pH-induced structural changes in bacteriorhodopsin studied by Fourier transform infrared spectroscopy

Száraz, Sándor; Oesterhelt, Dieter; Ormos, Pál

doi:10.1016/s0006-3495(94)80644-7

Cited by 99 publications

(68 citation statements)

References 39 publications

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“…Such high pK a values are consistent with the pK a estimate (Ͼ11) of Asp-96 in bacteriorhodopsin (33), which serves a similar function as Glu-242. In our model, we restrict the pK a of site 1 (Glu-242) in the oxidized state to vary between 9 and 12, such that its intrinsic free energy is bounded by Ϫ2.8 Ͻ G 1 0 Ͻ Ϫ6.9 kcal/mol.…”

Section: Resultssupporting

confidence: 84%

Kinetic gating of the proton pump in cytochrome c oxidase

Kim

Wikström

Hummer

2009

Proc. Natl. Acad. Sci. U.S.A.

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Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, reduces oxygen to water and uses the released energy to pump protons across a membrane. Here, we use kinetic master equations to explore the energetic and kinetic control of proton pumping in CcO. We construct models consistent with thermodynamic principles, the structure of CcO, experimentally known proton affinities, and equilibrium constants of intermediate reactions. The resulting models are found to capture key properties of CcO, including the midpoint redox potentials of the metal centers and the electron transfer rates. We find that coarse-grained models with two proton sites and one electron site can pump one proton per electron against membrane potentials exceeding 100 mV. The high pumping efficiency of these models requires strong electrostatic couplings between the proton loading (pump) site and the electron site (heme a), and kinetic gating of the internal proton transfer. Gating is achieved by enhancing the rate of proton transfer from the conserved Glu-242 to the pump site on reduction of heme a, consistent with the predictions of the water-gated model of proton pumping. The model also accounts for the phenotype of D-channel mutations associated with loss of pumping but retained turnover. The fundamental mechanism identified here for the efficient conversion of chemical energy into an electrochemical potential should prove relevant also for other molecular machines and novel fuel-cell designs.bioenergetics ͉ biological machines ͉ kinetic master equation ͉ respiration C ytochrome c oxidase (CcO) captures the energy from the reduction of oxygen to drive the translocation of protons across the inner mitochondrial (or bacterial) membrane (1-8). The resulting electrochemical gradient powers the production of ATP, the energy source of the cell. CcO takes up four electrons from the outside (P side) of the membrane and four protons from the inside (N side) for the reduction of one dioxygen molecule. Four additional protons are translocated across the membrane from the N side to the P side. Crystal structures of the enzyme from various organisms (3, 4, 9, 10) indicate the electron and proton pathways, formed by conserved residues and cofactors. Extensive experimental and theoretical studies have provided valuable information about these pathways, the reaction sequence, and energetic aspects of catalysis (5,7,9,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). However, some key aspects of the fundamental mechanisms governing the coupling between the chemical reaction and vectorial proton translocation are still unclear.Here, the objective is to explain how vectorial proton translocation is accomplished in CcO, with respect to both the driving forces (i.e., proton and electron affinities expressed as pK a values and midpoint redox potentials, respectively, and Coulombic couplings) and the kinetic control (i.e., the ''gating'' effects that result from a dependence of kinetic barriers on the microscopic states). Specifically, we aim to construct ...

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Section: Resultssupporting

confidence: 84%

Kinetic gating of the proton pump in cytochrome c oxidase

Kim

Wikström

Hummer

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…2A), such as bacterio-and halorhodopsin, facilitate light-driven proton and ion transport across the cell membrane. These residues exhibit a range of unusual pK a values that enable them to serve as electrostatic switches essential for light-driven proton pumping (29)(30)(31). Moreover, it is known that the ionization state of these residues can be regulated by changes in external pH (30,32).…”

Section: Significancementioning

confidence: 99%

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Isom¹,

Dohlman

2015

Proc. Natl. Acad. Sci. U.S.A.

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Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light-and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligandbinding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states.7-transmembrane receptor | G protein-coupled receptor | buried charge | structural bioinformatics | molecular evolution S even-transmembrane receptors (7TMRs) are present in all domains of life. In archaea and bacteria these receptors convert light energy into transmembrane ion gradients or intracellular signaling cascades. In humans 7TMRs comprise a family of more than 800 G protein-coupled receptors (GPCRs) that detect extracellular signals such as odorants, taste, light, hormones, and neurotransmitters. Although microbial and eukaryotic 7TMRs lack sequence similarity, their 3D folds share a degree of similarity that often is observed in remote evolutionary relationships identified by structure comparison algorithms (1).Recent breakthroughs in membrane protein crystallography have advanced our understanding of GPCR function by providing models of unactivated and activated receptor conformations (2-8). However, structure-based approaches for systematically quantifying differences between GPCR activation states are lacking. Here we introduce a computational approach that correlates GPCR activation with networks of electrostatic interactions in the receptor core. We show that membrane-spanning networks of ionizable residues, which we find are a common feature of microbial 7TMRs, also represent a unique signature of GPCR activation that is likely to be essential to receptor function.The molecular changes that accompany 7TMR activation were studied initially in bacteriorhodopsin and rhodopsin, the first microbial 7TMR and GPCR to be crystallized (9-11). More recently, a rapid expansion of GPCR structural information has revealed several molecular switches and structural features that are thought to be indicative of receptor activation. These include the ionic loc...

