Zitare, U.A.; Szuster, J.; Santalla, M.C.; Llases, M.E.; Morgada, M.N.; Vila, A.J.; Murgida, D.H. "Fine Tuning of Functional Features of the Cu A Site by Loop-Directed Mutagenesis" (2019) Inorganic Chemistry. 58(3):2149-2157
El editor solo permite decargar el artículo en su versión post-print desde el repositorio. Por favor, si usted posee dicha versión, enviela a
Consulte el artículo en la página del editor
Consulte la política de Acceso Abierto del editor


Here we report the spectroscopic and electrochemical characterization of three novel chimeric Cu A proteins in which either one or the three loops surrounding the metal ions in the Thermus thermophilus protein have been replaced by homologous human and plant sequences while preserving the set of coordinating amino acids. These conservative modifications mimic basic differences between Cu A sites from different organisms and allow for fine tuning the energy gap between alternative electronic ground states of Cu A. . This results in a systematic modulation of thermodynamic and kinetic electron transfer (ET) parameters and in the selection of one of two possible redox-active molecular orbitals, which differ in the ET reorganization energy by a factor of 2. Moreover, the ET mechanism is found to be frictionally controlled, and the modifications introduced into the different chimeras do not affect the frictional activation parameter. © 2019 American Chemical Society.


Documento: Artículo
Título:Fine Tuning of Functional Features of the Cu A Site by Loop-Directed Mutagenesis
Autor:Zitare, U.A.; Szuster, J.; Santalla, M.C.; Llases, M.E.; Morgada, M.N.; Vila, A.J.; Murgida, D.H.
Filiación:Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Instituto de Química Física de Los Materiales, Medio Ambiente y Energía, Universidad de Buenos Aires, CONICET, Buenos Aires, 1428, Argentina
Departamento de Química Biológica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR), Universidad Nacional de Rosario, CONICET, Rosario, 2000, Argentina
Univ Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes), UMR 6226, Rennes, F-35000, France
Palabras clave:copper; cytochrome c oxidase; chemistry; electrochemical analysis; electron transport; kinetics; metabolism; molecular model; thermodynamics; Thermus thermophilus; X ray crystallography; Copper; Crystallography, X-Ray; Electrochemical Techniques; Electron Transport; Electron Transport Complex IV; Kinetics; Models, Molecular; Thermodynamics; Thermus thermophilus
Página de inicio:2149
Página de fin:2157
Título revista:Inorganic Chemistry
Título revista abreviado:Inorg. Chem.
CAS:copper, 15158-11-9, 7440-50-8; cytochrome c oxidase, 72841-18-0, 9001-16-5; Copper; Electron Transport Complex IV


  • Yoshikawa, S., Shimada, A., Reaction Mechanism of Cytochrome c Oxidase (2015) Chem. Rev., 115, pp. 1936-1989
  • Pauleta, S.R., Dell'Acqua, S., Moura, I., Nitrous Oxide Reductase (2013) Coord. Chem. Rev., 257, pp. 332-349
  • Kroneck, P.M.H., Walking the Seven Lines: Binuclear Copper A in Cytochrome c Oxidase and Nitrous Oxide Reductase (2018) JBIC, J. Biol. Inorg. Chem., 23, pp. 27-39
  • Alvarez-Paggi, D., Zitare, U., Murgida, D.H., The Role of Protein Dynamics and Thermal Fluctuations in Regulating Cytochrome c/Cytochrome c Oxidase Electron Transfer (2014) Biochim. Biophys. Acta, Bioenerg., 1837, pp. 1196-1207
