Spectroelectrochemical insights into structural and redox properties of immobilized endonuclease III and its catalytically inactive mutant

- High electric fields, generated upon redox activation of DrEndoIII2, can alter its structural and thermodynamic properties.
- Desorption of ferrous DrEndoIII2 from the DNA substrate does not occur as the current mechanistic model proposes.
- Residues K132 and D150 are crucial for DrEndoIII2/DNA complex formation and ET.

Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII2). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex.

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