Redox-linked Proton Research Articles

Light-induced structural changes in cytochrome c oxidase: implication for the mechanism of electron and proton gating

We have investigated electrogenic events and absorbance changes following pulsed illumination of partly reduced cytochrome c oxidase in the absence of dioxygen and carbon monoxide (Hallén et al. (1993) FEBS Lett. 318, 134–138). In both types of experiment similar kinetics were observed; a rapid ( τ < 0.5 μs) change was followed by relaxations with time constants of approx. 7 μs and 80 μs. Both the time constant and the activation energy of the 80 μs component were, within the experimental error, the same as those of one of the steps in the reduction of dioxygen by reduced cytochrome c oxidase. The absorbance changes showed a rapid haem reduction, followed by reoxidation. They were affected by CN − and N − 3, ligands which bind in the binuclear centre of cytochrome c oxidase; the absorbance changes were quenched by CN − and in the presence of N − 3, the amplitude of the 7 μs component increased whereas that of the 80 μs decreased. Based on these findings, a model is proposed which involves electron transfer from Cu + B to Fe 3+ a 3 as a response to structural changes upon pulsed illumination. The same structural changes are also suggested to take place in the oxygen reduction. These changes may play an important role in the gating of electrons as well as protons, an obligatory feature of a redox-linked proton pump.

Primary sequence, oxidation-reduction potentials and tertiary-structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin.

Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfate-reducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidized/semiquinone forms at pH 6.7 and 25 degrees C is -40 mV, while the value for the semiquinone/hydroquinone forms (E1), at the same pH, -387 mV. E2 varies linearly with pH, while E1 is independent of pH at high values. However, at low pH (< 7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin [Moura, I., Moura, J.J.G., Bruschi, M. & LeGall, J. (1980) Biochim. Biophys. Acta 591, 1-8]. The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS. Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model [similarity, 48.6%; Watenpaugh, K.D., Sieker, L.C., Jensen, L.H., LeGall, J. & Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185-3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force-field calculations.

Characterization of the haem environment in Methylophilus methylotrophus ferricytochrome c″ by 1H‐NMR

Two-dimensional NMR techniques have been used to assign proton resonances in the haem cavity of Methylophilus methylotrophus cytochrome c", a monohaem protein with bis-histidinyl ligation which has been shown to couple electron and proton transfer. All the assignments were made directly for the oxidized paramagnetic form of the cytochrome. Nearly all of the haem protons (90%) and the protons of both axial ligands have been assigned; the side-chain protons from four other residues in the haem pocket have also been identified. The data indicate a highly symmetric unpaired-electron distribution in the haem group, which agrees with a perpendicular orientation of the axial imidazole planes. The two haem propionate groups have contrasting degrees of exposure to the solvent, with the propionate group at position 13 being highly exposed. To obtain information on the dynamics of the haem environment, measurements of the 1H/2H-exchange rates of amide protons located in the haem cavity were performed. The two faces of the haem are found to differ markedly with respect to water accessibility. All of this information, together with additional protein sequencing data, indicates that His52 remains attached upon reduction and that the redox-linked protonation occurs via a channel running through the haem cleft on the opposite face.

Vectorial chemistry in bioenergetics: cytochrome c oxidase as a redox-linked proton pump

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTVectorial chemistry in bioenergetics: cytochrome c oxidase as a redox-linked proton pumpBo G. MalmstroemCite this: Acc. Chem. Res. 1993, 26, 6, 332–338Publication Date (Print):June 1, 1993Publication History Published online1 May 2002Published inissue 1 June 1993https://doi.org/10.1021/ar00030a006RIGHTS & PERMISSIONSArticle Views175Altmetric-Citations118LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Discrete steps in dioxygen activation--the cytochrome oxidase/O2 reaction.

