Publication Date

2007

Document Type

Dissertation

Committee Members

Lawrence Prochaska (Advisor)

Degree Name

Doctor of Philosophy (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.

Page Count

269

Department or Program

Biomedical Sciences

Year Degree Awarded

2007

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