Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA

Abstract

Escherichia coli endonuclease III (EndoIII) and MutY are DNA glycosylases that contain [4Fe4S] clusters and that serve to maintain the integrity of the genome after oxidative stress. Electrochemical studies on highly oriented pyrolytic graphite (HOPG) revealed that DNA binding by EndoIII leads to a large negative shift in the midpoint potential of the cluster, consistent with stabilization of the oxidized [4Fe4S]³⁺ form. However, the smooth, hydrophobic HOPG surface is nonideal for working with proteins in the absence of DNA. In this work, we use thin film voltammetry on a pyrolytic graphite edge electrode to overcome these limitations. Improved adsorption leads to substantial signals for both EndoIII and MutY in the absence of DNA, and a large negative potential shift is retained with DNA present. In contrast, the EndoIII mutants E200K, Y205H, and K208E, which provide electrostatic perturbations in the vicinity of the cluster, all show DNA-free potentials within error of wild type; similarly, the presence of negatively charged poly-l-glutamate does not lead to a significant potential shift. Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the [4Fe4S] cluster, helping to explain why all DNA-binding proteins with [4Fe4S] clusters studied to date have similar DNA-bound potentials.

Figure 1

Representative CVs from EndoIII and MutY thin-films on a PGE electrode. A thin film containing only 75 μM EndoIII capped with Nafion gave a quasi-reversible signal with reductive and oxidative peaks centered, respectively, at 74 ± 11 and 162 ± 20 mV versus NHE (center; red trace). Notably, the addition of CNTs substantially amplified the signal, simplifying quantification (center; blue trace). Similarly, 25 μM MutY in the presence of CNTs yielded a signal with reductive and oxidative peaks at 100 ± 9.0 and 162 ± 3.0 mV versus NHE (bottom). CV scans were taken at a scan rate of 100 mV/s.

Figure 2

EndoIII and MutY thin-film voltammetry on a PGE electrode in the presence of DNA. By SQWV, the presence of DNA resulted in a −65 mV shift in the potential of 75 μM EndoIII (center), with a similar result for 25 μM MutY (bottom). Signals were very small due to surface passivation, but they were still readily apparent by SQWV. Unfortunately, CNTs led to inconsistent and unstable signals, likely interfering with DNA binding, so they could not be used to enhance the signals. Thin films were formed from several layers of a pre-mixed 1:1 protein/DNA solution, and were capped with Nafion. SQWV scans were taken at a frequency of 15 Hz with 0.025 V amplitude, and scans were from positive to negative potentials (indicated by the arrow).

Figure 3

Thin-film voltammetry of WT EndoIII with poly-L glutamate and comparison of WT with the mutants E200K, Y205H, and K208E. Unlike DNA, poly-L glutamate caused no significant potential shift even at 6 mM glutamate (top). 75 μM WT and mutant EndoIII in protein/CNT/Nafion thin films had nearly identical potentials, with CV peaks centered around 125 mV versus NHE (center); the similarity is even more apparent by DPV (bottom). Small shifts within the measurement error (10–15 mV) are still possible, but pale in comparison to the effect of DNA. All CVs shown were taken at 100 mV/s, while DPVs were taken at an amplitude of 0.05 V with a 0.5 s pulse period. Poly-L glutamate was pre-incubated with 75 μM EndoIII both with and without CNTs; due to the larger signals, the CVs shown are from films including CNTs.