Date of Award

2006

Degree Name

Chemistry

College

College of Science

Type of Degree

M.S.

Document Type

Thesis

First Advisor

Jaroslava Miksovska

Second Advisor

William Price

Third Advisor

Leslie Frost

Abstract

Lactoperoxidase (LPO) is a heme peroxidase found in tears, milk, saliva, and cervical fluids that has bactericidal activity. The bactericidal nature of LPO is due to its ability to oxidize halides and pseudohalides in the presence of H2O2. Full understanding of the catalytic cycle of LPO requires detailed characterization of the mechanism of ligand binding to the enzyme active site. This includes a dynamic and energetic description of conformational changes associated with heme-ligand interactions. Photoacoustic calorimetry is applied to determine the kinetics and magnitude of reaction volume and enthalpy changes for CO dissociation from CO-LPO. Time-resolved absorption spectroscopy was used to determine activation enthalpy and entropy of photodissociation of CO from CO-LPO as well. UV-visible spectroscopy indicated that, upon reduction of LPO with sodium dithionite, an intermediate state (Fe(II)LPOintermediate) forms, that biphasically relaxes to a final reduced state (Fe(II)LPOfinal). We show that the rate constant for conversion of Fe(II)LPOintermediate to Fe(II)LPOfinal is pH dependant and propose that Fe(II)LPOintermediate exists in two conformations. The conformation at neutral pH exhibits a fast conversion, where as at high pH the conformation with slow conversion is populated. We speculate that the protonation state of Arg372, that is located in the heme-binding pocket, may control the conversion rate. Transient absorption spectroscopy results show that CO rebinding to Fe(II)LPO, is pH dependant, with CO rebinding faster at higher pH. At pH=4.0 and 7.0, the rate constant of recombination of CO to LPO is 2.7x104 M-1·s-1 and 4.9x104 M-1·s-1, respectively. In 100 mM CAPS buffer (pH=10.0) recombination is biphasic with the fast rate constant 8.4x104 M-1·s-1 and the slow rate constant 1.3x104 M-1·s-1. Photoacoustic calorimetry results show that photodissociation of CO from Fe(II)LPO is exothermic at neutral pH (ΔH=-16.3±4.6kcal·mol-1) and acidic pH (ΔH=-12.5 ± 5.5kcal·mol-1), with volume change of ~3ml·mol-1. Photolysis of CO-Fe at pH=10.0 and in 500 mM NaCl buffers (pH=7.0) is an endothermic process with a change in volume of ~3 ml·mol-1. The small volume changes observed suggest that, in addition to photolysis of the CO-Fe bond and solvation of the CO molecule, other processes contribute to the observed volume and enthalpy changes. It is likely that dissociation of CO is associated with breakage of a salt bridge between Arg557 and the ring-D propionate group. Changes in the protein charge distribution are likely to cause electrostriction and therefore results in the smaller volume change. This is confirmed by the fact that, at pH=10.0 and 500 mM NaCl buffer (pH=7.0), the observed enthalpy change corresponds to the enthalpy of the CO-Fe bond whereas the enthalpy change is exothermic at pH=4.0 and 7.0.

Subject(s)

Heme.

Halides.

Peroxidase.

Chemistry, Physical and theoretical.

Photodissociation.

Photochemistry.

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