  • Williams, P.A., Blackburn, N.J., Sanders, D., Bellamy, H., Stura, E.A., Fee, J.A., McRee, D.E., The CuA Domain of Thermus Thermophilus ba3-Type Cytochrome c Oxidase at 1.6 A Resolution (1999) Nat. Struct. Biol., 6, pp. 509-516
  • Sousa, F.L., Alves, R.J., Ribeiro, M.A., Pereira-Leal, J.B., Teixeira, M., Pereira, M.M., The Superfamily of Heme-Copper Oxygen Reductases: Types and Evolutionary Considerations (2012) Biochim. Biophys. Acta, Bioenerg., 1817, pp. 629-637
  • Solomon, E.I., Xie, X., Dey, A., Mixed Valent Sites in Biological Electron Transfer (2008) Chem. Soc. Rev., 37, p. 623
  • Gray, H.B., Malmström, B.G., Williams, R.J.P., Copper Coordination in Blue Proteins (2000) JBIC, J. Biol. Inorg. Chem., 5, pp. 551-559
  • Ramirez, B.E., Malmstrom, B.G., Winkler, J.R., Gray, H.B., The Currents of Life: The Terminal Electron-Transfer Complex of Respiration (1995) Proc. Natl. Acad. Sci. U. S. A., 92, pp. 11949-11951
  • Fisher, O.S., Kenney, G.E., Ross, M.O., Ro, S.Y., Lemma, B.E., Batelu, S., Thomas, P.M., Kelleher, N.L., Characterization of a Long Overlooked Copper Protein from Methane- and Ammonia-Oxidizing Bacteria (2018) Nat. Commun., 9, p. 4276
  • Farrar, J.A., Neese, F., Lappalainen, P., Kroneck, P.M.H., Saraste, M., Zumft, W.G., Thomson, A.J., The Electronic Structure of Cu A : A Novel Mixed-Valence Dinuclear Copper Electron-Transfer Center (1996) J. Am. Chem. Soc., 118, pp. 11501-11514
  • Neese, F., Zumft, W.G., Antholine, W.E., Kroneck, P.M.H., The Purple Mixed-Valence Cu A Center in Nitrous-Oxide Reductase: EPR of the Copper-63-, Copper-65-, and Both Copper-65- and [ 15 N]Histidine-Enriched Enzyme and a Molecular Orbital Interpretation (1996) J. Am. Chem. Soc., 118, pp. 8692-8699
  • Bertini, I., Bren, K.L., Clemente, A., Fee, J.A., Gray, H.B., Luchinat, C., Malmström, B.G., Slutter, C.E., The Cu A Center of a Soluble Domain from Thermus Cytochrome ba 3 . An NMR Investigation of the Paramagnetic Protein (1996) J. Am. Chem. Soc., 118, pp. 11658-11659
  • Abriata, L.A., Alvarez-Paggi, D., Ledesma, G.N., Blackburn, N.J., Vila, A.J., Murgida, D.H., Alternative Ground States Enable Pathway Switching in Biological Electron Transfer (2012) Proc. Natl. Acad. Sci. U. S. A., 109, pp. 17348-17353
  • Olsson, M.H.M., Ryde, U., Geometry, Reduction Potential, and Reorganization Energy of the Binuclear Cu A Site, Studied by Density Functional Theory (2001) J. Am. Chem. Soc., 123, pp. 7866-7876
  • Gorelsky, S.I., Xie, X., Chen, Y., Fee, J.A., Solomon, E.I., The Two-State Issue in the Mixed-Valence Binuclear CuA Center in Cytochrome c Oxidase and N2O Reductase (2006) J. Am. Chem. Soc., 128, pp. 16452-16453
  • Gennari, M., Pécaut, J., Debeer, S., Neese, F., Collomb, M.-N., Duboc, C., A Fully Delocalized Mixed-Valence Bis-μ(Thiolato) Dicopper Complex: A Structural and Functional Model of the Biological CuA Center (2011) Angew. Chem., Int. Ed., 50, pp. 5662-5666
  • Houser, R.P., Young, V.G., Tolman, W.B., A Thiolate-Bridged, Fully Delocalized Mixed-Valence Dicopper(I,II) Complex That Models the Cu A Biological Electron-Transfer Site (1996) J. Am. Chem. Soc., 118, pp. 2101-2102
