The cytochrome ba ₃ oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping

Abstract

The heme-copper oxygen reductases are redox-driven proton pumps that generate a proton motive force in both prokaryotes and mitochondria. These enzymes have been divided into 3 evolutionarily related groups: the A-, B-and C-families. Most experimental work on proton-pumping mechanisms has been performed with members of the A-family. These enzymes require 2 proton input pathways (D-and K-channels) to transfer protons used for oxygen reduction chemistry and for proton pumping, with the D-channel transporting all pumped protons. In this work we use site-directed mutagenesis to demonstrate that the ba 3 oxygen reductase from Thermus thermophilus, a representative of the B-family, does not contain a D-channel. Rather, it utilizes only 1 proton input channel, analogous to that of the A-family K-channel, and it delivers protons to the active site for both O 2 chemistry and proton pumping. Comparison of available subunit I sequences reveals that the only structural elements conserved within the oxygen reductase families that could perform these functions are active-site components, namely the covalently linked histidinetyrosine, the Cu B and its ligands, and the active-site heme and its ligands. Therefore, our data suggest that all oxygen reductases perform the same chemical reactions for oxygen reduction and comprise the essential elements of the proton-pumping mechanism (e.g., the proton-loading and kinetic-gating sites). These sites, however, cannot be located within the D-channel. These results along with structural considerations point to the A-propionate region of the active-site heme and surrounding water molecules as the proton-loading site.cytochrome c oxidase ͉ respiratory enzymes

Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3fromThermus thermophilus

“…1), and then into the bulk aqueous phase. Finally, the structurally conserved water bridging propionate-A and propionate-D of the active-site heme have also been proposed to be the proton loading site (22,23), which is the model most favored by the results presented here.…”

“…Essentially, one is left with the six histidine ligands to the metals, the four propionate side chains of the hemes, and conserved internal water molecules (23). Several models of proton pumping have focused on one or more of the histidine ligands to Cu B (24)(25)(26)(27)(28)(29), but no compelling evidence has emerged.…”

“…Propionate-A of the active-site heme remains a viable candidate, and has been suggested based on several indirect lines of evidence (8,23,28,33). From measurements of the time-resolved generation of the transmembrane potential of the aa 3 -type oxygen reductase from Paracoccus denitrificans, Wikström and Verkhovsky (8) concluded that the most likely candidate for the proton loading site is propionate-A of heme a 3 .…”

Entrance of the proton pathway in cbb 3 -type heme-copper oxidases

“…Also, our results show that in the E25 P A and Q variants, even though the uptake of protons is severely slowed, it still occurs through the same path (from the inside), because the RCRs are still approximately 5 in the liposome-reconstituted E25 P A and Q variants. In the ba 3 HCuO from T. thermophilus, which belongs to the B family, it was also suggested that there is only one proton pathway spatially equivalent to the K pathway (17), and this pathway was shown to be used during the oxidation of the fully reduced enzyme (31) and also during reduction of the enzyme (17), supporting this pathway being the only functional proton pathway.…”

“…The residues constituting the D pathway are missing in cbb 3 s and cbb 3 s presumably have only one pathway (3,16), spatially analogous to the K pathway in the A type, for delivering protons from the cytosol to the catalytic site. This feature is shared between the C-and B-type oxidases (17) and has been proposed to be linked to the lower pumping stoichiometry observed in these HCuOs (n ¼ 2 in 2) (1, 18). In NOR, both the D and the K pathways are missing, and protons are supplied from the periplasmic side of the membrane in a reaction that does not conserve the energy available from NO reduction (19,20).…”