A chemically explicit model for the mechanism of proton pumping in heme–copper oxidases

102

185

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

Section: Discussionmentioning

confidence: 99%

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

Chang¹,

Hemp

Chen

et al. 2009

102

185

“…However, it is not known what residue binds the proton or if the same residue is used in each redox state (22)(23)(24)(25). The propionic acids on the A (PRA) or D (PRD) rings of heme a 3 (26) or His A334, which is a ligand to Cu B , have all been proposed to be the PLS (15,24,27,28).…”

Section: Significancementioning

confidence: 99%

Characterizing the proton loading site in cytochromecoxidase

Gunner

2014

Cytochrome c oxidase (CcO) uses the energy released by reduction of O 2 to H 2 O to drive eight charges from the high pH to low pH side of the membrane, increasing the electrochemical gradient. Four electrons and protons are used for chemistry, while four more protons are pumped. Proton pumping requires that residues on a pathway change proton affinity through the reaction cycle to load and then release protons. The protonation states of all residues in CcO are determined in MultiConformational Continuum Electrostatics simulations with the protonation and redox states of heme a, a 3 , Cu B , Y288, and E286 used to define the catalytic cycle. One proton is found to be loaded and released from residues identified as the proton loading site (PLS) on the P-side of the protein in each of the four CcO redox states. Thus, the same proton pumping mechanism can be used each time CcO is reduced. Calculations with structures of Rhodobacter sphaeroides, Paracoccus denitrificans, and bovine CcO derived by crystallography and molecular dynamics show the PLS functions similarly in different CcO species. The PLS is a cluster rather than a single residue, as different structures show 1-4 residues load and release protons. However, the proton affinity of the heme a 3 propionic acids primarily determines the number of protons loaded into the PLS; if their proton affinity is too low, less than one proton is loaded.MCCE | bioenergetics | pK a | proton transfer

“…(B, Inset) Exchange of chloride with bromide at the original carboxyl site in D132A crystal. The OH form a tight (2.6 Å) hydrogen bond that has been suggested to control the entrance of protons into the active site (3,14,27). Mutations in this pathway cause major loss of enzyme activity, but the remaining activity is still coupled to proton pumping (9,28,29), implying that this pathway is exclusively involved in substrate proton uptake.…”

Section: S142mentioning

confidence: 99%

“…A model has been proposed previously (27) that offers a rationale for a sequence of events that could produce the conformational changes seen in the crystallized reduced state and also provides an explanation for the incomplete changes in the Fig. 5.…”

Section: A Model To Account For Conformational Changes Seen In Reducedmentioning

confidence: 99%

Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

Liu

Qin

Ferguson‐Miller

2011

Self Cite

Crystal structures in both oxidized and reduced forms are reported for two bacterial cytochrome c oxidase mutants that define the D and K proton paths, showing conformational change in response to reduction and the loss of strategic waters that can account for inhibition of proton transfer. In the oxidized state both mutants of the Rhodobacter sphaeroides enzyme, D132A and K362M, show overall structures similar to wild type, indicating no long-range effects of mutation. In the reduced state, the mutants show an altered conformation similar to that seen in reduced wild type, confirming this reproducible, reversible response to reduction. In the strongly inhibited D132A mutant, positions of residues and waters in the D pathway are unaffected except in the entry region close to the mutation, where a chloride ion replaces the missing carboxyl and a 2-Å shift in N207 results in loss of its associated water. In K362M, the methionine occupies the same position as the original lysine, but K362-and T359-associated waters in the wild-type structure are missing, likely accounting for the severe inhibition. Spectra of oxidized frozen crystals taken during X-ray radiation show metal center reduction, but indicate development of a strained configuration that only relaxes to a native form upon annealing. Resistance of the frozen crystal to structural change clarifies why the oxidized conformation is observable and supports the conclusion that the reduced conformation has functional significance. A mechanism is described that explains the conformational change and the incomplete response of the D-path mutant.conformational gating | membrane protein structure | proton path mutants | X-ray reduction | water chain C ytochrome c oxidase (CcO) is a major contributor to and regulator of energy conservation in eukaryotic and many prokaryotic organisms. It uses four electrons from cytochrome c and four protons from the inner side of the membrane to reduce O 2 to water (1). In each catalytic cycle, four protons are also translocated across the membrane to generate a membrane potential, but the mechanistic details of coupling between proton pumping and the electron transfer events are still not fully understood (1, 2). Our recently solved crystal structures of the fully reduced form of Rhodobacter sphaeroides (Rs) CcO reveal conformational changes that were not previously observed (3), introducing mechanistic possibilities for coupling and gating of proton transfer, the significance of which we further elucidate in this report.Bacterial forms of CcO are similar in many respects to the mammalian mitochondrial CcO and their simpler subunit composition and ease of mutation have made them useful model systems for studying the mechanism of energy conservation, assuming it to be conserved. It has become increasingly clear, however, that within the large superfamily of heme-copper oxidases some bacterial forms may have solved the proton pumping problem in different ways (4, 5), and even those most closely related to eukaryotic oxidases (the aa ...