Gerald Alter (Committee Member), Lawrence Prochaska (Advisor), Yong-jie Xu (Committee Member)
Master of Science (MS)
Cytochrome c oxidase (COX) is the final electron acceptor in mitochondrial respiratory chain and in many bacterial species including Rhodobacter sphaeroides. Electron transfer is coupled with the pumping of protons across the membrane. Previous work has shown that reaction of beef COX with dicyclohexylcarbodiimide (DCCD) resulted in an inhibition of proton translocation by covalently binding to the conserved amino acid residue E90 located in a nonpolar region of subunit III (SIII). E90 is involved in a bonding pair with another conserved residue H212, possibly connected by a salt bridge or a hydrogen bond in the three dimensional structure of SIII. Our goal was to test whether the retention of the E90-H212 linkage and the spatial arrangements of these amino acid residues were critical for electron transfer and proton pumping activities of the enzyme. This work analyzes the functional role of these amino acids through the creation of three mutants in SIII-H212E, E90H, and E90H/H212E. SDS-PAGE verified all mutants contained SI-III. The first mutant, H212E, bacteria cultures grew significantly faster as compared to wild-type; while the other two mutant culture grew at comparable rates to wild-type. Additionally, the visible absorbance spectrum of H212E mutation in bacterial membranes showed little or no heme aa3 oxidase expression while the other two mutants exhibited properties similar to wild-type. Conversely, the spectrum of isolated and purified COX-SIII E90H mutant protein displayed a red shift while the double mutation COX-III E90H/H212E resembled that of wild-type. Electron transfer activity of two purified mutant proteins E90H and E90H/H212E revealed decreases in steady-state activities approximately 40% and 15% respectively. Lastly, the two mutants displayed a slight alkaline shift in pKa value for electron transfer activity. In summary, these results imply that the absolute positions of E90 and H212 are essential to COX activity. The single mutations resulting in two like charges caused changes in COX activity whereas the double mutation that retained the native salt bridge did not. Additionally, the increase in pKa may suggest the environment around the active site is perturbed, which is reflected by the decrease in electron transfer activity.
Department or Program
Department of Biochemistry and Molecular Biology
Year Degree Awarded
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