Publication Date
2014
Document Type
Dissertation
Committee Members
Gerald M. Alter (Committee Member), Heather A. Hostetler (Committee Member), J. Ashot Kozak (Committee Member), Lawrence J. Prochaska (Advisor), Michael L. Raymer (Committee Member)
Degree Name
Doctor of Philosophy (PhD)
Abstract
Cytochrome c oxidase (COX) catalyzes the oxidation of ferrocytochrome c and the reduction of oxygen to water while concomitantly translocating protons across the inner mitochondrial membrane. The catalytic core of COX consists of three subunits that are conserved from the bacterial to the mitochondrial forms of the enzyme. Subunits I and II (SUI and SUII) contain the metal centers where electrons are transferred and oxygen binds for reduction. Subunit III (SUIII) does not contain any metals and has an unknown function. It contains three conserved histidine residues (3, 7 and 10) that are surface exposed and are in close proximity to the mouth of the D-channel, which delivers protons to both the catalytic and pump loading sites. Additionally, SUIII contains conserved binding sites for phospholipids. The loss of phospholipids decreases electron transfer activity; thus, they must have a functional and structural role in COX.
A triple histidine mutation (to glutamine) in SUIII was created in Rhodobacter sphaeroids, a bacterial model of the mitochondrion. SDS-PAGE shows that half of the enzyme lost SUIII. The mutant COX retained half of the wild-type electron transfer activity and exhibited turnover-induced suicide inactivation. Visible absorbance spectroscopy during steady-state turnover indicates that 20 % more electrons accumulate at heme a in the mutant. In addition, when reconstituted into liposomes, the mutant enzyme pumps protons at half the efficiency of wild-type. Our results indicate that although the mutation does not perturb the catalytic site, as verified by pH dependence, it slows electron transfer activity indirectly by slowing proton uptake through the D-channel. Taken together, the results show that the three histidine residues in SUIII stabilize the interactions between SUI and SUIII and serve as a proton collecting antenna for the entry point of the D-channel in SUI.
To study the function of the phospholipids, phospholipase A2 and detergent were used to extract them from COX. This was confirmed by measuring phosphate content using ICP-MS. Electron transfer activity was decreased 30-50 % and the enzyme exhibited suicide inactivation, both were reversible by the addition of exogenous lipids, most specifically by cardiolipin and long chain fatty acids. Limited proteolysis by a-chymotrypsin of the delipidated COX exhibited a faster digestion rate of SUI as compared to control. This indicates that a conformational change allowed SUI to be more labile for chymotrypsin digestion in the absence of lipids. COX was also labeled with a fluorophore (IAEDANS), which specifically links to SUIII. Fluorescence rotational rate, as measured by anisotropy, was faster in the delipidated COX, which suggests an increased flexibility in SUIII. Additionally, fluorescence energy transfer between IAEDANS and a fluorescently labeled cardiolipin revealed that cardiolipin binds in the v-shaped cleft of SUIII in the delipidated COX, and that it shifts closer to the active site during catalytic turnover. In conclusion, these results show that the phospholipids regulate events occurring during electron transfer activity by maintaining the structural integrity of the enzyme at the active site.
Page Count
246
Department or Program
Biomedical Sciences
Year Degree Awarded
2014
Copyright
Copyright 2014, some rights reserved. My ETD may be copied and distributed only for non-commercial purposes and may not be modified. All use must give me credit as the original author.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
