).
Figure 5.
Figure 5.
Biophysical and electrochemical characterization of native and nitrosylated EndoIII. (Top left) Cyclic voltammograms of buffer (dotted), native EndoIII (black), and nitrosylated EndoIII (red) on a DNA-modified gold electrode. (Top middle) Results of electrophoretic mobility shift assays plotted as EndoIII-bound dsDNA as a function of EndoIII concentration. Open circles and error bars represent the mean and standard error (n = 5), respectively. Solid lines represent Hill function fitting used to calculate dissociation constants. (Top right) Circular dichroism spectra of buffer (dotted), native EndoIII (black), and nitrosylated (red) EndoIII. (Bottom left) Differential pulse voltammograms of background-subtracted native (black) and nitrosylated (red) EndoIII on an edge-plane graphite electrode. Samples used in circular dichroism and graphite electrochemistry experiments did not contain DNA. (Bottom right) Representation of a DNA-modified gold electrode surface with native EndoIII (left) and nitrosylated EndoIII (right). Small black lines represent mercaptohexanol used to passivate the gold electrode surface. DNA-bound redox activity is not observed for nitrosylated EndoIII despite having a similar DNA-binding affinity to native EndoIII.
Scheme 1.
Scheme 1.
Cluster Nitrosylation Followed by Reduction by Dithionite As Visualized through Conversions between EPR-Active and EPR-Silent States
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