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 M. Mitchell, Younkyoo Kim, Michelle A. Pressler, Christos Georgiou, Gerald T. Babcock, James O. Alben, Shelagh Ferguson-Miller, Robert B. Gennis

Research output: Contribution to journalArticlepeer-review


The aa 3-type cytochrome c oxidase from Rhodobacter sphaeroides is closely related to eukaryotic cytochrome c oxidases. Analysis of site directed mutants identified the ligands of heme a, heme a 3, and Cu(B) [Hosler et al. (1993) J. Bioenerg. Biomembr. 25, 121-133], which have been confirmed by high-resolution structures of homologous oxidases [Iwata et al. (1995) Nature 376, 660; Tsukihara et al. (1995) Science 269, 1069; (1996) 272, 1136]. Since the protons used to form water originate from the inner side of the membrane, and the heme a 3-Cu(B) center is located near the outer surface, the protein must convey these substrate protons to the oxygen reduction site. Transmembrane helix VIII in subunit I is close to this site and contains several conserved polar residues that could function in a rate- determining proton relay system. To test this role, apolar residues were substituted for T352, T359, and K362 in helix VIII and the mutants were characterized in terms of activity and structure. Mutation of T352, near Cu(B), strongly decreases enzyme activity and disrupts the spectral properties of the heme a 3-Cu(B) center. Mutation of T359, below heme a 3, substantially reduces oxidase activity with only minor effects on metal center structure. Two mutations of K362, ~15 Å below the axial ligand of heme α 3, are inactive, make heme a 3 difficult to reduce, and cause changes in the resonance Raman signal specific for the iron-histidine bond to heme a 3. The results are consistent with a key role for T352, T359, and K362 in oxidase activity and with 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.

Original languageEnglish (US)
Pages (from-to)10776-10783
Number of pages8
Issue number33
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry

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