Title

Mutagenic and Spectroscopic Investigation of pH Dependent CooA DNA Binding

Author

Brian R. Weaver, Valparaiso University

Date of Award

Spring 2017

Project Type

Departmental Honors Paper/Project

College

Arts and Sciences

Department

Chemistry

First Advisor

Robert W. Clark

Abstract

The carbon monoxide (CO) sensing heme protein, CooA, is a transcription factor which exists in several bacteria that utilize CO as an energy source. CooA positively regulates the expression of coo genes in the presence of CO such that the corresponding proteins may metabolize CO. The present studies have yielded the unexpected result that Fe(III) CooA binds DNA tightly at pH < 7, deviating from all previously reported work which indicate that CooA DNA binding is initiated only when the exogenous CO effector reacts with the Fe(II) CooA heme. This observation suggests that the disruption of one or more salt bridges upon effector binding may be a critical feature of the normal CooA activation mechanism. To test this possibility, several protein variants that eliminated a selected salt bridge for the CooA homolog from Rhodospirillum rubrum were prepared via site-directed mutagenesis. Samples of these variant proteins, which were overexpressed in Escherichia coli, were then characterized by spectroscopic methods and functional assays to investigate the impact these mutations had on CooA heme coordination structure and DNA-binding activity. Results of this work are presented in light of the accepted CooA activation mechanism.

Recommended Citation

Weaver, Brian R., "Mutagenic and Spectroscopic Investigation of pH Dependent CooA DNA Binding" (2017). Chemistry Honors Papers. 2.
https://scholar.valpo.edu/chem_honors/2