Spectroscopic Characterization of the Function and Mechanism of Dehaloperoxidase

Abstract

The research presented in this dissertation focused on the effects of substrate binding on dehaloperoxidase, (DHP). Using Resonance Raman, (RR), UV-Visible spectroscopy, (UV-VIS), electron spin resonance, (ESR) and cyclic voltammetry, (CV) techniques we have shown that substrate indeed does bind to DHP and, in doing so, the spin state of the iron heme is affected. The binding of substrate by DHP is significant since DHP has a globin fold, and traditional globins do not have a substrate binding sites. In addition to DHP's globin function, DHP is a peroxidase; thus it is capable of converting halogenated phenols to less halogenated quinones. The change in spin state of the heme iron which is observed when substrate binds to DHP indicates that the substrate binding acts as a trigger to switch DHP from a globin to a peroxidase. Using RR has shown that DHP has a spin state between those of horse heart myoglobin, (Mb) and horseradish peroxidase, (HRP) and it is this spin state which allows for the globin and peroxidase activities of DHP. ESR and X-band experiments were use to measure the zero field splitting parameters of DHP with and without bound substrate and from the X-band experiments it is clear that the binding of the substrate increases the population of the high-spin state of the iron in DHP. CV of the oxyDHP⁄deoxyDHP couple has a redox potential of +440 mV verses NHE. When substrate is bound to oxyDHP, the redox potential shifts to increasingly positive values as a function of substrate concentration. In spite of the aforementioned methods employed to measure the effects of substrate binding on DHP, no detectable difference in the Soret band is observed upon substrate binding.