Glutamates 99 and 107 in transmembrane helix III of subunit I of cytochrome bd are critical for binding of the heme b595-d binuclear center and enzyme activity

Cytochrome bd is a quinol oxidase of Escherichia coli under microaerophilic growth conditions. Coupling of the release of protons to the periplasm by quinol oxidation to the uptake of protons from the cytoplasm for dioxygen reduction generates a proton motive force. On the basis of sequence analysis, glutamates 99 and 107 conserved in transmembrane helix III of subunit I have been proposed to convey protons from the cytoplasm to heme d at the periplasmic side. To probe a putative proton channel present in subunit I of E. coli cytochrome bd, we substituted a total of 10 hydrophilic residues and two glycines conserved in helices I and III-V and examined effects of amino acid substitutions on the oxidase activity and bound hemes. We found that Ala or Leu mutants of Arg9 and Thr15 in helix I, Gly93 and Gly100 in helix III, and Ser190 and Thr194 in helix V exhibited the wild-type phenotypes, while Ala and Gln mutants of His126 in helix IV retained all hemes but partially lost the activity. In contrast, substitutions of Thr26 in helix I, Glu99 and Glu107 in helix III, Ser140 in helix IV, and Thr187 in helix V resulted in the concomitant loss of bound heme b(558) (T187L) or b(595)-d (T26L, E99L/A/D, E107L/A/D, and S140A) and the activity. Glu99 and Glu107 mutants except E107L completely lost the heme b(595)-d center, as reported for heme b(595) ligand (His19) mutants. On the basis of this study and previous studies, we propose arrangement of transmembrane helices in subunit I, which may explain possible roles of conserved hydrophilic residues within the membrane.