The kinetic constraints that are imposed on cytochrome oxidase in its dual function as the terminal oxidant in the respiratory process and as a redox-linked proton pump provide a unique opportunity to investigate the molecular details of biological O2 activation. By using flow/flash techniques, it is possible to visualize individual steps in the O2-binding and reduction process, and results from a number of spectroscopic investigations on the oxidation of reduced cytochrome oxidase by O2 are now available. In this article, we use these results to synthesize a reaction mechanism for O2 activation in the enzyme and to simulate time-concentration profiles for a number of intermediates that have been observed experimentally. Kinetic manifestation of the consequences of coupling exergonic electron transfer to endergonic proton translocation emerge from this analysis. Energetic efficiency in this process apparently requires that potentially toxic intermediate oxidation states of dioxygen accumulate to substantial concentration during the reduction reaction.

Effect of diethyl pyrocarbonate modification on spectral and steady-state kinetic properties of bovine heart cytochrome oxidase.

The histidine-specific reagent diethyl pyrocarbonate has been used to chemically modify bovine heart cytochrome oxidase. Thirty-two of sixty-seven histidine residues of cytochrome oxidase are accessible to modification by diethyl pyrocarbonate. Effects on the Soret and alpha bands of the heme spectrum indicate disturbance in the environment of one or both of the heme groups. However, diethyl pyrocarbonate modification does not alter the 830-nm absorbance band, suggesting that the environment of CuA is unchanged. Maximal modification of cytochrome oxidase by diethyl pyrocarbonate results in loss of 85-90% of the steay-state electron transfer activity, which can be reversed by hydroxylamine treatment. However, modification of the first 20 histidines does not alter either activity or the heme spectrum, but only when 32 residues have been modified are the activity and heme spectral changes complete. The steady-state kinetic profile of fully modified oxidase is monophasic; the phase corresponding to tight cytochrome c binding and low turnover is retained, whereas the high turnover phase is abolished. Proteoliposomes incorporated with modified oxidase have a 65% lower respiratory control ratio and 40% lower proton pumping stoichiometry than liposomes containing unmodified oxidase. These results are discussed in terms of a redox-linked proton pumping model for energy coupling via cytochrome oxidase.

Redox and flavin‐binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough)

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3-4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild-type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N-terminal methionine residue. Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is -143 mV (25 degrees C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is -440 mV. The effects of pH on the observed potentials were examined; E2 varies linearly with pH (slope = -59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox-linked protonation of the flavodoxin hydroquinone. D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25 degrees C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd = 0.72 microM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25 degrees C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1 = -262 mV, E2 = -193 mV; 25 degrees C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.

Redox-linked proton translocation by NADH-ubiquinone reductase (complex I).

The respiratory chain NADH-ubiquinone reductase (EC 1.6.5.3) of mitochondria, which is also called complex I, links electron transfer from NADH to ubiquinone with translocation of 4 or 5 protons across the inner membrane. The enzyme comprises some 30 different subunits, one FMN, a still not extactly defined number of iron-sulfur clusters, and probabty one form of bound ubiquinone as internal redox groups. It is because of this enormous complexity, that NADH-ubiquinone reductase has remained the least understood among the proton translocating complexes of mitochondria (for reviews see Hatefi, 1985; Ragan, 1987; Weiss et al., 1991). More recent biochemical investigations in combination with electron microscopic studies (Friedrich et al., 1989; Wang et al., 1991; Tuschen et al., 1990; G. Hofhaus, H. Weiss, and K. Leonard, unpublished results) have revealed that complex I is constructed of two distinct parts. They are assembled and probably emerged in evolution independently of each other, are arranged in a different manner with regard to the mitochondrial inner membrane, and contribute distinct segments to the electron pathway in the complex (for review see Weiss et al., 1991). Furthermore, by sequence comparison, related electron transfer segments have been discovered in completely different bacterial enzymes (Friedrich et al., 1990; Pilkington et al., 1991;

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase exhibit proton translocating activity in the presence of gramicidin

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa 3 in the Soret region (430 nm); gramicidin had no effect on the heme aa 3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each ionophore gave a similar apparent proton translocated to electron transferred stoichiometry ( H + e − ratio) of 0.66 ± 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H + e − ratio decreased to 0.280 ± 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons.

Cytochrome oxidase as a proton pump.

The general structure of cytochrome oxidase is reviewed and evidence that the enzyme acts as a redox-linked proton pump outlined. The overall H+/e- stoichiometry of the pump is discussed and results [Wikström (1989), Nature 338, 293] which suggest that only the final two electrons which reduce the peroxide adduct to water are coupled to protein translocated are considered in terms of the restrictions they place on pump mechanisms. "Direct" and "indirect" mechanisms for proton translocation are discussed in the context of evidence for redox-linked conformational changes in the enzyme, the role of subunit III, and the nature of the CuA site.

Redox-linked proton translocation by direct-coupled ligand conduction.

The term "direct-coupled" is considered in the context of redox-linked proton translocation mechanisms, and the origins of this concept, its philosophical implications, applications, and contributions to the development of bioenergetics, are discussed.

H+/e− stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes Rate-dependent changes of the stoichiometry in the cytochrome c oxidase vesicles

The H+/e− stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e− flow and H+ translocation. It is shown that the ←H+/e− ratio for redox-linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.

Nature and functional implications of the cytochrome a3 transients after photodissociation of CO-cytochrome oxidase.

Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (approximately 1 microseconds) as the post-photodissociation CU+B-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CuB to Fea3 when CO binds to CuB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The time-resolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CuB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fea3 pair. We speculate as to the identity of the ligand that is transferred between CuB and Fea3 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.

ChemInform Abstract: Cytochrome c Oxidase as a Redox‐Linked Proton Pump

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Structural and functional characteristics of polypeptide subunits of the bovine heart ubiquinol—cytochrome‐c reductase complex

Structural and functional characteristics of subunits of bovine heart cytochrome-c reductase have been investigated by controlled digestion of soluble and membrane-reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits. The results obtained show that the N-terminal segments of core protein II and the 14-kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe-S protein, located at the outer C-periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N-terminal region. Proteolytic cleavage of 7-11 residues from the N-terminal segment of the 14-kDa protein is apparently associated with decoupling of redox-linked proton pumping. Partial digestion of core protein II, the 6.4-kDa protein, and the C-terminal region of the 9.2-kDa protein, is without effect on the redox and proton-motive activity of the complex.

Structure-Function Relationships in Iron and Manganese Superoxide Dismutases

Using the complete sequences for MnSOD from Thermus thermophilus and for FeSOD from E. coli, structural models for both oxidized enzymes have been refined, the Mn protein to an R of 0.186 for all data between 10.0 and 1.8 A, and the Fe protein to an R of 0.22 for data between 10.0 and 2.5 A. The results of the refinements support the presence of a solvent as a fifth ligand to Mn(III) and Fe(III) and a coordination geometry that is close to trigonal bipyramidal. The putative substrate-entry channel is comprised of residues from both subunits of the dimer; several basic residues that are conserved may facilitate approach of O2-, while other conserved residues maintain interchain packing interactions. Analysis of the azide complex of Fe(III) dismutase suggests that during turnover O2- binds to the metal at a sixth coordination site without displacing the solvent ligand. Because crystals reduced with dithionite show no evidence for displacement of the protein ligands, the redox-linked proton acceptor (C. Bull and J.A. Fee (1985), Journal of the American Chemistry Society 107, 3295-3304) is unlikely to be one of the histidines which bind the metal ion. Structural, kinetic, titration, and spectroscopic data can be accommodated in a mechanistic scheme which accounts for the differential titration behaviour of the Fe(III) and Fe(II) enzymes at neutral and high pH.

Cytochrome c oxidase as a redox-linked proton pump

Some general principles for redox-linked proton pumps are described and applied to cytochrome oxidase. A reaction scheme obeying these principles is described. The scheme includes electron transfer between the redox centers of the oxidase, the mechanism of dioxygen reduction, protonation and deprotonation steps for both scalar and vectorial protons, as well as input-output transitions for electrons and protons. It is consistent with a large amount of kinetic information.

Cytochrome c″ isolated from Methylophilus methylotrophus. An example of bis-histidine-co-ordinated Fe3+ haem, with near-perpendicular orientation of the ligands

Cytochrome c'' (Methylophilus methylotrophus) is a soluble protein, Mr 15,000, possessing one haem which is high-spin in the reduced state but switches to a low-spin form on oxidation. Low-temperature electron-paramagnetic-resonance spectroscopy of the oxidized state shows a low-spin signal at gz = 3.65 with a folded line-shape typical of a haem of low rhombicity, and the near-infrared magnetic-circular-dichroism (m.c.d.) spectra reveal an unusually intense (delta epsilon = 400 M-1.cm-1 at 5 T, 4.2 K) charge-transfer band at 1560 nm, establishing that the oxidized haem is co-ordinated by two His residues in a near-perpendicular orientation. This conformation is well established for transmembrane b cytochromes, but this appears to be the first example in a water-soluble cytochrome. The low-temperature m.c.d. spectra of the reduced form of the protein confirms that the haem contains a high-spin Fe2+ ligated by one His residue. The redox-linked spin-state change releases a His group. Since this residue is likely to bind a proton at pH values less than 6.5, this cytochrome may provide a useful model of a molecular mechanism of a redox-linked proton uptake and release process.

Effect of chemical modification of lysine amino groups on redox and protonmotive activity of bovine heart cytochrome c oxidase reconstituted in phospholipid membranes

A study is presented of the effect of chemical modification of lysine amino groups on the redox and protonmotive activity of bovine heart cytochrome c oxidase. Treatment of soluble oxidase with succinic acid anhydride resulted in succinylation of lysines in all the subunits of the enzyme. The consequent change of surface charges from positive to negative resulted in inversion of the orientation of the reconstituted enzyme from right-side-out to inside-out. Reconstitution of the oxidase in phospholipid vesicles prevented succinylation of subunits III and Vb and depressed that of other subunits with the exception of subunits II and IV which were predominantly labeled in a concentration-dependent manner by succinic acid anhydride. This modification of lysines produced a decoupling effect on redox-linked proton ejection, which was associated with a decrease of the respiratory control exerted by the delta pH component of PMF. The decoupling effect was directly shown to be exerted at the level of the pH-dependent rate-limiting step in intramolecular electron flow located on the oxygen side of heme a.

Protonmotive activity of cytochrome c oxidase: control of oxidoreduction of the heme centers by the protonmotive force in the reconstituted beef heart enzyme

This paper contributes to the characterization of partial steps of electron and proton transfer in mitochondrial cytochrome c oxidase with respect to their membrane arrangement and involvement in energy-linked protonmotive activity. It is shown that delta psi controls electron flow from cytochrome c to heme a is consistent with the view that the latter center is buried in the membrane in a central position. The pressure exerted by delta psi on oxidation of heme alpha 3 by O2 indicates also that this center is buried in the membrane at some distance from the inner side and is consistent with observations showing that protons consumed in the reduction of O2 to H2O derive from the inner space. Electron flow from heme alpha to heme alpha 3 is shown to be specifically controlled by delta pH and in particular by the pH of the inner phase. Analysis of the effect of DCCD treatment of oxidase vesicles reveals that concentrations of this reagent which result in selective modification of subunit III (Prochaska et al., 1981) produce inhibition of redox-linked proton release. Higher concentrations of DCCD which result also in modification of subunits II and IV (Prochaska et al., 1981) cause inhibition of the pH-dependent electron-transfer step from heme alpha to heme alpha 3.