Abstract
Neuroglobin (Ngb) is a small globular protein that binds diatomic ligands like oxygen, carbon monoxide (CO) and nitric oxide at a heme prosthetic group. We have performed FTIR spectroscopy in the infrared stretching bands of CO and flash photolysis with monitoring in the electronic heme absorption bands to investigate structural heterogeneity at the active site of Ngb and its effects on CO binding and migration at cryogenic temperatures. Four CO stretching bands were identified; they correspond to discrete conformations that differ in structural details and CO binding properties. Based on a comparison of bound-state and photoproduct IR spectra of the wild-type protein, Ngb distal pocket mutants and myoglobin, we have provided structural interpretations of the conformations associated with the different CO bands. We have also studied ligand migration to the primary docking site, B. Rebinding from this site is governed by very low enthalpy barriers (∼1 kJ/mol), indicating an extremely reactive heme iron. Moreover, we have observed ligand migration to a secondary docking site, C, from which CO rebinding involves higher enthalpy barriers.
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References
Stryer, L.: Biochemistry, 4th ed., Freeman, San Francisco, 1995.
Nienhaus, G.U. and Young, R.D.: Protein dynamics, VCH, New York, 1996.
Frauenfelder, H., Sligar, S.G. and Wolynes, P.G.: The Energy Landscapes and Motions of Proteins, Science 254 (1991), 1598–1603.
Ansari, A., Berendzen, J., Bowne, S.F., Frauenfelder, H., Iben, I.E., Sauke, T.B., Shyamsunder, E. and Young, R.D.: Protein States and Proteinquakes, Proc. Natl. Acad. Sci. U.S.A. 82 (1985), 5000–5004.
Ansari, A., Berendzen, J., Braunstein, D., Cowen, B.R., Frauenfelder, H., Hong, M.K., Iben, I.E., Johnson, J.B., Ormos, P., Sauke, T.B., et al.: Rebinding and Relaxation in the Myoglobin Pocket, Biophys. Chem. 26 (1987), 337–355.
Andrews, B.K., Romo, T., Clarage, J.B., Pettitt, B.M. and Phillips, G.N., Jr.: Characterizing Global Substates of Myoglobin, Structure 6 (1998), 587–594.
Garcia, A.E., Blumenfeld, R., Hummer, G. and Krumhansl, J.A.: Multi-Basin Dynamics of a Protein in a Crystal Environment, Physica D 107 (1997), 225–239.
Becker, O.M. and Karplus, M.: The Topology of Multidimensional Potential Energy Surfaces: Theory and Application to Peptide Structure and Kinetics, J. Chem. Phys. 106 (1997), 1495–1517.
McMahon, B.H., Müller, J.D., Wraight, C.A. and Nienhaus, G.U.: Electron Transfer and Protein Dynamics in the Photosynthetic Reaction Center, Biophys. J. 74 (1998), 2567–2587.
Thorn, L.D. and Wiersma, D.A.: Real Time Observation of Low-Temperature Protein Motions, Phys. Rev. Lett. 74 (1995), 2138–2141.
Hofmann, C., Aartsma, T.J., Michel, H. and Kohler, J.: Direct Observation of Tiers in the Energy Landscape of a Chromoprotein: A Single-Molecule Study, Proc. Natl. Acad. Sci. U.S.A. 100 (2003), 15534–15538.
Braunstein, D.P., Chu, K., Egeberg, K.D., Frauenfelder, H., Mourant, J.R., Nienhaus, G.U., Ormos, P., Sligar, S.G., Springer, B.A. and Young, R.D.: Ligand Binding to Heme Proteins: III. FTIR Studies of His-E7 and Val-E11 Mutants of Carbonmonoxymyoglobin, Biophys. J. 65 (1993), 2447–2454.
Li, T., Quillin, M.L., Phillips, G.N., Jr. and Olson, J.S.: Structural Determinants of the Stretching Frequency of CO Bound to Myoglobin, Biochemistry 33 (1994), 1433–1446.
Vojtechovsky, J., Chu, K., Berendzen, J., Sweet, R.M. and Schlichting, I.: Crystal Structures of Myoglobin-Ligand Complexes at Near-Atomic Resolution, Biophys. J. 77 (1999), 2153–2174.
