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

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. Metalloproteins containing Fe-S clusters constitute an important group due to several important functions including electron transfer, H 2 evolution, regulation, and catalysis 1,2. In particular, there are proteins actively involved in the base excision repair (BER) pathway, interacting with DNA for the search of oxidized/damaged bases and its consequently excising them from the genome 3-5. The E. coli DNA glycosylases EndoIII and MutY are homologues and are involved in the removal of damaged pyrimidines and the removal of mismatched adenine from the A:8-oxoguanine (8-oxoG) pair, respectively 6,7. Among the six different superfamilies of DNA glycosylases, EndoIII, a bifunctional DNA glycosylase, was the first-discovered enzyme. It belongs to one of the two superfamilies, called helix-hairpin-helix (HhH), which contain Fe-S clusters 7,8. This name was assigned on the basis of the secondary structural element present in these enzymes that is essential for DNA binding 9. Other members of this superfamily include MutY, Endonuclease III-like protein 1 (hNTHL1), human MutY homolog DNA glycosylase (MUTYH), and 3-methyladenine glycosylase II (AlkA) 10,11. Earlier characterization revealed that both EndoIII and MutY contain [4Fe4S] clusters 12 ; initially, however, it was not easy to show that the cluster is redox active under physiological conditions, and hence it was basically assigned a structural role 13. But the quest to prove that these DNA glycosylases detect a single damaged base within an enormous number of normal bases and the specific role of Fe-S clusters in these processes has led to several important revelations abou...

Radiation-Induced Effect on Spin-Selective Electron Transfer through Self-Assembled Monolayers of ds-DNA

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Infrared Nanospectroscopy Reveals DNA Structural Modifications upon Immobilization onto Clay Nanotubes

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