Date of Award

December 2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Peter Geissinger

Second Advisor

Jorg C. Woehl

Committee Members

Dennis W. Bennett, Nicholas S. Silvaggi, David N. Frick

Keywords

Electric Field, Heme Proteins, Hole-burning Spectroscopy, Perturbation Theory, Porphyrins, Stark Spectroscopy

Abstract

Heme proteins have diverse functions as well as varied structures but share the same organic, conjugated cofactor. Similarly varied approaches have been taken to deduce how heme can take on different roles based on its protein environment. A unique approach is to view the protein matrix as a constellation of point charges that generates a defined, reproducible, net internal electric field that has influence over the electronic properties of the heme cofactor. This work considers how porphyrins, the basic chromophore building block of heme, can be used as a native spectroscopic sensor of internal electric field at the active site of heme proteins.

First, a number of approaches to model the electrostatic nature of protein structure are described. One approach based on Coulomb’s law is used to estimate the net electric field in myoglobin, easily placing the internal electric field on the order of MV/cm.

A closer inspection of myoglobin structure reveals that slight changes in position or strategic mutations can cause appreciable change in the field magnitude and direction. Then, the idea of a porphyrin probe is further developed, followed by a theoretical and spectral characterization of porphyrins substituted into heme proteins for use in emission spectroscopy as non-emissive heme must be replaced by other porphyrin analogs with higher quantum yield.

Once the porphyrin–protein system has been established as the guest–host system of interest, the hole-burning Stark spectroscopy method was used to quantitatively measure the magnitude and direction of the internal electric field vector generated by the protein. The collected Stark spectra had a more established classical analysis method for analysis, but a major aspect of this work is a quantum-mechanical analysis method that has been advanced for more practical and widespread usage. This novel quantum-mechanical approach to the method has promise for greater accuracy for internal electric field determination as well as the ability to resolve the field into spatial components in order to determine not just field magnitude but also direction. The results from the new analysis of experimental data for myoglobin of the in-plane components of the field places both at 1.7 MV/cm. Finally, two ab initio excited-state methods, CIS and TDDFT, were used to calculate electronic state energies and transition dipole moment values in support of this new quantum-mechanical analysis method. The two methods are described thoroughly with presentation of benefits and drawbacks to each method.

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