Johnson, J.B., Lamb, D.C., Frauenfelder, H., Müller, J.D., McMahon, B., Nienhaus, G.U. and Young, R.D.: Ligand Binding to Heme Proteins. VI. Interconversion of Taxonomic Substates in Carbonmonoxymyoglobin, Biophys. J. 71 (1996), 1563–1573.
Müller, J.D., McMahon, B.H., Chien, E.Y., Sligar, S.G. and Nienhaus, G.U.: Connection between the Taxonomic Substates and Protonation of Histidines 64 and 97 in Carbonmonoxy Myoglobin, Biophys. J. 77 (1999), 1036–1051.
Kriegl, J.M., Nienhaus, K., Deng, P., Fuchs, J. and Nienhaus, G.U.: Ligand Dynamics in a Protein Internal Cavity, Proc. Natl. Acad. Sci. U.S.A. 100 (2003), 7069–7074.
Alben, J.O., Beece, D., Bowne, S.F., Doster, W., Eisenstein, L., Frauenfelder, H., Good, D., McDonald, J.D., Marden, M.C., Moh, P.P., Reinisch, L., Reynolds, A.H., Shyamsunder, E. and Yue, K.T.: Infrared Spectroscopy of Photodissociated Carboxymyoglobin at Low Temperatures, Proc. Natl. Acad. Sci. U.S.A. 79 (1982), 3744–3748.
Nienhaus, K., Deng, P., Kriegl, J.M. and Nienhaus, G.U.: Structural Dynamics of Myoglobin: The Effect of Internal Cavities on Ligand Migration and Binding, Biochemistry 42 (2003), 9647–9658.
Nienhaus, K., Deng, P., Kriegl, J.M. and Nienhaus, G.U.: Structural Dynamics of Myoglobin: Spectroscopic and Structural Characterization of Ligand Docking Sites in Myoglobin Mutant L29W, Biochemistry 42 (2003), 9633–9646.
Nienhaus, K., Deng, P., Olson, J.S., Warren, J.J. and Nienhaus, G.U.: Structural Dynamics of Myoglobin: Ligand Migration and Binding in Valine 68 Mutants, J. Biol. Chem. 278 (2003), 42532–42544.
Nienhaus, G.U., Mourant, J.R., Chu, K. and Frauenfelder, H.: Ligand Binding to Heme Proteins: The Effect of Light on Ligand Binding in Myoglobin, Biochemistry 33 (1994), 13413–13430.
Ostermann, A., Waschipky, R., Parak, F.G. and Nienhaus, G.U.: Ligand Binding and Conformational Motions in Myoglobin, Nature 404 (2000), 205–208.
Lim, M., Jackson, T.A. and Anfinrud, P.A.: Mid-Infrared Vibrational Spectrum of CO after Photodissociation from Heme: Evidence for a Ligand Docking Site in the Heme Pocket of Hemoglobin and Myoglobin, J. Chem. Phys. 102 (1995), 4355–4366.
Lim, M., Jackson, T.A. and Anfinrud, P.A.: Ultrafast Rotation and Trapping of Carbon Monoxide Dissociated from Myoglobin, Nat. Struct. Biol. 4 (1997), 209–214.
Tilton, R.F., Jr., Kuntz, I.D., Jr. and Petsko, G.A.: Cavities in proteins: Structure of a Metmyoglobin-Xenon Complex Solved to 1.9 Å, Biochemistry 23 (1984), 2849–2857.
Burmester, T., Weich, B., Reinhardt, S. and Hankeln, T.: A Vertebrate Globin Expressed in the Brain, Nature 407 (2000), 520–523.
Venis, S.: Neuroglobin might Protect Brain Cells During Stroke, Lancet 358 (2001), 2055.
Sun, Y., Jin, K., Mao, X.O., Zhu, Y. and Greenberg, D.A.: Neuroglobin is Up-Regulated by and Protects Neurons from Hypoxic-Ischemic Injury, Proc. Natl. Acad. Sci. U.S.A. 98 (2001), 15306–15311.
