A combined Far-FTIR, FTIR Spectromicroscopy, and DFT Study of the Effect of DNA Binding on the [4Fe4S] Cluster Site in EndoIII

Ayaz Hassan,Filipe C D A Lima,João C P De Souza,Lucyano J A Macedo,Frank N Crespilho

doi:10.1038/s41598-020-58531-4

Ayaz Hassan, Filipe C D A Lima + Show 3 more

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https://doi.org/10.1038/s41598-020-58531-4

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Abstract

Endonuclease III (EndoIII) is a DNA glycosylase that contains the [4Fe4S] cluster, which is essential for the protein to bind to damaged DNA in a process called base excision repair (BER). Here we propose that the change in the covalency of Fe–S bonds of the [4Fe4S] cluster caused by double-stranded (ds)-DNA binding is accompanied by a change in their strength, which is due to alterations of the electronic structure of the cluster. Micro-FTIR spectroscopy in the mid-IR region and FTIR spectroscopy in the far IR (450 and 300 cm−1) were used independently to study the structural changes in EndoIII and the behavior of the [4Fe4S] cluster it contains, in the native form and upon its binding to ds-DNA. Structural changes in the DNA itself were also examined. The characteristics vibrational modes, corresponding to Fe–S (sulfide) and Fe–S (thiolate) bonds were identified in the cluster through far IR spectroscopy as well through quantum chemistry calculations. Based on the experimental results, these vibrational modes shift in their spectral positions caused by negatively charged DNA in the vicinity of the cluster. Modifications of the Fe–S bond lengths upon DNA binding, both of the Fe–S (sulfide) and Fe–S (thiolate) bonds in the [4Fe4S] cluster of EndoIII are responsible for the stabilization of the cluster towards higher oxidation state (3+), and hence its redox communication along the ds-DNA helix.

Highlights

Metalloproteins containing Fe–S clusters constitute an important group due to several important functions including electron transfer, H2 evolution, regulation, and catalysis[1,2]
FTIR imaging allowed us to spectroscopically examine Endonuclease III (EndoIII) and Double-stranded DNA (ds-DNA) with a lateral resolution that was only limited by the wavelength-dependent diffraction of the IR light, with a pixel resolution of about 2.5 μm in the reflectance mode, making it possible to work with a minute amount of sample[29,30]
We have evaluated the vibrational properties of the [4Fe4S] cluster and other parts of the EndoIII enzyme, with and without its interaction with ds-DNA, using FTIR spectroscopy in the mid- and far-IR

Summary

Introduction

Metalloproteins containing Fe–S clusters constitute an important group due to several important functions including electron transfer, H2 evolution, regulation, and catalysis[1,2]. Solomon and co-workers reported that the presence of DNA in the proximity of the [4Fe4S] cluster destabilized the S 3p orbital energy and increased the covalency (α2) of the Fe–S bond by increasing the S 3p character in the metal 3d antibonding orbital (Ψ*) This increase in the Fe–S bond covalency stabilizes the oxidized state of the cluster more than the reduced state, decreasing the redox potential as previously mentioned. Far infrared (FIR) spectroscopy (below 500 cm−1) is a technique to elucidate the structural properties of compounds whose bonds vibrate at low frequency (energy); in particular, in the structural study of inorganic complexes, information about metal-ligand bond vibrations may be obtained[21] This technique has received great attention in recent years as a probe of the redox co-factor of Fe–S cluster-containing metalloenzymes due to the rich chemical information contained in these metal centers[22]

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