scholarly journals Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site

2015 ◽  
Vol 112 (11) ◽  
pp. 3397-3402 ◽  
Author(s):  
Christoph von Ballmoos ◽  
Nathalie Gonska ◽  
Peter Lachmann ◽  
Robert B. Gennis ◽  
Pia Ädelroth ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Time Constant ◽  
Thermus Thermophilus ◽  
Proton Translocation ◽  
Proton Pumping ◽  
Wild Type ◽  
Structural Variant ◽  
In The Wild ◽  
Membrane Bound ◽  
Proton Uptake

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative “pump site” was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

scholarly journals Transmembrane Charge Separation during the Ferryl-oxo → Oxidized Transition in a Nonpumping Mutant of CytochromecOxidase

2004 ◽  
Vol 279 (50) ◽  
pp. 52558-52565 ◽  
Author(s):  
Sergey A. Siletsky ◽  
Ashtamurthy S. Pawate ◽  
Kara Weiss ◽  
Robert B. Gennis ◽  
Alexander A. Konstantinov
Keyword(s):  
Electron Transfer ◽  
Aqueous Phase ◽  
Proton Translocation ◽  
Proton Pumping ◽  
K Channel ◽  
Wild Type ◽  
Time Resolved ◽  
In The Wild ◽  
Solvent Isotope ◽  
Charge Translocation

The N139D mutant of cytochromecoxidase fromRhodobacter sphaeroidesretains full steady state oxidase activity but completely lacks proton translocation coupled to turnover in reconstituted liposomes (Pawate, A. S., Morgan, J., Namslauer, A., Mills, D., Brzezinski, P., Ferguson-Miller, S., and Gennis, R. B. (2002)Biochemistry41, 13417–13423). Here, time-resolved electron transfer and vectorial charge translocation in the ferryl-oxo → oxidized transition (transfer of the 4th electron in the catalytic cycle) have been studied with the N139D mutant using ruthenium(II)-tris-bipyridyl complex as a photoactive single-electron donor. With the wild type oxidase, the flash-induced generation of Δφ in the ferryl-oxo → oxidized transition begins with rapid vectorial electron transfer from CuAto heme a (τ ∼15 μs), followed by two protonic phases, referred to as the intermediate (0.4 ms) and slow electrogenic phases (1.5 ms). In the N139D mutant, only a single protonic phase (τ ∼0.6 ms) is observed, which was associated with electron transfer from heme a to the heme a3/CuBsite and decelerates ∼4-fold in D2O. With the wild type oxidase, such a high H2O/D2O solvent isotope effect is characteristic of only the slow (1.5 ms) phase. Presumably, the 0.6-ms electrogenic phase in the N139D mutant reports proton transfer from the inner aqueous phase to Glu-286, replacing the “chemical” proton transferred from Glu-286 to the heme a3/CuBsite. The transfer occurs through the D-channel, because it is observed also in the N139D/K362M double mutant in which the K-channel is blocked. It is concluded that the intermediate electrogenic phase observed in the wild type enzyme is missing in the N139D mutant and is because of translocation of the “pumped” proton from Glu-286 to the D-ring propionate of heme a3or to release of this proton to the outer aqueous phase. Significantly, with the wild type oxidase, the protonic electrogenic phase associated with proton pumping (∼0.4 ms) precedes the electrogenic phase associated with the oxygen chemistry (∼1.5 ms).

Molecular architecture of the proton diode of cytochrome c oxidase

2008 ◽  
Vol 36 (6) ◽  
pp. 1169-1174 ◽  
Author(s):  
Peter Brzezinski ◽  
Joachim Reimann ◽  
Pia Ädelroth
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Proton Transfer ◽  
Cytochrome C Oxidase ◽  
Proton Translocation ◽  
Short Review ◽  
Proton Pumping ◽  
Molecular Architecture ◽  
Electrochemical Gradient ◽  
Membrane Bound

CytcO (cytochrome c oxidase) is a membrane-bound multisubunit protein which catalyses the reduction of O2 to H2O. The reaction is arranged topographically so that the electrons and protons are taken from opposite sides of the membrane and, in addition, it is also linked to proton pumping across the membrane. Thus the CytcO moves an equivalent of two positive charges across the membrane per electron transferred to O2. Proton transfer through CytcO must be controlled by the protein to prevent leaks, which would dissipate the proton electrochemical gradient that is maintained across the membrane. The molecular mechanism by which the protein controls the unidirectionality of proton-transfer (cf. proton diode) reactions and energetically links electron transfer to proton translocation is not known. This short review summarizes selected results from studies aimed at understanding this mechanism, and we discuss a possible mechanistic principle utilized by the oxidase to pump protons.

scholarly journals Five decades of research on mitochondrial NADH-quinone oxidoreductase (complex I)