  • Gennari, M., Pécaut, J., Collomb, M.-N., Duboc, C., A Copper Thiolate Centre for Electron Transfer: Mononuclear vs. Dinuclear Complexes (2012) Dalton Trans, 41, p. 3130
  • Zitare, U., Alvarez-Paggi, D., Morgada, M.N., Abriata, L.A., Vila, A.J., Murgida, D.H., Reversible Switching of Redox-Active Molecular Orbitals and Electron Transfer Pathways in Cu A Sites of Cytochrome c Oxidase (2015) Angew. Chem., Int. Ed., 54, pp. 9555-9559
  • Morgada, M.N., Abriata, L.A., Zitare, U., Alvarez-Paggi, D., Murgida, D.H., Vila, A.J., Control of the Electronic Ground State on an Electron-Transfer Copper Site by Second-Sphere Perturbations (2014) Angew. Chem., Int. Ed., 53, pp. 6188-6192
  • Tsai, M.-L., Hadt, R.G., Marshall, N.M., Wilson, T.D., Lu, Y., Solomon, E.I., Axial Interactions in the Mixed-Valent Cu A Active Site and Role of the Axial Methionine in Electron Transfer (2013) Proc. Natl. Acad. Sci. U. S. A., 110, pp. 14658-14663
  • Hwang, H.J., Lu, Y., PH-Dependent Transition between Delocalized and Trapped Valence States of a CuA Center and Its Possible Role in Proton-Coupled Electron Transfer (2004) Proc. Natl. Acad. Sci. U. S. A., 101, pp. 12842-12847
  • Xie, X., Gorelsky, S.I., Sarangi, R., Garner, D.K., Hwang, H.J., Hodgson, K.O., Hedman, B., Solomon, E.I., Perturbations to the Geometric and Electronic Structure of the Cu A Site: Factors That Influence Delocalization and Their Contributions to Electron Transfer (2008) J. Am. Chem. Soc., 130, pp. 5194-5205
  • Ledesma, G.N., Murgida, D.H., Ly, H.K., Wackerbarth, H., Ulstrup, J., Costa-Filho, A.J., Vila, A.J., The Met Axial Ligand Determines the Redox Potential in Cu A Sites (2007) J. Am. Chem. Soc., 129, pp. 11884-11885
  • Alvarez-Paggi, D., Abriata, L.A., Murgida, D.H., Vila, A.J., Native Cu A Redox Sites Are Largely Resilient to PH Variations within a Physiological Range (2013) Chem. Commun., 49, pp. 5381-5383
  • Alvarez-Paggi, D., Zitare, U.A., Szuster, J., Morgada, M.N., Leguto, A.J., Vila, A.J., Murgida, D.H., Tuning of Enthalpic/Entropic Parameters of a Protein Redox Center through Manipulation of the Electronic Partition Function (2017) J. Am. Chem. Soc., 139, pp. 9803-9806
  • Zaballa, M.-E., Abriata, L.A., Donaire, A., Vila, A.J., Flexibility of the Metal-Binding Region in Apo-Cupredoxins (2012) Proc. Natl. Acad. Sci. U. S. A., 109, pp. 9254-9259
  • Morgada, M.N., Abriata, L.A., Cefaro, C., Gajda, K., Banci, L., Vila, A.J., Loop Recognition and Copper-Mediated Disulfide Reduction Underpin Metal Site Assembly of CuA in Human Cytochrome Oxidase (2015) Proc. Natl. Acad. Sci. U. S. A., 112, pp. 11771-11776
  • Soulimane, T., Buse, G., Bourenkov, G.P., Bartunik, H.D., Huber, R., Than, M.E., Structure and Mechanism of the Aberrant ba3-cytochrome c Oxidase from Thermus Thermophilus (2000) EMBO J., 19, pp. 1766-1776
  • Andersson, R., Safari, C., Dods, R., Nango, E., Tanaka, R., Yamashita, A., Nakane, T., Båth, P., Serial Femtosecond Crystallography Structure of Cytochrome c Oxidase at Room Temperature (2017) Sci. Rep., 7, p. 4518
  • Gough, J., Chothia, C., The Linked Conservation of Structure and Function in a Family of High Diversity (2004) Structure, 12, pp. 917-925
  • Steffens, G.J., Buse, G., Studies on Cytochrome c Oxidase, IV[1 - 3]. Primary Structure and Function of Subunit II (1979) Hoppe-Seylers Z. für Physiol. Chem., 360, pp. 613-619
