Site-Directed Mutagenesis of Highly Conserved Residues in Helix VIII of Subunit I of the Cytochrome bo Ubiquinol Oxidase from Escherichia coli: An Amphipathic Transmembrane Helix That May Be Important in Conveying Protons to the Binuclear Center

Cytochrome bo from Escherichia coli is a ubiquinol oxidase which is a member of the superfamily of heme-copper respiratory oxidases. This superfamily, which includes the eukaryotic cytochrome c oxidases, has in common a bimetallic center consisting of a high-spin heme component and a copper atom (Cu_B) which is the site where molecular oxygen is reduced to water. Subunit I, which contains all the amino acid ligands to the metal components of the binuclear center, has 15 putative transmembrane spanning helices, of which 12 are common to the entire superfamily. Transmembrane helix VIII has been noted to contain highly conserved polar residues that fall along one face of the helix. These residues could, in principle, be important components of a pathway providing a conduit for protons from the cytoplasm to gain access to the binuclear center. These conserved residues include Thr352, Thr359, and Lys362. In addition, Pro358, in the middle of this transmembrane helix, is totally conserved in the superfamily. Some substitutions for Thr352 (Ala, Asn) result in major perturbations at the binuclear center as judged by the low-temperature Fourier transform infrared (FTIR) absorbance difference spectroscopy of the CO adducts. Whereas Thr352Ala is inactive enzymatically, both Thr352Asn and Thr352Ser have substantial activity. Substitutions for Thr359 (Ala or Ser) also do not perturb the spectroscopic properties of the binuclear metal center, but the Thr359Ala mutant is devoid of enzyme activity. Changing the neighboring Pro358 to Ala has no detectable effect on the properties of the oxidase. However, all substitutions for Lys362 (Leu, Met, Gln, or Arg) are inactive. Whereas the Lys362Met has a wild-type FTIR spectrum of the CO adduct, the Lys362Gln shows substantial perturbation to the metal center. In summary, the data suggest that residues in helix VIII are in the immecdiate vicinity of the binuclear center and show that some of the polar residues within helix VIII are functionally essential, independent of any role in maintaining the structural integrity of the metal centers. A distinct possibility is that these residues are important components of a proton and/or water conducting channel from the cytoplasmic surface to the site where oxygen is reduced to water at the heme-copper center.