Sun, Y., Jin, K., Peel, A., Mao, X.O., Xie, L. and Greenberg, D.A.: Neuroglobin Protects the Brain from Experimental Stroke in vivo, Proc. Natl. Acad. Sci. U.S.A. 100 (2003), 3497–3500.
Wakasugi, K., Nakano, T. and Morishima, I.: Oxidized Human Neuroglobin Acts as a Heterotrimeric Galpha Protein Guanine Nucleotide Dissociation Inhibitor, J. Biol. Chem. 278 (2003), 36505–36512.
Vallone, B., Nienhaus, K., Matthes, A., Brunori, M. and Nienhaus, G.U.: The Structure of Carbonmonoxy Neuroglobin Reveals a Heme-Sliding Mechanism for Control of Ligand Affinity, Proc. Natl. Acad. Sci. U.S.A., in press.
Kriegl, J.M., Bhattacharyya, A.J., Nienhaus, K., Deng, P., Minkow, O. and Nienhaus, G.U.: Ligand Binding and Protein Dynamics in Neuroglobin, Proc. Natl. Acad. Sci. U.S.A. 99 (2002), 7992–7997.
Pesce, A., Dewilde, S., Nardini, M., Moens, L., Ascenzi, P., Hankeln, T., Burmester, T. and Bolognesi, M.: Human Brain Neuroglobin Structure Reveals a Distinct Mode of Controlling Oxygen Affinity, Structure (Camb.) 11 (2003), 1087–1095.
Vallone, B., Nienhaus, K., Brunori, M. and Nienhaus, G.U.: The Structure of Murine Neuroglobin: Novel Pathways for Ligand Migration and Binding, Proteins 56 (2004), 85–92.
Nienhaus, K., Kriegl, J.M. and Nienhaus, G.U.: Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding, J. Biol. Chem. 279 (2004), 22944–22952.
Dewilde, S., Kiger, L., Burmester, T., Hankeln, T., Baudin-Creuza, V., Aerts, T., Marden, M.C., Caubergs, R. and Moens, L.: Biochemical Characterization and Ligand Binding Properties of Neuroglobin, a Novel Member of the Globin Family, J. Biol. Chem. 276 (2001), 38949–38955.
Trent, J.T., Watts, R.A. and Hargrove, M.S.: Human Neuroglobin, a Hexacoordinate Hemoglobin that Reversibly Binds Oxygen, J. Biol. Chem. 276 (2001), 30106–30110.
Uno, T., Ryu, D., Tsutsumi, H., Tomisugi, Y., Ishikawa, Y., Wilkinson, A.J., Sato, H. and Hayashi, T.: Residues in the Distal Heme Pocket of Neuroglobin: Implications for the Multiple Ligand Binding Steps, J. Biol. Chem. 279 (2003), 5886–5893.
Lamb, D.C., Nienhaus, K., Arcovito, A., Draghi, F., Miele, A.E., Brunori, M. and Nienhaus, G.U.: Structural Dynamics of Myoglobin: Ligand Migration Among Protein Cavities Studied by Fourier Transform Infrared/Temperature Derivative Spectroscopy, J. Biol. Chem. 277 (2002), 11636–11644.
Nienhaus, G.U. and Nienhaus, K.: Infrared Study of Carbon Monoxide Migration among Internal Cavities of Myoglobin Mutant L29W, J. Biol. Phys. 28 (2002), 163–172.
Berendzen, J. and Braunstein, D.: Temperature-Derivative Spectroscopy: A Tool for Protein Dynamics, Proc. Natl. Acad. Sci. U.S.A. 87 (1990), 1–5.
Mourant, J.R., Braunstein, D.P., Chu, K., Frauenfelder, H., Nienhaus, G.U., Ormos, P. and Young, R.D.: Ligand Binding to Heme Proteins: II. Transitions in the Heme Pocket of Myoglobin, Biophys. J. 65 (1993), 1496–1507.
Young, R.D. and Bowne, S.F.: Conformational Substates and Barrier Height Distributions in Ligand Binding to Heme Proteins, J. Chem. Phys. 81 (1984), 3730–3737.
Lamb, D.C., Kriegl, J., Kastens, K. and Nienhaus, G.U.: Quantum-Mechanical Tunneling of Water in Heme Proteins, J. Phys. Org. Chem. 13 (2000), 1–5.