  • Abriata, L.A., Vila, A.J., Dal Peraro, M., Molecular Dynamics Simulations of Apocupredoxins: Insights into the Formation and Stabilization of Copper Sites under Entatic Control (2014) JBIC, J. Biol. Inorg. Chem., 19, pp. 565-575
  • Muresanu, L., Pristovsek, P., Löhr, F., Maneg, O., Mukrasch, M.D., Rüterjans, H., Ludwig, B., Lücke, C., The Electron Transfer Complex between Cytochrome C552 and the CuA Domain of the Thermus Thermophilus ba3 Oxidase. A Combined NMR and Computational Approach (2006) J. Biol. Chem., 281, pp. 14503-14513
  • Gamelin, D.R., Randall, D.W., Hay, M.T., Houser, R.P., Mulder, T.C., Canters, G.W., De Vries, S., Solomon, E.I., Spectroscopy of Mixed-Valence Cu A -Type Centers: Ligand-Field Control of Ground-State Properties Related to Electron Transfer (1998) J. Am. Chem. Soc., 120, pp. 5246-5263
  • Debeer George, S., Metz, M., Szilagyi, R.K., Wang, H., Cramer, S.P., Lu, Y., Tolman, W.B., Solomon, E.I., A Quantitative Description of the Ground-State Wave Function of Cu A by X-Ray Absorption Spectroscopy: Comparison to Plastocyanin and Relevance to Electron Transfer (2001) J. Am. Chem. Soc., 123, pp. 5757-5767
  • Andrew, C.R., Fraczkiewicz, R., Czernuszewicz, R.S., Lappalainen, P., Saraste, M., Sanders-Loehr, J., Identification and Description of Copper-Thiolate Vibrations in the Dinuclear Cu A Site of Cytochrome c Oxidase (1996) J. Am. Chem. Soc., 118, pp. 10436-10445
  • Clark, K.M., Tian, S., Van Der Donk, W.A., Lu, Y., Probing the Role of the Backbone Carbonyl Interaction with the Cu A Center in Azurin by Replacing the Peptide Bond with an Ester Linkage (2017) Chem. Commun., 53, pp. 224-227
  • Kyte, J., Doolittle, R.F., A Simple Method for Displaying the Hydropathic Character of a Protein (1982) J. Mol. Biol., 157, pp. 105-132
  • Battistuzzi, G., Borsari, M., Cowan, J.A., Eicken, C., Loschi, L., Sola, M., Redox Chemistry and Acid-Base Equilibria of Mitochondrial Plant Cytochromes c (1999) Biochemistry, 38, pp. 5553-5562
  • Battistuzzi, G., Borsari, M., Loschi, L., Sola, M., Redox Thermodynamics, Acid-Base Equilibria and Salt-Induced Effects for the Cucumber Basic Protein. General Implications for Blue-Copper Proteins (1997) JBIC, J. Biol. Inorg. Chem., 2, pp. 350-359
  • Bertrand, P., Mbarki, O., Asso, M., Blanchard, L., Guerlesquin, F., Tegoni, M., Control of the Redox Potential in C-Type Cytochromes: Importance of the Entropic Contribution (1995) Biochemistry, 34, pp. 11071-11079
  • Battistuzzi, G., Bellei, M., Borsari, M., Canters, G.W., De Waal, E., Jeuken, L.J.C., Ranieri, A., Sola, M., Control of Metalloprotein Reduction Potential: Compensation Phenomena in the Reduction Thermodynamics of Blue Copper Proteins (2003) Biochemistry, 42, pp. 9214-9220
  • Battistuzzi, G., Borsari, M., Di Rocco, G., Ranieri, A., Sola, M., Enthalpy/Entropy Compensation Phenomena in the Reduction Thermodynamics of Electron Transport Metalloproteins (2004) JBIC, J. Biol. Inorg. Chem., 9, pp. 23-26
  • Blackburn, N.J., De Vries, S., Barr, M.E., Houser, R.P., Tolman, W.B., Sanders, D., Fee, J.A., X-Ray Absorption Studies on the Mixed-Valence and Fully Reduced Forms of the Soluble Cu A Domains of Cytochrome c Oxidase (1997) J. Am. Chem. Soc., 119, pp. 6135-6143