2018 ◽  
Vol 399 (11) ◽  
pp. 1249-1264 ◽  
Author(s):  
Tomoko Ohnishi ◽  
S. Tsuyoshi Ohnishi ◽  
John C. Salerno
Keyword(s):  
Electron Transfer ◽  
Respiratory Chain ◽  
Complex I ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Mitochondrial Respiratory Chain ◽  
Proton Pumping ◽  
Entry Site ◽  
Quinone Oxidoreductase ◽  
Terminal Electron Acceptor

AbstractNADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson’s and Alzheimer’s, and it is proposed to be involved in aging. Complex I has one non-covalently bound FMN, eight to 10 iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membranein situanalysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone (SQ) state. In the aerobic steady state of respiration the semi-ubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, SQ signals have been resolved which were designated SQNfand SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the SQ radicals and their possible part in proton pumping.

scholarly journals Cooperation among c-subunits of FoF1-ATP synthase in rotation-coupled proton translocation

2021 ◽  
Author(s):  
Noriyo Mitome ◽  
Shintaroh Kubo ◽  
Sumie Ohta ◽  
Hikaru Takashima ◽  
Yuto Shigefuji ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Proton Pump ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Wild Type ◽  
Single Chain ◽  
Proton Release ◽  
Biochemical Assays ◽  
Proton Uptake ◽  
Fof1 Atp Synthase

In FoF1-ATP synthase, proton translocation through Fo drives rotation of the c-subunit oligomeric ring relative to the a-subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different c-subunits contribute to proton release to and proton uptake from the a-subunit. However, no studies have demonstrated cooperativity among c-subunits toward FoF1-ATP synthase activity. Here, we addressed this using Bacillus PS3 ATP synthase harboring c-ring with various combinations of wild-type and cE56D, enabled by genetically fused single-chain c-ring. ATP synthesis and proton pump activities were significantly decreased by a single cE56D mutation and further decreased by double cE56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among c-subunits. Similar results were obtained for proton transfer-coupled molecular simulations. Simulations revealed that prolonged proton uptake in mutated c-subunits is shared between two c-subunits, explaining the cooperation observed in biochemical assays.

scholarly journals Direct Observation of Electrically Conductive Pili Emanating from Geobacter sulfurreducens

2021 ◽  
Author(s):  
Xinying Liu ◽  
David Jeffrey Fraser Walker ◽  
Stephen Nonnenmann ◽  
Dezhi Sun ◽  
Derek R. Lovley
Keyword(s):  
Electron Transfer ◽  
Electron Transport ◽  
Long Range ◽  
Geobacter Sulfurreducens ◽  
Metal Corrosion ◽  
Extracellular Electron Transfer ◽  
Wild Type ◽  
Electrically Conductive ◽  
In The Wild ◽  
Pilin Gene

Geobacter sulfurreducens is a model microbe for elucidating the mechanisms for extracellular electron transfer in several biogeochemical cycles, bioelectrochemical applications, and microbial metal corrosion. Multiple lines of evidence previously suggested that electrically conductive pili (e-pili) are an essential conduit for long-range extracellular electron transport in G. sulfurreducens. However, it has recently been reported that G. sulfurreducens does not express e-pili and that filaments comprised of multi-heme c-type cytochromes are responsible for long-range electron transport. This possibility was directly investigated by examining cells, rather than filament preparations, with atomic force microscopy. Approximately 90 % of the filaments emanating from wild-type cells had a diameter (3 nm) and conductance consistent with previous reports of e-pili harvested from G. sulfurreducens or heterologously expressed in E. coli from the G. sulfurreducens pilin gene. The remaining 10% of filaments had a morphology consistent with filaments comprised of the c-type cytochrome OmcS. A strain expressing a modified pilin gene designed to yield poorly conductive pili expressed 90 % filaments with a 3 nm diameter, but greatly reduced conductance, further indicating that the 3 nm diameter conductive filaments in the wild-type strain were e-pili. A strain in which genes for five of the most abundant outer-surface c-type cytochromes, including OmcS, was deleted yielded only 3 nm diameter filaments with the same conductance as in the wild-type. These results demonstrate that e-pili are the most abundant conductive filaments expressed by G. sulfurreducens, consistent with previous functional studies demonstrating the need for e-pili for long-range extracellular electron transfer.

Lipids affect the charge stabilization in wild-type and mutant reaction centers of Rhodobacter sphaeroides R-26

1999 ◽  
Vol 26 (5) ◽  
pp. 465 ◽  
Author(s):  
László Nagy ◽  
Elfrida Fodor ◽  
Júlia Tandori ◽  
László Rinyu ◽  
Tibor Farkas
Keyword(s):  
Electron Transfer ◽  
Rhodobacter Sphaeroides ◽  
Electrostatic Interactions ◽  
Reaction Centers ◽  
Wild Type ◽  
Photosynthetic Membrane ◽  
Protein Ratio ◽  
Intracytoplasmic Membranes ◽  
In The Wild ◽  
Charge Stabilization

