Studies of Cytochrome C Oxidases From Rhodobacter Sphaeroides and Vibrio Cholerae
Han, Huazhi
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https://hdl.handle.net/2142/87914
Description
Title
Studies of Cytochrome C Oxidases From Rhodobacter Sphaeroides and Vibrio Cholerae
Author(s)
Han, Huazhi
Issue Date
2010
Doctoral Committee Chair(s)
Gennis, Robert B.
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
Cytochrome c oxidase is the terminal enzyme to accept electrons from cytochrome c in the respiratory chain of mitochondria, bacteria and archaea. It couples the reducing oxygen to water and pumping the protons across the membrane. The second largest group, C-type, is also known as cbb3 cytochrome c oxidase which is almost exclusively found in the Proteobacteria. All the current studies show that cytochrome cbb3 oxidases apply quite distinct mechanism for receiving electrons and pumping protons. The work in this thesis clearly demonstrates that B- and C-type oxidases have less energy coupling efficiency and share a similar pumping mechanism other than A-type family members. Subunits CcoO and CcoP in cbb 3--type oxidase comprise of one and two heme c respectively. The binding motif for heme c is characterized to be the sequence of CXXCH. Site-directed mutagenesis studies of the various combined mutants of the motif show that the cysteine and histidine residues are critical for the incorporation of heme c into cbb3 enzymes from Rhodobacter sphaeroides and Vibrio cholerae. Results also show that every heme c is involved in the electron translocations in a consequential way and is important for the activity and stability of the enzyme complex. Sequence alignment and structural modeling have shown that cbb 3--type oxidase contains only one proton channel (K-channel analogue) which consists of its own conserved residues. The putative K-channel related residues were tested by site-directed mutagenesis and all shown to be important for catalytic activity. ICP-OES analysis in this study demonstrates that only two calcium ions exist as the redox inactive metal groups. One identified Ca2+ connects subunit CcoN and CcoO and bridges heme b and heme b3 through the propionate groups. Site-directed mutagenesis studies of the binding residues E180 (subunit CcoN) and S105 (subunit CcoO, R. sphaeroides numbering) indicate that they are essential for the activity of the enzyme and recruitment of both calcium ions and CuB. Moreover, mutagenesis study of another conserved nearby residue E183 suggests that this acidic residue is also required for the correct incorporation of calcium and copper ions and necessary for the fully functional enzyme.
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