Insights into the Mechanism of Proton Transport in Cytochrome c Oxidase

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139

The influenza A virus M2 channel (AM2) is crucial in the viral life cycle. Despite many previous experimental and computational studies, the mechanism of the activating process in which proton permeation acidifies the virion to release the viral RNA and core proteins is not well understood. Herein the AM2 proton permeation process has been systematically characterized using multiscale computer simulations, including quantum, classical, and reactive molecular dynamics methods. We report, to our knowledge, the first complete free-energy profiles for proton transport through the entire AM2 transmembrane domain at various pH values, including explicit treatment of excess proton charge delocalization and shuttling through the His37 tetrad. The free-energy profiles reveal that the excess proton must overcome a large freeenergy barrier to diffuse to the His37 tetrad, where it is stabilized in a deep minimum reflecting the delocalization of the excess charge among the histidines and the cost of shuttling the proton past them. At lower pH values the His37 tetrad has a larger total charge that increases the channel width, hydration, and solvent dynamics, in agreement with recent 2D-IR spectroscopic studies. The proton transport barrier becomes smaller, despite the increased charge repulsion, due to backbone expansion and the more dynamic pore water molecules. The calculated conductances are in quantitative agreement with recent experimental measurements. In addition, the free-energy profiles and conductances for proton transport in several mutants provide insights for explaining our findings and those of previous experimental mutagenesis studies.T he influenza type A virus is a highly pathogenic RNA virus that causes flu in birds and mammals (1). The influenza A M2 (AM2) protein (2) contains a homotetramer channel that transports protons across the viral membrane and acidifies the virion interior, enabling the dissociation of the viral matrix proteins, which is a crucial step in viral replication (3). The protein has been the target of antiviral drugs amantadine and rimantadine (4, 5). Much effort has been devoted to discovering the structure and proton transport (PT) mechanism of the AM2 channel, resulting in many crystal structures available in the protein data bank (6-14). Based on the crystal structures and electrophysiology experiments, several PT models have been suggested. These mechanisms can be divided into two main categories, delineated by the role of the four histidine residues (a.k.a. the His37 tetrad) that reside in the middle of the AM2 transmembrane domain (AM2/TM) (Fig. 1A), which has been experimentally shown (15) to account for the proton permeation behavior of the full AM2 protein. The "shutter" mechanism (16,17) suggests that the His37 tetrad works as a gate. At low pH the gate opens due to the electrostatic repulsion between the biprotonated, positively charged histidine residues. The excess proton is then transferred through continuous water wire via the Grotthuss mechanism, without changing the pro...

Section: Methodsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza A M2 proton channel

Liang

Swanson

et al. 2014

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139

“…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). The energetics and pathway of proton pumping has been simulated assuming the PRD of heme a 3 is the PLS (26,(29)(30)(31)(32). Because there are many ionizable groups on the P-side, the PLS could also be made up of a cluster of residues rather than a single site (SI Appendix, Fig.…”

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

“…Critical components of these enzymes include proton-conducting channels, which are conserved structures within each major family of HCOs (A, B, and C families) (4)(5)(6). These proton channels are defined by conserved polar residues, as well as internal water molecules that provide a hydrogen-bonded pathway for proton diffusion within the protein by a Grotthus-type mechanism (7)(8)(9). The channels are required to provide pathways both for chemical protons, which are consumed at the enzyme active site to form water, and for pumped protons, which are transported across the membrane.…”

mentioning

confidence: 99%

Conformational coupling between the active site and residues within the K ^C -channel of the Vibrio cholerae cbb ₃ -type (C-family) oxygen reductase

Ahn¹,

Mahinthichaichan²,

Lee³

et al. 2014

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K C -channel is a conserved glutamate in subunit III. However, the majority of the K C -channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K Cchannel, was found to depend on the conformation of Y241 Vc , located in subunit I at the interface with subunit III. Mutations of Y241 Vc (to A/F/H/S) in the Vibrio cholerae cbb 3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b 3 . The data suggest a linkage between residues lining the K C -channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241 Vc and the active site crosslinked Y255 Vc , as well as two Cu B histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.oxygen reductase | proton pathway | bioenergetics | Vibrio cholerae | cbb 3