Crystallographic and protein engineering studies of the structure and function of cytochrome c oxidase from Rhodobacter sphaeroides

Cytochrome c oxidase (CcO) is the terminal enzyme of mitochondrial and bacterial respiratory chains. It accepts electrons from cytochrome c and reduces oxygen to water. Driven by this process, protons are translocated across the mitochondrial inner membrane of eukaryotes or the periplasmic membrane of prokaryotes, forming a transmembrane proton gradient that is used for the synthesis of ATP. Cytochrome c oxidase from Rhodobacter sphaeroides (RsC cO), a homologue of mammalian CcO, is used as the research model for structural and functional studies in this thesis.;A RsCcO-EYFP (Enhance Yellow Fluorescent Protein) fusion protein was created to increase the hydrophilic portion of CcO and form better crystal contacts. Activity and proton pumping assays showed that the fusion protein was fully active. Reconstituted vesicle results also showed that the pH-sensitive EYFP in this fusion protein could be used as a pH indicator for the study of the proton pumping mechanism. Two mutants of RsCcO with different shortened subunit I C-termini were constructed and crystallized. The 16 residue deletion mutant (PJL33) showed lower activity at high pH and weak proton pumping, suggesting that the deletion affected the stability of the subunit III interaction with the enzyme. The crystal structure of PJL33 (2.10 A) also showed a conformational change at the new C-terminus, which could contribute to the activity changes. The 6 residue deletion mutant (PJL49) showed normal activity except a diminished proton pumping function. The crystal of PJL49 diffracted to 2.5 A. In neither case did the removal of flexible regions result in higher resolution four subunit crystals, as hoped. High resolution crystal structures were obtained of the two mutants that define the proton uptake pathways in RsC cO, D132A and K362M. In the oxidized crystal of D132A (2.15A), the mutation caused no change in overall structure, but a localized conformational change in the D132 region. A chloride ion replaced the D132 carboxyl position and the backbone of residues 130 to 135 shifted, causing a conformational change of N207 and loss of its bound water. These changes could block proton uptake in the D-pathway and account for the strong inhibition. The oxidized crystal structure of the proton path mutant, K362M (2.30 A), showed no significant overall structural changes, in spite of major inhibition, except for the loss of two waters adjacent to K362 and T359. This result supports the critical role for water molecules in proton uptake. Crystal structures of reduced forms of both K362M (2.50 A) and D132A (2.15 A) showed similar conformational changes as seen in WT, supporting the importance of redox-induced conformational effects. Spectra taken during X-ray radiation of the oxidized crystals showed reduction of the metal centers, but indicated a strained configuration that only relaxes to a native reduced form upon annealing. The results explain the ability to observe conformational differences between oxidized and reduced crystal forms.