Alben, J.O., Beece, D., Bowne, S.F., Eisenstein, L., Frauenfelder, H., Good, D., Marden, M., Moh, P.P., Reinisch, L., Reynolds, A.H. and Yue, K.T.: Isotope Effect in Molecular Tunneling, Phys. Rev. Lett. 44 (1980), 1157–1160.
Steinbach, P.J., Chu, K., Frauenfelder, H., Johnson, J.B., Lamb, D.C., Nienhaus, G.U., Sauke, T.B. and Young, R.D.: Determination of Rate Distributions from Kinetic Experiments, Biophys. J. 61 (1992), 235–245.
Steinbach, P.J., Ansari, A., Berendzen, J., Braunstein, D., Chu, K., Cowen, B.R., Ehrenstein, D., Frauenfelder, H., Johnson, J.B., Lamb, D.C., Luck, S., Mourant, J.R., Nienhaus, G.U., Ormos, P., Philipp, R., Xie, A. and Young, R.D.: Ligand Binding to Heme Proteins: Connection Between Dynamics and Function, Biochemistry 30 (1991), 3988–4001.
Austin, R.H., Beeson, K.W., Eisenstein, L., Frauenfelder, H. and Gunsalus, I.C.: Dynamics of Ligand Binding to Myoglobin, Biochemistry 14 (1975), 5355–5373.
Ormos, P., Szaraz, S., Cupane, A. and Nienhaus, G.U.: Structural Factors Controlling Ligand Binding to Myoglobin: A Kinetic Hole-Burning Study, Proc. Natl. Acad. Sci. U.S.A. 95 (1998), 6762–6767.
Phillips, G.N., Jr., Teodoro, M.L., Li, T., Smith, B. and Olson, J.S.: Bound CO is a Molecular Probe of Electrostatic Potential in the Distal Pocket of Myoglobin, J. Phys. Chem. B 103 (1999), 8817–8829.
Nienhaus, K., Olson, J.S., Franzen, S. and Nienhaus, G.U.: The Origin of Stark Splitting in the Initial Photoproduct State of MbCO, J. Am. Chem. Soc. 127 (2005), 40–41.
Yang, F. and Phillips, G.N., Jr.: Crystal Structures of CO-, Deoxy- and Met-Myoglobins at Various pH Values, J. Mol. Biol. 256 (1996), 762–774.
Alben, J.O. and Caughey, W.S.: An Infrared Study of Bound Carbon Monoxide in the Human Red Blood Cell, Isolated Hemoglobin, and Heme Carbonyls, Biochemistry 7 (1968), 175–183.
Ray, G.B., Li, X.-Y., Ibers, J.A., Sessler, J.L. and Spiro, G.S.: How Far can Proteins Bend the FeCO Unit, J. Am. Chem. Soc. 116 (1994), 162–176.
Couture, M., Burmester, T., Hankeln, T. and Rousseau, D.L.: The Heme Environment of Mouse Neuroglobin. Evidence for the Presence of two Conformations of the Heme Pocket, J. Biol. Chem. 276 (2001), 36377–36382.
Bhattacharya, S., Sukits, S.F., MacLaughlin, K.L. and Lecomte, J.T.: The Tautomeric State of Histidines in Myoglobin, Biophys. J. 73 (1997), 3230–3240.
Yeh, S.R., Couture, M., Ouellet, Y., Guertin, M. and Rousseau, D.L.: A Cooperative Oxygen Binding Hemoglobin from Mycobacterium Tuberculosis. Stabilization of Heme Ligands by a Distal Tyrosine Residue, J. Biol. Chem. 275 (2000), 1679–1684.
Schlichting, I., Berendzen, J., Phillips, G.N., Jr. and Sweet, R.M.: Crystal Structure of Photolysed Carbonmonoxy-Myoglobin, Nature 371 (1994), 808–812.
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Nienhaus, K., Nienhaus, G.U. A Spectroscopic Study of Structural Heterogeneity and Carbon Monoxide Binding in Neuroglobin. J Biol Phys 31, 417–432 (2005). https://doi.org/10.1007/s10867-005-0173-0
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DOI: https://doi.org/10.1007/s10867-005-0173-0