Date of Award

5-2018

Document Type

Thesis campus only

Department

Chemistry

First Advisor

Laura Hunsicker-Wang

Abstract

The respiratory electron transport chain is the final set of steps in the production of adenosine tripohosphate (ATP), the main form of energy used by cells. The final complex in the chain, cytochrome c oxidase, couples electron transfer with proton translocation, which contributes to the electrochemical gradient that drives the production of ATP. The transferred electrons are then used to reduce molecular oxygen to water. CuA is the initial electron acceptor in cytochrome c oxidase. It consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine, and a carbonyl in the peptide backbone of a nearby glutamine. The two ligating histidines are of particular interest as their protonation state may affect the ability of the center to accept electrons, and they may be involved in the movement of protons through cytochrome c oxidase. To test for the presence of reactive ligating histidines, a portion of cytochrome c oxidase from the bacteria Thermus thermophilus that contains the CuA site (the TtCuA protein) was reacted with the chemical modifier diethyl pyrocarbonate and observed through UV-Visible and circular dichroism spectroscopies at pH 5.0-9.0. Additionally, the mutant protein with the non-ligating histidines removed (H40A/H117A) was similarly tested. Results from both proteins indicate that diethyl pyrocarbonate reacts with one of the two ligating histidines. The existence of the reactive ligating histidine suggests that this residue may play a role in modulating the electronic and redox properties of TtCuA through kinetically-controlled proton exchange with the solvent. Continuing studies aim to identify general characteristics of reactive ligating histidines through DEPC studies on the metalloproteins Sco and azurin, both of which contain a mononuclear copper center.

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