Biochemical Studies on the Catalytic Cycle of Cytochrome C Oxidase in Rhodobacter Sphaeroides

134 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2000.Cytochrome c oxidase from Rhodobacter sphaeroides exhibits properties similar to those of mitochondrial bovine oxidase. The X-ray crystal structure of cytochrome c oxidase from Paracoccus denitrificans reveals two proton channels leading to the binuclear center. (Iwata, S., Ostermeier, C., Ludwig, B., & Michel, H. (1995) Nature 376, 660--669) One channel is referred to as the K-channel after the highly conserved lysine-362 located within it. The other channel is similarly referred to as the D-channel after its highly conserved aspartic acid-132 located at its entrance. The mutation of K362 to methionine inhibited reduction of the binuclear center and proton uptake. It has been shown that the K-channel is responsible for delivering protons to the binuclear center upon reduction during the first step in the catalytic cycle. Peroxide was used as a tool to investigate intermediates in the second half of the catalytic cycle. It was also shown that a proton from the K-channel was responsible for determining which spectroscopic intermediates get formed upon reaction with peroxide at low pH. Residues around the binuclear center were characterized with Electron Paramagnetic Resonance spectroscopy and resonance Raman spectroscopy. The role of tyrosine-288 was discussed in terms of the presence of a covalent linkage between Y288 and H284 and the formation of a tyrosyl radical species in the catalytic cycle of cytochrome c oxidase.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD