FEBS Letters xxx (2015) xxx–xxx
journal homepage: www.FEBSLetters.org
Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants Thomas Meyer a, Frédéric Melin a, Oliver-M.H. Richter b, Bernd Ludwig b, Aimo Kannt c,1, Hanne Müller c, Hartmut Michel c, Petra Hellwig a,⇑ a
Chimie de la Matière Complexe UMR 7140, Laboratoire de Bioélectrochimie et Spectroscopie, CNRS-Université de Strasbourg, 1 rue Blaise Pascal, 67070 Strasbourg, France Institute of Biochemistry, Molecular Genetics, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany c Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Max-von-Laue-Str. 3, D-60438 Frankfurt/Main, Germany b
a r t i c l e
i n f o
Article history: Received 26 November 2014 Revised 5 January 2015 Accepted 7 January 2015 Available online xxxx Edited by Peter Brzezinski Keywords: Cytochrome c oxidase Zn2+ inhibition Proton pumping Bioelectrochemistry
a b s t r a c t Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn2+ ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The concerted movement of protons and electrons is a common feature of many energy-transducing complexes including the photosynthetic reaction center (RC), the cytochrome (cyt) bc1 complex (cyt bc1), the NADH ubiquinone oxidoreductase (complex I) or cyt c oxidase (CcO). These enzymes generate an electrochemical proton gradient across the membrane by the vectorial uptake and release of protons at the lipid bilayer in a manner coupled to electron transfer and which is then used to drive the synthesis of ATP. Earlier observations have indicated that transition metal binding in energy transducing components can be used to identify residues involved in proton transfer pathways in proton translocating enzymes. It was reported that transition metal ions such as Zn2+ and Cd2+ can inhibit the proton transfer activity of bacterial RCs [1,2]. Zn2+ binding to the RC from Rhodobacter sphaeroides, decreases the rate of Abbreviations: cyt bc1, bc1 complex; cyt, cytochrome; CcO, cytochrome c oxidase; complex I, NADH ubiquinone oxidoreductase; RC, reaction center; Tris, tris(hydroxymethyl) aminoethane ⇑ Corresponding author. 1 Present address: Sanofi-Aventis Deutschland GmbH, Diabetes R&D, Industriepark Hoechst, H824, D-65926 Frankfurt am Main, Germany.
proton transfer to the acidic residue named E212, becoming a rate-limiting step [2]. The X-ray crystal structure of the RC with bound Zn2+ revealed that the Zn2+ binding cluster D124, H126 and H128 of the RC subunit H is involved in the pathway of the first proton delivery to QB- at the entry point [3]. Also the inhibition of proton translocation by Zn2+ in the bc1 complex (complex III) [4,5] and inhibition of proton pumping in complex I [6] have been described. The IC50 for the inhibition of the respiratory complexes were estimated to be in the mid-micromolar range for complexes I and IV and in the mid nanomolar range for complex III. Under physiological conditions, the levels of free Zn2+ in mitochondria are close to zero. However, pathologic conditions may increase Zn2+ concentrations, thus inhibiting oxidative phosphorylation basically at the level of complex III [7]. It is not clear whether the inhibition of complexes I and IV are of physiological relevance. As for complex IV, it was shown that zinc inhibits the proton translocation and causes selective uncoupling from electron transfer [8–10] and it can thus be used as a tool to investigate proton pumping. Two separate proton pathways have been suggested for the bacterial CcO, the so-called D- and K-pathways [11–14], leading from the N-(negative) side (cytoplasmic side) of the membrane towards the heme-copper binuclear site. As identified in the
http://dx.doi.org/10.1016/j.febslet.2015.01.014 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Meyer, T., et al. Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.01.014
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T. Meyer et al. / FEBS Letters xxx (2015) xxx–xxx
structure of the CcO from Paracoccus denitrificans, studied here, the D-pathway starts at D124 (P. denitrificans CcO amino acid numbering), close to the cytoplasmic surface of CcO [15,16]. It appears to be the only pathway required when the fully reduced CcO reacts with molecular oxygen [14]. The K-pathway, named after the conserved amino acid K354, is located close to the protein/lipid interface and leads straight to the active site. This pathway may be involved in the delivery of the first one or two protons during the reduction of the oxidized enzyme. These proton uptake pathways were studied using Zn2+ and Cd2+ binding experiments describing the action of the cations from inside the liposomes on the entrance proton channels [17]. The metal binding sites have been studied by anomalous difference Fourier analyses of R. sphaeroides CcO crystals in the presence of cadmium revealing a binding site in the proposed initial proton donor/acceptor of the K pathway, E101 (E78 in P. denitrificans) of subunit II [18]. The number of Zn2+ binding sites and their affinities has been studied in detail. It was reported that the presumed inhibition of the exit pathway by Zn2+ with apparent Ki of 5–10 lM was observed only in the highly energized membrane, while 1 mM Zn2+ was required to observe inhibition from the P-side in the deenergized state [19]. As shown later on by Kuznetsova et al. [20] at least two Zn inhibitory sites are located at the opposite sides of the membrane. Binding of Zn2+ with the exit proton pathway (from the P-side of the membrane) in the oxidized CcO required either very long preincubation with lM concentrations of Zn2+ and the enzyme turning over, or mM concentrations of Zn2+ [19,20]. Vygodina et al. [21] reported that the inhibition of the exit pathway may occur rapidly with Ki of about 1 lM, provided some high potential non-heme center (e.g. CuB) is reduced prior to onset of the reaction with oxygen. Here we apply electrochemical experiments on Zn2+ inhibited CcO for the study on these mutants. The technique is based on porous three-dimensional gold nanoparticle electrodes obtained by drop-casting a concentrated gold colloid on the surface of a polycrystalline gold substrate. These electrodes allow both an exceptionally high surface coverage and a reversible direct electron transfer with the immobilized enzyme [22–24]. After immobilization of CcO, the catalytic current and the oxygen reduction potential can be investigated in presence of oxygen [23].
2.3. Electrochemistry A 2 mm diameter polycrystalline gold electrode was polished with diamond paste and activated in a 0.1 M H2SO4 solution by maintaining the potential at +2.21 V for 5 s, 0.14 V for 10 s and cycling 100 times between +0.01 V and +1.71 V at 4 V/s. To verify the cleanliness of the surface, a last scan between +0.01 V and +1.71 V was performed. The resulting electrode was subsequently modified by deposition of 3 drops of 3 ll concentrated gold nanoparticles solution. Each drop was allowed to dry under air. Then, the electrode was immersed in a 1:1 ethanolic solution of mercaptohexanol and hexanethiol (1 mM) and kept at 4 °C overnight. The resulting electrode was rinsed with ethanol and dried under argon. Then 3 ll of a 100 lM protein sample were deposited on the surface and left overnight under argon at 4 °C. Finally, the electrode was rinsed with fresh buffer to remove the excess of non-adsorbed protein. The measurements were performed in a standard three electrode cell connected to a Princeton Applied Research VERSASTAT 4 potentiostat. An AgCl/Ag 3 M NaCl reference electrode was used together with a platinum wire as counter electrode. All the potentials mentioned in this article are referred to a standard hydrogen electrode (SHE). All the voltamograms were recorded with a scan rate of 0.02 V s 1. The samples were first studied anaerobically and then cycled in the presence of oxygen. 2.4. Zinc inhibition Zinc inhibition was carried out by adding (0–2 ml) of a 2 mM buffered ZnCl2 solution to the 20 ml initial Tris buffer solution. 10 min equilibration time was required before the measurement. In control experiments 2 mM cyanide was added (see Suppl. Materials) leading to a loss of the catalytic current. IC50 were determined from the plots of catalytic current vs Zn2+ concentration. 3. Results 3.1. Electrocatalytic reaction of variants in the proton pathways Fig. 1 shows the catalytic current obtained for wild type (black line), K354M (dotted line), D124N (dark grey) variants and the D124N/K354 double variant (light gray) of CcO from P. denitrificans.
2. Materials and methods 2.1. Gold nanoparticles synthesis 15 nm gold nanoparticles were synthesized following a procedure defined by Turkevich et al. [25] and refined by Frens [26]. Briefly, 125 ml of a 1 mM solution of HAuCl4 was allowed to boil before addition of 13 ml of a 39 mM sodium citrate aqueous solution. After appearance of the red dark color, the solution was maintained under boiling for 10 min before cooling at room temperature. Finally, gold nanoparticles were centrifuged at 10 000 rpm for 30 min in order to remove 95% of the supernatant to obtain a concentrated solution of gold nanoparticles. 2.2. Protein preparation Wild type, K354M, D124N and D124N/K354M mutant enzyme CcO from P. denitrificans have been purified as previously described [27,28]. To perform protein film voltammetry, depletion of the amount of detergent of the samples was necessary. To decrease the amount of detergent of the samples, the stock protein was diluted with 50 mM Tris buffer for the zinc inhibition measurements or with 50 mM phosphate buffer. The sample was then reconcentrated to 100 lM for gold surface deposition.
Fig. 1. Voltamograms of WT (black), K354M (light gray), D124N (red), D124N/ K354M (blue) CcO from P. denitrificans at pH 7 (v = 0.02 V/s, 20 °C). The reduction potentials are 0.18 V for wild type, 0.28 V for the K354M and D124N variants and 0.31 V for the double variant (potential vs SHE).
Please cite this article in press as: Meyer, T., et al. Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.01.014
T. Meyer et al. / FEBS Letters xxx (2015) xxx–xxx
The reduction of oxygen in the wild type CcO from P. denitrificans immobilized on gold nanoparticles takes place at – 0.18 V [23]. In the different variants studied, a down shift of the reduction potential to values around – 0.28 V takes place, pointing towards a slower reduction. This effect is found for both, the K354M variant where the K path is blocked, and the D124N variant at the entry site of the D path. Interestingly even in the double mutant (D124N/ K354) a small current is still observed, and the reduction takes place at 0.31 V. The more negative potential seen for the mutant enzymes may be explained due a redox dependent protonation that is blocked in the variant. Originally it would have been expected that the oxygen reaction in variants with a blocked proton path would not be possible. In the double mutant, both the chemical and the pumped protons would not be available. It may not be excluded, however, that a small number of protons may still be transferred in the mutants, and this would be enough to enable a slow oxygen reaction. We would expect though, that the curves observed would be reduced more dramatically. However, the oxygen reaction could take place, if protons may arrive from the exit site. This possibility was discussed for the CcO from R. sphaeroides [19,29] and specific experimental evidence of proton uptake at the active site for the F ? O state transition was reported [30,31]. Proton transfer reactions in the O ? E transition of P. denitrificans CcO revealed H+-uptake and release steps during single-electron injection into oxidized CcO [32]. Interestingly, electron transfer to CuA induced proton uptake at the opposite membrane surface. Furthermore it was
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reported that in the D124N and K354M variants initial proton uptake was reduced but not blocked. We may suggest that similarly, protons arrive via the exit side here and even if the reduction of the oxygen is significantly slowed down the mutation would not hinder the turnover. 3.2. Electrocatalytic reaction in the presence of Zn2+ In order to further analyse this possibility, both the proton pathways have been blocked by adding Zn2+. As can be seen in Fig. 2, the catalytic current decreases upon subsequent addition of Zn2+; the loss in intensity correlating with the quantity of the inhibitor added. The inset shows the plots of the catalytic current vs Zn2+. After several cycles a curve can be seen, that corresponds to the cyclovoltamogram in anaerobic conditions, where only electron transfer takes place. Clearly the inhibition of both, proton entry paths and proton exit paths, with an excess of Zn2+ disables the oxygen reaction as no substrate protons are available. On the basis of the Zn2+ titration the IC50 can be calculated to be 5 lM for wild type, a value that corresponds to previous reports [17,8]. It may also be considered, that the high potential non-heme center (CuB) is reduced prior to onset of the reaction with oxygen leading to the rapid inhibition of the exit pathway [21]. The electrochemical approach can thus be used to determine the IC50 value of inhibitors. The same experiment was thus performed with the D124N mutant enzyme, where an IC50 value of 4 lM was found. This aspartate residue is thus not a binding partner for Zn2+. For the K354M mutant the catalytic current was too low for a clear analysis. 4. Conclusions From the data observed here and in line with previous studies on proton paths in CcO, it can be suggested that even if the two input proton pathways are blocked for proton translocation, protons can also arrive from the exit side of the CcO. This observed back-leak may be able to support oxidase activity as protons would be transferred to residue(s) that are supplied by proton uptake from the inside through the D/K pathway during turnover. The control of proton back-leak through the oxidase has been suggested as a mechanism of regulating the efficiency of CcO [29,33]. The arrival of a small number of protons through the D and K pathways, however, cannot be completely ruled out. Acknowledgements We are grateful to the Institut Universitaire de France (IUF), the Centre International de Recherche aux Frontières de la Chimie (FRC, Strasbourg) the Centre National de la Recherche Scientifique (CNRS), the University of Strasbourg, the Max Planck Society and the Cluster of Excellence Frankfurt (‘‘macromolecular complexes’’) for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.01. 014. References
Fig. 2. Voltamograms of WT and D124N CcO from P. denitrificans from 0 lM Zn2+ (bold light gray) to 200 lM Zn2+ (bold black). Measurements were done at pH 7 (v = 0.02 V/s, 20 °C).
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Please cite this article in press as: Meyer, T., et al. Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.01.014
journal homepage: www.FEBSLetters.org
Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants Thomas Meyer a, Frédéric Melin a, Oliver-M.H. Richter b, Bernd Ludwig b, Aimo Kannt c,1, Hanne Müller c, Hartmut Michel c, Petra Hellwig a,⇑ a
Chimie de la Matière Complexe UMR 7140, Laboratoire de Bioélectrochimie et Spectroscopie, CNRS-Université de Strasbourg, 1 rue Blaise Pascal, 67070 Strasbourg, France Institute of Biochemistry, Molecular Genetics, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany c Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Max-von-Laue-Str. 3, D-60438 Frankfurt/Main, Germany b
a r t i c l e
i n f o
Article history: Received 26 November 2014 Revised 5 January 2015 Accepted 7 January 2015 Available online xxxx Edited by Peter Brzezinski Keywords: Cytochrome c oxidase Zn2+ inhibition Proton pumping Bioelectrochemistry
a b s t r a c t Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn2+ ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The concerted movement of protons and electrons is a common feature of many energy-transducing complexes including the photosynthetic reaction center (RC), the cytochrome (cyt) bc1 complex (cyt bc1), the NADH ubiquinone oxidoreductase (complex I) or cyt c oxidase (CcO). These enzymes generate an electrochemical proton gradient across the membrane by the vectorial uptake and release of protons at the lipid bilayer in a manner coupled to electron transfer and which is then used to drive the synthesis of ATP. Earlier observations have indicated that transition metal binding in energy transducing components can be used to identify residues involved in proton transfer pathways in proton translocating enzymes. It was reported that transition metal ions such as Zn2+ and Cd2+ can inhibit the proton transfer activity of bacterial RCs [1,2]. Zn2+ binding to the RC from Rhodobacter sphaeroides, decreases the rate of Abbreviations: cyt bc1, bc1 complex; cyt, cytochrome; CcO, cytochrome c oxidase; complex I, NADH ubiquinone oxidoreductase; RC, reaction center; Tris, tris(hydroxymethyl) aminoethane ⇑ Corresponding author. 1 Present address: Sanofi-Aventis Deutschland GmbH, Diabetes R&D, Industriepark Hoechst, H824, D-65926 Frankfurt am Main, Germany.
proton transfer to the acidic residue named E212, becoming a rate-limiting step [2]. The X-ray crystal structure of the RC with bound Zn2+ revealed that the Zn2+ binding cluster D124, H126 and H128 of the RC subunit H is involved in the pathway of the first proton delivery to QB- at the entry point [3]. Also the inhibition of proton translocation by Zn2+ in the bc1 complex (complex III) [4,5] and inhibition of proton pumping in complex I [6] have been described. The IC50 for the inhibition of the respiratory complexes were estimated to be in the mid-micromolar range for complexes I and IV and in the mid nanomolar range for complex III. Under physiological conditions, the levels of free Zn2+ in mitochondria are close to zero. However, pathologic conditions may increase Zn2+ concentrations, thus inhibiting oxidative phosphorylation basically at the level of complex III [7]. It is not clear whether the inhibition of complexes I and IV are of physiological relevance. As for complex IV, it was shown that zinc inhibits the proton translocation and causes selective uncoupling from electron transfer [8–10] and it can thus be used as a tool to investigate proton pumping. Two separate proton pathways have been suggested for the bacterial CcO, the so-called D- and K-pathways [11–14], leading from the N-(negative) side (cytoplasmic side) of the membrane towards the heme-copper binuclear site. As identified in the
http://dx.doi.org/10.1016/j.febslet.2015.01.014 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Meyer, T., et al. Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.01.014
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structure of the CcO from Paracoccus denitrificans, studied here, the D-pathway starts at D124 (P. denitrificans CcO amino acid numbering), close to the cytoplasmic surface of CcO [15,16]. It appears to be the only pathway required when the fully reduced CcO reacts with molecular oxygen [14]. The K-pathway, named after the conserved amino acid K354, is located close to the protein/lipid interface and leads straight to the active site. This pathway may be involved in the delivery of the first one or two protons during the reduction of the oxidized enzyme. These proton uptake pathways were studied using Zn2+ and Cd2+ binding experiments describing the action of the cations from inside the liposomes on the entrance proton channels [17]. The metal binding sites have been studied by anomalous difference Fourier analyses of R. sphaeroides CcO crystals in the presence of cadmium revealing a binding site in the proposed initial proton donor/acceptor of the K pathway, E101 (E78 in P. denitrificans) of subunit II [18]. The number of Zn2+ binding sites and their affinities has been studied in detail. It was reported that the presumed inhibition of the exit pathway by Zn2+ with apparent Ki of 5–10 lM was observed only in the highly energized membrane, while 1 mM Zn2+ was required to observe inhibition from the P-side in the deenergized state [19]. As shown later on by Kuznetsova et al. [20] at least two Zn inhibitory sites are located at the opposite sides of the membrane. Binding of Zn2+ with the exit proton pathway (from the P-side of the membrane) in the oxidized CcO required either very long preincubation with lM concentrations of Zn2+ and the enzyme turning over, or mM concentrations of Zn2+ [19,20]. Vygodina et al. [21] reported that the inhibition of the exit pathway may occur rapidly with Ki of about 1 lM, provided some high potential non-heme center (e.g. CuB) is reduced prior to onset of the reaction with oxygen. Here we apply electrochemical experiments on Zn2+ inhibited CcO for the study on these mutants. The technique is based on porous three-dimensional gold nanoparticle electrodes obtained by drop-casting a concentrated gold colloid on the surface of a polycrystalline gold substrate. These electrodes allow both an exceptionally high surface coverage and a reversible direct electron transfer with the immobilized enzyme [22–24]. After immobilization of CcO, the catalytic current and the oxygen reduction potential can be investigated in presence of oxygen [23].
2.3. Electrochemistry A 2 mm diameter polycrystalline gold electrode was polished with diamond paste and activated in a 0.1 M H2SO4 solution by maintaining the potential at +2.21 V for 5 s, 0.14 V for 10 s and cycling 100 times between +0.01 V and +1.71 V at 4 V/s. To verify the cleanliness of the surface, a last scan between +0.01 V and +1.71 V was performed. The resulting electrode was subsequently modified by deposition of 3 drops of 3 ll concentrated gold nanoparticles solution. Each drop was allowed to dry under air. Then, the electrode was immersed in a 1:1 ethanolic solution of mercaptohexanol and hexanethiol (1 mM) and kept at 4 °C overnight. The resulting electrode was rinsed with ethanol and dried under argon. Then 3 ll of a 100 lM protein sample were deposited on the surface and left overnight under argon at 4 °C. Finally, the electrode was rinsed with fresh buffer to remove the excess of non-adsorbed protein. The measurements were performed in a standard three electrode cell connected to a Princeton Applied Research VERSASTAT 4 potentiostat. An AgCl/Ag 3 M NaCl reference electrode was used together with a platinum wire as counter electrode. All the potentials mentioned in this article are referred to a standard hydrogen electrode (SHE). All the voltamograms were recorded with a scan rate of 0.02 V s 1. The samples were first studied anaerobically and then cycled in the presence of oxygen. 2.4. Zinc inhibition Zinc inhibition was carried out by adding (0–2 ml) of a 2 mM buffered ZnCl2 solution to the 20 ml initial Tris buffer solution. 10 min equilibration time was required before the measurement. In control experiments 2 mM cyanide was added (see Suppl. Materials) leading to a loss of the catalytic current. IC50 were determined from the plots of catalytic current vs Zn2+ concentration. 3. Results 3.1. Electrocatalytic reaction of variants in the proton pathways Fig. 1 shows the catalytic current obtained for wild type (black line), K354M (dotted line), D124N (dark grey) variants and the D124N/K354 double variant (light gray) of CcO from P. denitrificans.
2. Materials and methods 2.1. Gold nanoparticles synthesis 15 nm gold nanoparticles were synthesized following a procedure defined by Turkevich et al. [25] and refined by Frens [26]. Briefly, 125 ml of a 1 mM solution of HAuCl4 was allowed to boil before addition of 13 ml of a 39 mM sodium citrate aqueous solution. After appearance of the red dark color, the solution was maintained under boiling for 10 min before cooling at room temperature. Finally, gold nanoparticles were centrifuged at 10 000 rpm for 30 min in order to remove 95% of the supernatant to obtain a concentrated solution of gold nanoparticles. 2.2. Protein preparation Wild type, K354M, D124N and D124N/K354M mutant enzyme CcO from P. denitrificans have been purified as previously described [27,28]. To perform protein film voltammetry, depletion of the amount of detergent of the samples was necessary. To decrease the amount of detergent of the samples, the stock protein was diluted with 50 mM Tris buffer for the zinc inhibition measurements or with 50 mM phosphate buffer. The sample was then reconcentrated to 100 lM for gold surface deposition.
Fig. 1. Voltamograms of WT (black), K354M (light gray), D124N (red), D124N/ K354M (blue) CcO from P. denitrificans at pH 7 (v = 0.02 V/s, 20 °C). The reduction potentials are 0.18 V for wild type, 0.28 V for the K354M and D124N variants and 0.31 V for the double variant (potential vs SHE).
Please cite this article in press as: Meyer, T., et al. Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.01.014
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The reduction of oxygen in the wild type CcO from P. denitrificans immobilized on gold nanoparticles takes place at – 0.18 V [23]. In the different variants studied, a down shift of the reduction potential to values around – 0.28 V takes place, pointing towards a slower reduction. This effect is found for both, the K354M variant where the K path is blocked, and the D124N variant at the entry site of the D path. Interestingly even in the double mutant (D124N/ K354) a small current is still observed, and the reduction takes place at 0.31 V. The more negative potential seen for the mutant enzymes may be explained due a redox dependent protonation that is blocked in the variant. Originally it would have been expected that the oxygen reaction in variants with a blocked proton path would not be possible. In the double mutant, both the chemical and the pumped protons would not be available. It may not be excluded, however, that a small number of protons may still be transferred in the mutants, and this would be enough to enable a slow oxygen reaction. We would expect though, that the curves observed would be reduced more dramatically. However, the oxygen reaction could take place, if protons may arrive from the exit site. This possibility was discussed for the CcO from R. sphaeroides [19,29] and specific experimental evidence of proton uptake at the active site for the F ? O state transition was reported [30,31]. Proton transfer reactions in the O ? E transition of P. denitrificans CcO revealed H+-uptake and release steps during single-electron injection into oxidized CcO [32]. Interestingly, electron transfer to CuA induced proton uptake at the opposite membrane surface. Furthermore it was
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reported that in the D124N and K354M variants initial proton uptake was reduced but not blocked. We may suggest that similarly, protons arrive via the exit side here and even if the reduction of the oxygen is significantly slowed down the mutation would not hinder the turnover. 3.2. Electrocatalytic reaction in the presence of Zn2+ In order to further analyse this possibility, both the proton pathways have been blocked by adding Zn2+. As can be seen in Fig. 2, the catalytic current decreases upon subsequent addition of Zn2+; the loss in intensity correlating with the quantity of the inhibitor added. The inset shows the plots of the catalytic current vs Zn2+. After several cycles a curve can be seen, that corresponds to the cyclovoltamogram in anaerobic conditions, where only electron transfer takes place. Clearly the inhibition of both, proton entry paths and proton exit paths, with an excess of Zn2+ disables the oxygen reaction as no substrate protons are available. On the basis of the Zn2+ titration the IC50 can be calculated to be 5 lM for wild type, a value that corresponds to previous reports [17,8]. It may also be considered, that the high potential non-heme center (CuB) is reduced prior to onset of the reaction with oxygen leading to the rapid inhibition of the exit pathway [21]. The electrochemical approach can thus be used to determine the IC50 value of inhibitors. The same experiment was thus performed with the D124N mutant enzyme, where an IC50 value of 4 lM was found. This aspartate residue is thus not a binding partner for Zn2+. For the K354M mutant the catalytic current was too low for a clear analysis. 4. Conclusions From the data observed here and in line with previous studies on proton paths in CcO, it can be suggested that even if the two input proton pathways are blocked for proton translocation, protons can also arrive from the exit side of the CcO. This observed back-leak may be able to support oxidase activity as protons would be transferred to residue(s) that are supplied by proton uptake from the inside through the D/K pathway during turnover. The control of proton back-leak through the oxidase has been suggested as a mechanism of regulating the efficiency of CcO [29,33]. The arrival of a small number of protons through the D and K pathways, however, cannot be completely ruled out. Acknowledgements We are grateful to the Institut Universitaire de France (IUF), the Centre International de Recherche aux Frontières de la Chimie (FRC, Strasbourg) the Centre National de la Recherche Scientifique (CNRS), the University of Strasbourg, the Max Planck Society and the Cluster of Excellence Frankfurt (‘‘macromolecular complexes’’) for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.01. 014. References
Fig. 2. Voltamograms of WT and D124N CcO from P. denitrificans from 0 lM Zn2+ (bold light gray) to 200 lM Zn2+ (bold black). Measurements were done at pH 7 (v = 0.02 V/s, 20 °C).
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Please cite this article in press as: Meyer, T., et al. Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants. FEBS Lett. (2015), http://dx.doi.org/10.1016/j.febslet.2015.01.014