  • Zitare, U.A., Szuster, J., Scocozza, M.F., Espinoza-Cara, A., Leguto, A.J., Morgada, M.N., Vila, A.J., Murgida, D.H., The Role of Molecular Crowding in Long-Range Metalloprotein Electron Transfer: Dissection into Site- and Scaffold-Specific Contributions (2019) Electrochim. Acta, 294, pp. 117-125
  • Fujita, K., Nakamura, N., Ohno, H., Leigh, B.S., Niki, K., Gray, H.B., Richards, J.H., Mimicking Protein-Protein Electron Transfer: Voltammetry of Pseudomonas Aeruginosa Azurin and the Thermus Thermophilus Cu A Domain at ω-Derivatized Self-Assembled-Monolayer Gold Electrodes (2004) J. Am. Chem. Soc., 126, pp. 13954-13961
  • Marcus, R.A., On the Theory of Electron-Transfer Reactions. VI. Unified Treatment for Homogeneous and Electrode Reactions (1965) J. Chem. Phys., 43, pp. 679-701
  • Marcus, R.A., Chemical and Electrochemical Electron-Transfer Theory (1964) Annu. Rev. Phys. Chem., 15, pp. 155-196
  • Laviron, E., General Expression of the Linear Potential Sweep Voltammogram in the Case of Diffusionless Electrochemical Systems (1979) J. Electroanal. Chem. Interfacial Electrochem., 101, pp. 19-28
  • Monari, S., Battistuzzi, G., Bortolotti, C.A., Yanagisawa, S., Sato, K., Li, C., Salard, I., Ranieri, A., Understanding the Mechanism of Short-Range Electron Transfer Using an Immobilized Cupredoxin (2012) J. Am. Chem. Soc., 134, pp. 11848-11851
  • Leguto, A.J., Smith, M., Morgada, M.N., Zitare, U.A., Murgida, D.H., Lancaster, K.M., Vila, A.J., Dramatic Electronic Perturbations of CuA Centers via Subtle Geometric Changes (2019) J. Am. Chem. Soc., , press
  • Marcus, R.A., Sutin, N., Electron Transfers in Chemistry and Biology (1985) Biochim. Biophys. Acta, Rev. Bioenerg., 811, pp. 265-322
  • Sumi, H., Marcus, R.A., Dynamical Effects in Electron Transfer Reactions (1986) J. Chem. Phys., 84, pp. 4894-4914
  • Khoshtariya, D.E., Dolidze, T.D., Shushanyan, M., Davis, K.L., Waldeck, D.H., Van Eldik, R., Fundamental Signatures of Short- and Long-Range Electron Transfer for the Blue Copper Protein Azurin at Au/SAM Junctions (2010) Proc. Natl. Acad. Sci. U. S. A., 107, pp. 2757-2762
  • Kuimova, M.K., Mapping Viscosity in Cells Using Molecular Rotors (2012) Phys. Chem. Chem. Phys., 14, pp. 12671-12686


---------- APA ----------
Zitare, U.A., Szuster, J., Santalla, M.C., Llases, M.E., Morgada, M.N., Vila, A.J. & Murgida, D.H. (2019) . Fine Tuning of Functional Features of the Cu A Site by Loop-Directed Mutagenesis. Inorganic Chemistry, 58(3), 2149-2157.
---------- CHICAGO ----------
Zitare, U.A., Szuster, J., Santalla, M.C., Llases, M.E., Morgada, M.N., Vila, A.J., et al. "Fine Tuning of Functional Features of the Cu A Site by Loop-Directed Mutagenesis" . Inorganic Chemistry 58, no. 3 (2019) : 2149-2157.
---------- MLA ----------
Zitare, U.A., Szuster, J., Santalla, M.C., Llases, M.E., Morgada, M.N., Vila, A.J., et al. "Fine Tuning of Functional Features of the Cu A Site by Loop-Directed Mutagenesis" . Inorganic Chemistry, vol. 58, no. 3, 2019, pp. 2149-2157.
---------- VANCOUVER ----------
Zitare, U.A., Szuster, J., Santalla, M.C., Llases, M.E., Morgada, M.N., Vila, A.J., et al. Fine Tuning of Functional Features of the Cu A Site by Loop-Directed Mutagenesis. Inorg. Chem. 2019;58(3):2149-2157.