Studies on critical proton pathway residues in the AA3-type cytochrome c oxidase from Rhodobacter sphaeroides

Aerobic organisms derive energy needed to sustain life from foodstuffs through the process of cellular respiration. Respiration is performed by a series of soluble and multi-subunit membrane proteins that form the 'electron-transport chain', found in the inner mitochondrial membrane of eukaryotes and the cytoplasmic membrane of aerobic prokaryotes. Cytochrome c oxidase, the terminal member of this respiratory chain, catalyzes the four-electron reduction of oxygen to water and uses the free energy of this reaction to translocates protons across the membrane. The oxidase accomplishes oxygen reduction by drawing electron and protons from the opposite sides of the membrane and transfers the pumped protons against the membrane potential through very specific routes within the enzyme. Several years of research have thrown some light on the electron and the proton-input pathways, catalytic cycle intermediates and the timing of the proton-pumping steps. However, the coupling of the oxygen chemistry to proton pumping is not yet understood at a molecular level and several grey areas like proton/water-exit channel, residues controlling the unidirectionality of the pump and a universal proton pumping mechanism are being actively researched. The focus of this thesis is on amino-acid residues in the catalytic subunit-I that are important to the delivery of protons to the active site for the reduction of oxygen and proton pumping. Chapter 2 describes the characteristics of mutations at the residue Met-107 near the critical proton transferring residue in the D-pathway, Glu-286. The aim of the study is to check if Met-107 has a role in assisting Glu-286 in the delivery of protons. Chapter 3 describes the characteristics of mutations at Glu-286 with respect to their effects on the environment of the active-site. This study aims to provide experimental evidence for the long range hydrogen-bonded connectivity between Glu-286 and the active site. Chapter 4 adds to the experimental evidence on the well studied residue Lys-362, for its critical role in transferring protons through the K-pathway. The question of the number of protons delivered through this pathway has been addressed in this chapter. Chapter 5 describes some of the conditions required for the formation of the unique covalent cross-link found at the active site of all heme-copper oxidases. We have also speculated on the radical chemistry at the active site that may have lead to its formation.