The effect of lipids on stabilization of electrons on the secondary quinone was studied in reaction centers (RC) of herbicide-sensitive and -resistant (L229Ile → Met) Rhodobacter sphaeroides R-26. The lipid concentration and the lipid/protein ratio of the intracytoplasmic membranes (ICM) were larger in the mutant RCs than in the wild-type. The free energy changes of Q A – Q B → Q A Q B – electron transfer were ΔG 0 = –57 meV, –69 meV, –85 meV for the wild-type and ΔG 0 = 0 meV, –15 meV, –46 meV for the mutant at pH = 8.0, in detergent, liposome and ICM, respectively. The differences in the stabilization energies of both strains decreased from the detergent via proteoliposome to chromatophore. We conclude that the energetics of the interquinone electron transfer depends on the environment of the reaction center. The steric and/or electrostatic interactions of the environment and Q B pocket can modulate the energetics of the charge stabilization over large distances. The interaction may have crucial importance on coupling the electron transport in the photosynthetic membrane to the anabolic/catabolic processes taking place in the cells.

A Ca-Induced Na-Current in Paramecium

1980 ◽  
Vol 88 (1) ◽  
pp. 305-326
Author(s):  
YOSHIRO SAIMI ◽  
CHING KUNG
Keyword(s):  
Voltage Clamp ◽  
Time Constant ◽  
Negative Resistance ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Na Current ◽  
Inward Current ◽  
In The Wild ◽  
Current Flows ◽  
Sustained Inward Current

Under a voltage clamp, step depolarization and repolarization can induce a sustained inward current and a tail inward current in Paramecium tetraurelia bathed in a solution containing 8 mM-Na+. These currents are best seen in the ‘paranoiac’ mutant. The I-V plot of the sustained inward current can have a region of negative resistance around −20 mV. This current is absent when Na+ is excluded from the bath solution, and it increases as the Na+ concentration increases from 2 to 8 mM. Injection of Na+ into the cell suppresses this inward current. This current develops very slowly, reaching its maximum seconds after the step depolarization and decays with a time constant of hundreds of milliseconds after the repolarization. This slow current is dependent on Ca2+. It can be suppressed by reduction or deletion of external Ca2+ or by iontophoretic injection of EGTA. ‘Pawn’ mutants with defective Caconductance also lack this current. We conclude that Paramecium has a Ca-induced conductance through which the Na-current flows. Although more prominent in the ‘paranoiac’ mutant, this Ca-induced Na-current is also seen in the wild type. This conductance may function in generating plateau depolarizations lasting seconds or even minutes and the corresponding prolonged backward swimming away from sources of irritation and stress.

scholarly journals Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase

1996 ◽  
Vol 317 (2) ◽  
pp. 425-431 ◽  
Author(s):  
Glen HUGHES ◽  
Michael A. HARRISON ◽  
Yong-In KIM ◽  
David E. GRIFFITHS ◽  
Malcolm E. FINBOW ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Fluorescence Enhancement ◽  
Atp Hydrolysis ◽  
Dissociation Constants ◽  
Proton Translocation ◽  
Vacuolar Atpase ◽  
Proton Pumping ◽  
Wild Type ◽  
Expression Studies

The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H+-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement or affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites for organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the vacuolar H+-ATPase.

scholarly journals Improper Localization of the OmcS Cytochrome May Explain the Inability of thexapD-Deficient Mutant ofGeobacter sulfurreducensto Reduce Fe(III) Oxide

2017 ◽  
Author(s):  
Kelly A. Flanagan ◽  
Ching Leang ◽  
Joy E. Ward ◽  
Derek R. Lovley
Keyword(s):  
Electron Transfer ◽  
Electron Transport ◽  
Type Strain ◽  
Wild Type Strain ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Outer Cell ◽  
Wild Type ◽  
Redox Active ◽  
In The Wild

AbstractExtracellular electron transfer through a redox-active exopolysaccharide matrix has been proposed as a strategy for extracellular electron transfer to Fe(III) oxide byGeobacter sulfurreducens,based on the phenotype of axapD-deficient strain. Central to this model was the assertion that thexapD-deficient strain produced pili decorated with the multi-hemec-type cytochrome OmcS in manner similar to the wild-type strain. Further examination of thexapD-deficient strain with immunogold labeling of OmcS and transmission electron microscopy revealed that OmcS was associated with the outer cell surface rather than pili. PilA, the pilus monomer, could not be detected in thexapD-deficient strain under conditions in which it was readily detected in the wild-type strain. Multiple lines of evidence in previous studies have suggested that long-range electron transport to Fe(III) oxides proceeds through electrically conductive pili and that OmcS associated with the pili is necessary for electron transfer from the pili to Fe(III) oxides. Therefore, an alternative explanation for the Fe(III) oxide reduction phenotype of thexapD-deficientstrain is that the pili-OmcS route for extracellular electron transport to Fe(III) oxide has been disrupted in thexapD-deficient strain.

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