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“…(31) We next sought to apply this host-induced shift in effective basicity to promote reaction chemistry. We focused on the hydrolysis of orthoformates, HC(OR) 3 We probed the reaction mechanism using triethyl orthoformate as the substrate at pH 11.0 and 50 °C. First-order substrate consumption was observed under stoichiometric conditions ( Figure S4).…”

Section: ]mentioning

confidence: 99%

“…In order to test for competitive inhibition for the hydrolysis of orthoformates with 1, the rates of hydrolysis of triethyl orthoformate were measured in the presence of a varying amount of the strongly-binding inhibitor NPr 4 + (-ΔG° = 2.7(2) kcal/mol). The lower binding constant of NPr 4 + with respect to NEt 4 + facilitates the competitive binding experiments by allowing for the weakly binding substrate, HC(OEt) 3 , to more readily compete for the binding cavity of 1. By varying the concentration of substrate for each amount of inhibitor, the saturation curves were compared using an Eadie-Hofstee plot (Figure 4).…”

Section: ]mentioning

confidence: 99%

Acid Catalysis in Basic Solution: A Supramolecular Host Promotes Orthoformate Hydrolysis

Pluth

Bergman

Raymond

2007

Science

749

414

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Though many enzymes can promote chemical reactions by tuning substrate properties purely through the electrostatic environment of a docking cavity, this strategy has proven challenging to mimic in synthetic host-guest systems. Here we report a highly-charged, water soluble, metal-ligand assembly with a hydrophobic interior cavity that thermodynamically stabilizes protonated substrates and consequently catalyzes the normally acidic hydrolysis of orthoformates in basic solution, with rate accelerations of up to 890-fold. The catalysis reaction obeys Michaelis-Menten kinetics, exhibits competitive inhibition, and the substrate scope displays size selectivity consistent with the constrained binding environment of the molecular host.Synthetic chemists have long endeavored to design host molecules capable of selectively binding slow-reacting substrates and catalyzing their chemical reactions. While synthetic catalysts are often site-specific and require certain properties of the substrate to insure catalysis, enzymes are often able to modify basic properties of the bound substrate such as pK a in order to enhance reactivity. Two common motifs used by nature to activate otherwise unreactive compounds are the precise arrangement of hydrogen-bonding networks and electrostatic interactions between the substrate and adjacent residues of the protein.(1) Precise arrangement of hydrogen bonding networks near the active sites of proteins can lead to well-tuned pK a -matching,(2) and can result in pK a shifts of up to eight units, as shown in bacteriorhodopsin.(3) Similarly, purely electrostatic interactions can greatly favor charged states and have been responsible for pK a shifts of up to five units for acetoacetate decarboxylase.(4) Attempts have been made to isolate the contributions of electrostatic versus covalent interactions to such pK a shifts; however this remains a difficult challenge experimentally. This challenge emphasizes the importance of synthesizing host molecules that, like enzyme cavities, can enhance binding of small molecular guests and, in a few cases, catalyze chemical reactions. (5-7) Supramolecular assemblies with available functional groups have been used to generate solution-state pK a shifts of up to two pK a units (8-11) and to catalyze chemical reactions.(12, 13) Synthetic hosts often rely on hydrogen-bonding or ion-dipole interactions for guest inclusion, and numerous studies have investigated the effects of charge on guest binding affinities in supramolecular host-guest systems.(14, 15) We report here a synthetic supramolecular host assembly that relies exclusively on electrostatic and hydrophobic interactions for thermodynamic stabilization of protonated substrates. As nature has exploited pK a shifts to activate otherwise unreactive substrates toward catalysis, this stabilization is exploited to promote acid-catalyzed hydrolyses in strongly basic solution.During the past decade, the Raymond group has reported the formation and guesthosting properties of supramolecular assemblies of the...

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pH-induced structural changes in bacteriorhodopsin studied by Fourier transform infrared spectroscopy

Cited by 99 publications

References 39 publications

Kinetic gating of the proton pump in cytochrome c oxidase

Kinetic gating of the proton pump in cytochrome c oxidase

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Acid Catalysis in Basic Solution: A Supramolecular Host Promotes Orthoformate Hydrolysis

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