Polar residues in Helix VIII of Subunit I of Cytochrome c Oxidase Influence the Activity and the Structure of the Active Site

Jonathan P. Hosler, James P. Shapleigh, David C. Mitchell, Younkyoo Kim, Michelle Pressler, Christos Georgiou, Gerald T. Babcock, James O. Alben, Shelagh Ferguson-Miller and Robert B.Gennnis Biochemistry 35 10776-10783 (1996)

SUMMARY: To test the role of several conserved polar residues in TM helix VIII in a rate-determining proton relay apolar residues were substituted for T352, T359 and K362 in the aa3 oxidase from Rhodobacter sphaeroides. Mutation of T352, near CuB, strongly decreases enzyme activity and disrupts the spectral characteristics of the a3-CuB center. Mutations of T359, below heme a3, substantially reduce activity with only minor effects on metal center structure. Two mutations of K362, approx. 1.5 nm below the axial ligand of heme a3, are inactive, make heme a3 difficult to reduce and cause changes in resonance Raman modes specific for the heme a3-histidine bond. The results are consistent with a key role for T352, T359 and K362 in oxidase activity and the involvement of T359 and K362 in proton transfer through a relay system now plausibly identified in the crystal structure. However the characteristics of the K362 mutants raise some questions about the assignment of this as the substrate proton channel.