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wright1183979032.pdf (12.47 MB)
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Investigating the Role of Subunit III in the Structure and Function of Rhodobacter Sphaeroides Cytochrome C Oxidase
Author Info
Geyer, R. Ryan
Permalink:
http://rave.ohiolink.edu/etdc/view?acc_num=wright1183979032
Abstract Details
Year and Degree
2007, Doctor of Philosophy (PhD), Wright State University, Biomedical Sciences PhD.
Abstract
The role of subunit III (SIII) in cytochrome c oxidase structure and function was investigated using enzyme isolated from the bacterium Rhodobacter sphaeroides. Energy minimization calculations suggested that in the absence of SIII, subunit I (SI) adopted a more open conformation. This observation was tested through the use of limited proteolysis using, á-chymotrypsin. The results showed that in the absence of SIII the solution structures of wild-type and I/II oxidase were not significantly different, and that proteolysis occurred exclusively at the N and C-termini of SI. Upon inactivation of I/II oxidase by catalytic turnover, and subsequent digestion with the protease á-chymotrypsin it was concluded that SI underwent significant conformational changes. This change in conformation was probably due to CuB, located in the active site, dissociating from SI, upon turnover induced inactivation. A similar digestion pattern was observed in a mutant lacking CuB, which indicated that the large scale change in conformation was due to the loss of CuB from the active site. The functional role of SIII was also investigated by predicting and mutating interhelical hydrogen bonds within SIII. A cluster of amino acids consisting of Glu90, His212, and Tyr246 form a highly conserved triad of interhelical connectivity, perhaps linked by hydrogen bonds. Mutation of Glu90 to an alanine (E90A) resulted in a significant loss of SIII content, accompanied by a phenotype of turnover induced inactivation and reduced proton pumping efficiency, when reconstituted in phospholipid vesicles. Mutation of His212 to either an alanine or phenyalanine (H212A and H212F) resulted in wild-type expression of SIII and when reconstituted into a phospholipid vesicle, these mutants exhibited turnover induced inactivation as well as reduced proton pumping efficiency. Mutation of Tyr246 to a phenyalanine (Y246F) resulted in a mutant that was indistinguishable from wild-type enzyme. Therefore, it was concluded that Glu90 and His212 were involved in an interhelical hydrogen bond, and Y246 did not participate in this interaction. The functional data also suggests that the Glu90-His212 interaction may function to regulate electron transfer activity under certain conditions. In a second study the C-terminal helix of SIII, helix 7 was deleted from SIII via mutagenesis.
Committee
Lawrence Prochaska (Advisor)
Pages
269 p.
Subject Headings
Chemistry, Biochemistry
Keywords
membrane protein
;
limited proteolysis
;
site-directed mutagenesis
;
mass spectrometry
;
heme protein
;
cytochrome c oxidase
;
stopped flow spectroscopy
;
absorbance spectroscopy
Recommended Citations
Refworks
EndNote
RIS
Mendeley
Citations
Geyer, R. R. (2007).
Investigating the Role of Subunit III in the Structure and Function of Rhodobacter Sphaeroides Cytochrome C Oxidase
[Doctoral dissertation, Wright State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=wright1183979032
APA Style (7th edition)
Geyer, R..
Investigating the Role of Subunit III in the Structure and Function of Rhodobacter Sphaeroides Cytochrome C Oxidase.
2007. Wright State University, Doctoral dissertation.
OhioLINK Electronic Theses and Dissertations Center
, http://rave.ohiolink.edu/etdc/view?acc_num=wright1183979032.
MLA Style (8th edition)
Geyer, R.. "Investigating the Role of Subunit III in the Structure and Function of Rhodobacter Sphaeroides Cytochrome C Oxidase." Doctoral dissertation, Wright State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=wright1183979032
Chicago Manual of Style (17th edition)
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Document number:
wright1183979032
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Copyright Info
© 2007, all rights reserved.
This open access ETD is published by Wright State University and OhioLINK.