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 su perfamily, which includes the eukaryotic cytochrome c oxidases, has in common a bimetallic center consisting of a high-spin heme component a nd a copper atom (Cu(B)) which is the site where molecular oxygen is r educed to water. Subunit I, which contains all the amino acid ligands to the metal components of the binuclear center, has 15 putative trans membrane spanning helices, of which 12 are common to the entire superf amily. Transmembrane helix VIII has been noted to contain highly conse rved polar residues that fall along one face of the helix. These resid ues could, in principle, be important components of a pathway providin g a conduit for protons from the cytoplasm to gain access to the binuc lear center. These conserved residues include Thr352, Thr359, and Lys3 62. 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 jud ged by the low-temperature Fourier transform infrared (FTIR) absorbanc e difference spectroscopy of the CO adducts. Whereas Thr352Ala is inac tive enzymatically, both Thr352Asn and Thr352Ser have substantial acti vity. Substitutions for Thr359 (Ala or Ser) also do not perturb the sp ectroscopic properties of the binuclear metal center, but the Thr359Al a mutant is devoid of enzyme activity. Changing the neighboring Pro358 to Ala has no detectable effect on the properties of the oxidase. How ever, all substitutions for Lys362 (Leu, Met, Gln, or Arg) are inactiv e. Whereas the Lys362Met has a wild-type FTIR spectrum of the CO adduc t, the Lys362Gln shows substantial perturbation to the metal center. I n summary, the data suggest that residues in helix VIII are in the imm ediate vicinity of the binuclear center and show that some of the pola r residues within helix VIII are functionally essential, independent o f any role in maintaining the structural integrity of the metal center s. A distinct possibility is that these residues are important compone nts of a proton and/or water conducting channel from the cytoplasmic s urface to the site where oxygen is reduced to water at the heme-copper center.