Use of mutagenesis, novel detergents, and lipid analysis to optimize conditions for achieving high resolution 3-dimensional crystal structure of Rhodobacter sphaeroides cytochrome c oxidase

Aerobic respiration is the most common means by which non-photosynthetic organisms convert carbohydrates, fats and proteins, into a biologically useful form of energy. In this process, electrons are released and passed through a series of enzymes known as the respiratory electron transport chain. This process is coupled to proton translocation from the mitochondrial matrix to the intermembrane space, leading to generation of a membrane potential, which can be used to drive the synthesis of ATP.; In mammalian systems the ability to produce heat, instead of ATP, and to respond to signals of too much stored energy, are important physiological functions. In the case of cytochrome c oxidase, knowing the proton and electron pathways and the mechanism of coupling may lead us to an understanding of how oxidase can be uncoupled, and thus contribute to the controlled production of heat instead of stored energy (fat). Understanding the mechanisms of electron transfer and proton pumping at a molecular level requires a structure at high resolution.; Rhodobacter sphaeroides cytochrome c oxidase was used as a good model for the mitochondrial enzyme, and many efforts were made to optimize conditions for achieving a high resolution structure. Different purification protocols were used, and the enzyme purified with Ni 2+-NTA affinity chromatography followed by Mono Q ion-exchange chromatography was the most suitable for crystallization.; In order to standardize the crystallization conditions, associated membrane lipids were analyzed by thin layer chromatography, mass spectrometry and phosphate analysis. PE, PC, PG, sulfolipid and ornithine lipids were found in the membranes and at all purification steps. The two major lipid components in R.s. cells, PE and PC, were also found in the re-dissolved crystal. These results suggest the importance of these membrane lipids, and their retention, for getting a high resolution structure.; As another approach to improve the crystallization of cytochrome c oxidase, the protein was stabilized and the hydrophilic surface was increased by forming a covalently linked Cc/CcO complex. For this study R.s. cytochrome c₂ was engineered by putting in the missing lysine 13 that is found to be important for high affinity binding. This step improved the binding between cytochrome c and the oxidase significantly. Site-directed mutagenesis was used to form cysteine mutants on cytochrome c to cross-linked with cysteine mutants on subunit II of the oxidase. These mutations were made based on a computational model of the binding site between cytochrome c and cytochrome c oxidase. Purification and yield of cytochrome c mutants was improved significantly when we introduced a 5 histidine-tag at the C-terminus of cytochrome c and used Ni2+-NTA affinity chromatography as a purification system. All mutants have wild type characteristics and the formation of the complex was accomplished.; The results of these studies further our understanding of the nature of the Cc/CcO complex and define the lipid content of the purified oxidase through to the crystal form. The lipid content of the purified enzyme is representative of the R.s. membranes, suggesting the importance of a number of different lipids in the native structure.