Molecular mechanisms for generating transmembrane proton gradients

Self Cite

112

Cytochrome c oxidase contributes to the transmembrane proton gradient by removing two protons from the high-pH side of the membrane each time the binuclear center active site is reduced. One proton goes to the binuclear center, whereas the other is pumped to the low-pH periplasmic space. Glutamate 286 (Glu286) has been proposed to serve as a transiently deprotonated proton donor. Using unrestrained atomistic molecular dynamics simulations, we show that the size of and water distribution in the hydrophobic cavity that holds Glu286 is controlled by the protonation state of the propionic acid of heme a 3 , a group on the proton outlet pathway. Protonation of the propionate disrupts hydrogen bonding to two side chains, allowing a loop to swing open. Continuum electrostatics and atomistic free-energy perturbation calculations show that the resultant changes in hydration and electrostatic interactions lower the Glu proton affinity by at least 5 kcal/mol. These changes in the internal hydration level occur in the absence of major conformational transitions and serve to stabilize needed transient intermediates in proton transport. The trigger is not the protonation of the Glu of interest, but rather the protonation of a residue ∼10 Å away. Thus, unlike local water penetration to stabilize a new charge, this finding represents a specific role for water molecules in the protein interior, mediating proton transfers and facilitating ion transport.proton pumping | pK a W ater is essential to the structure, dynamics, and function of biomolecules (1, 2), and its role in protein folding, association (3), and dynamics (4, 5) has been well documented. The highly polar and polarizable water molecules play diverse roles in protein interiors. Water can aid catalysis in enzyme active sites (6-8). Water or water chains are often observed in proteins that are (9, 10) proton or ion transporters or pumps (11)(12)(13)(14). Internal cavities holding functional water molecules are believed to have a fairly constant level of hydration throughout the protein reaction cycle, unless significant conformational changes occur (15). Water penetration in response to the ionization or reduction of internal groups has been extensively discussed (16,17), although it is usually described as part of protein's local dielectric response.Cytochrome c oxidase (CcO) adds to the transmembrane proton gradient through proton transport coupled to electron transfer reactions (12,18,19). In the overall reaction, electrons from four cytochromes c are transferred to oxygen to make two water molecules at the binuclear center (BNC). The four protons needed for chemistry are bound only from the high-pH, N side of the membrane. Coupled to the process, four more protons are transferred across the membrane from the high-to low-pH (P) side of the membrane. Thus, eight charges are transferred across the membrane as each O 2 is reduced.Glu286 is a required, conserved residue that is expected to transfer protons from the D channel either to the BNC or the proton-loading site (...

Section: Microscopic Qm/mm-ti Pk′ 7 Calculations and The Effect Of Cmentioning

confidence: 99%

Changing hydration level in an internal cavity modulates the proton affinity of a key glutamate in cytochromecoxidase

Goyal

Yang

et al. 2013

Self Cite

112

“…Programs that use a continuum electrostatics (CE) analysis of protein interactions with Monte Carlo sampling have shed light on the Boltzmann distribution of protonation states at equilibrium in a given protein structure (6,34). CE calculations have followed proton transfers in BR trapped in the ground and later intermediate structures (22,23) and in other proteins (35,36).…”

Section: Significancementioning

confidence: 99%

“…The Schiff base (SB), BR-D85, and D212 form the central cluster and E194 and E204 comprise the extracellular cluster, whereas BR-D96 on the inner side of the protein transfers a proton from the cytoplasm to the SB (14,21). Each of these groups change charge as BR pumps protons from the highpH cytoplasm to the low-pH extracellular space (Table 1) (6,21). In HR, six of the buried BR ionizable residues are conserved.…”

mentioning

confidence: 99%

“…The transmembrane electrochemical gradient generated by ion and proton pumps drives key processes such as ATP synthesis and nerve transmission (5). Proton and ion transfer processes, protein-protein interactions, and enzyme catalysis often rely on charged amino acids (6). In homologous proteins, key amino acids can be replaced by bound ions that introduce enhanced sensitivity to ion concentration and pH.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Halorhodopsin pumps Cl^–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

Song¹,

Gunner

2014

Key mutations differentiate the functions of homologous proteins. One example compares the inward ion pump halorhodopsin (HR) and the outward proton pump bacteriorhodopsin (BR). Of the nine essential buried ionizable residues in BR, six are conserved in HR. However, HR changes three BR acids, D85 in a central cluster of ionizable residues, D96, nearer the intracellular, and E204, nearer the extracellular side of the membrane to the small, neutral amino acids T111, V122, and T230, respectively. In BR, acidic amino acids are stationary anions whose proton affinity is modulated by conformational changes, establishing a sequence of directed binding and release of protons. Multiconformation continuum electrostatics calculations of chloride affinity and residue protonation show that, in reaction intermediates where an acid is ionized in BR, a Cl -is bound to HR in a position near the deleted acid. In the HR ground state, Cl -binds tightly to the central cluster T111 site and weakly to the extracellular T230 site, recovering the charges on ionized BR-D85 and neutral E204 in BR. Imposing key conformational changes from the BR M intermediate into the HR structure results in the loss of Cl -from the central T111 site and the tight binding of Cl -to the extracellular T230 site, mirroring the changes that protonate BR-D85 and ionize E204 in BR. The use of a mobile chloride in place of D85 and E204 makes HR more susceptible to the environmental pH and salt concentrations than BR. These studies shed light on how ion transfer mechanisms are controlled through the interplay of protein and ion electrostatics.

“…The gradient is formed by protons transferred from the high (N-) to low (P-) pH side of the membrane through proteins that carry out exergonic electron transfer reactions (3,4). The protons move because the redox reactions modify the proton affinity of residues and cofactors by pK a shifts of the redox active species, by changing the long-range electrostatic potential or through induced conformational changes (4). The accessibility of sites to N-and P-sides of the membrane may also change to ensure that protons are taken up from the correct side in each step.…”

mentioning

confidence: 99%

Characterizing the proton loading site in cytochromecoxidase

Gunner

2014

Self Cite

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