Rapid release of retinal from a cone visual pigment following photoactivation.

As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated release of retinal from a short-wavelength-sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy. The half-time (t(1/2)) of release of retinal from VCOP was 7.1 s, 250-fold faster than that of rhodopsin. VCOP exhibited pH-dependent release kinetics, with the t(1/2) decreasing from 23 to 4 s with the pH decreasing from 4.1 to 8, respectively. However, the Arrhenius activation energy (E(a)) for VCOP derived from kinetic measurements between 4 and 20 °C was 17.4 kcal/mol, similar to the value of 18.5 kcal/mol for rhodopsin. There was a small kinetic isotope (D(2)O) effect in VCOP, but this effect was smaller than that observed in rhodopsin. Mutation of the primary Schiff base counterion (VCOP(D108A)) produced a pigment with an unprotonated chromophore (λ(max) = 360 nm) and dramatically slowed (t(1/2) ~ 6.8 min) light-dependent retinal release. Using homology modeling, a VCOP mutant with two substitutions (S85D and D108A) was designed to move the counterion one α-helical turn into the transmembrane region from the native position. This double mutant had a UV-visible absorption spectrum consistent with a protonated Schiff base (λ(max) = 420 nm). Moreover, the VCOP(S85D/D108A) mutant had retinal release kinetics (t(1/2) = 7 s) and an E(a) (18 kcal/mol) similar to those of the native pigment exhibiting no pH dependence. By contrast, the single mutant VCOP(S85D) had an ~3-fold decreased retinal release rate compared to that of the native pigment. Photoactivated VCOP(D108A) had kinetics comparable to those of a rhodopsin counterion mutant, Rho(E113Q), both requiring hydroxylamine to fully release retinal. These results demonstrate that the primary counterion of cone visual pigments is necessary for efficient Schiff base hydrolysis. We discuss how the large differences in retinal release rates between rod and cone visual pigments arise, not from inherent differences in the rate of Schiff base hydrolysis but rather from differences in the properties of noncovalent binding of the retinal chromophore to the protein.

[1]  S. Yokoyama.  Molecular evolution of vertebrate visual pigments , 2000, Progress in Retinal and Eye Research.

[2]  T. Lamb,et al.  Phototransduction, dark adaptation, and rhodopsin regeneration the proctor lecture. , 2006, Investigative ophthalmology & visual science.

[3]  R. Birge,et al.  Regulation of phototransduction in short-wavelength cone visual pigments via the retinylidene Schiff base counterion. , 2001, Biochemistry.

[4]  R. Birge,et al.  Glutamic acid 181 is negatively charged in the bathorhodopsin photointermediate of visual rhodopsin. , 2011, Journal of the American Chemical Society.

[5]  S. Kawamura,et al.  Molecular Mechanisms Characterizing Cone Photoresponses † , 2007, Photochemistry and photobiology.

[6]  R. Lefkowitz,et al.  Crystallizing Thinking about the β2-Adrenergic Receptor , 2008, Molecular Pharmacology.

[7]  Y. Shichida,et al.  Difference in molecular properties between chicken green and rhodopsin as related to the functional difference between cone and rod photoreceptor cells. , 1995, Biochemistry.

[8]  Y. Fukada,et al.  Is chicken green-sensitive cone visual pigment a rhodopsin-like pigment? A comparative study of the molecular properties between chicken green and rhodopsin. , 1994, Biochemistry.

[9]  D. Oprian,et al.  Characterization of rhodopsin congenital night blindness mutant T94I. , 2003, Biochemistry.

[10]  T. M. Esdaille,et al.  Dark Light, Rod Saturation, and the Absolute and Incremental Sensitivity of Mouse Cone Vision , 2010, The Journal of Neuroscience.

[11]  T. Lamb,et al.  Dark adaptation and the retinoid cycle of vision , 2004, Progress in Retinal and Eye Research.

[12]  Patrick Scheerer,et al.  Crystal structure of the ligand-free G-protein-coupled receptor opsin , 2008, Nature.

[13]  Edoardo Rovida,et al.  The First Steps , 2013 .

[14]  D. Farrens,et al.  Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis* , 2004, Journal of Biological Chemistry.

[15]  Marcus Elstner,et al.  The retinal conformation and its environment in rhodopsin in light of a new 2.2 A crystal structure. , 2004, Journal of molecular biology.

[16]  H. Khorana,et al.  Structure and Function in Rhodopsin , 1995, The Journal of Biological Chemistry.

[17]  E. Pugh,et al.  Lessons from photoreceptors: turning off g-protein signaling in living cells. , 2010, Physiology.

[18]  K. Yau,et al.  Role of visual pigment properties in rod and cone phototransduction , 2003, Nature.

[19]  Oliver P. Ernst,et al.  Crystal structure of metarhodopsin II , 2011, Nature.

[20]  Daniel J. Sandberg,et al.  Conserved residues in the extracellular loops of short-wavelength cone visual pigments. , 2011, Biochemistry.

[21]  K. Yau,et al.  Activation of Visual Pigments by Light and Heat , 2011, Science.

[22]  R. W. Rodieck.  The First Steps in Seeing , 1998 .

[23]  J Nathans,et al.  Determinants of visual pigment absorbance: identification of the retinylidene Schiff's base counterion in bovine rhodopsin. , 1990, Biochemistry.

[24]  T. Mielke,et al.  Electron crystallography reveals the structure of metarhodopsin I , 2004, The EMBO journal.

[25]  Steven O. Smith.  Structure and activation of the visual pigment rhodopsin. , 2010, Annual review of biophysics.

[26]  D. Farrens,et al.  Assessing structural elements that influence Schiff base stability: mutants E113Q and D190N destabilize rhodopsin through different mechanisms , 2003, Vision Research.

[27]  D. M. Starace,et al.  Activation of Transducin by a Xenopus Short Wavelength Visual Pigment* , 1997, The Journal of Biological Chemistry.

[28]  R A Mathies,et al.  Vibrationally coherent photochemistry in the femtosecond primary event of vision. , 1994, Science.

[29]  R. Birge,et al.  Regulation of photoactivation in vertebrate short wavelength visual pigments: protonation of the retinylidene Schiff base and a counterion switch. , 2007, Biochemistry.

[30]  R. Birge,et al.  Spectral tuning of deep red cone pigments. , 2008, Biochemistry.

[31]  K. Yau.  Introducing Trevor D. Lamb and Edward N. Pugh, Jr, the 2006 Recipients of the Proctor Medal , 2006 .

[32]  Yoshinori Shichida,et al.  Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[34]  A. Cooper,et al.  Mechanism of retinal schiff base formation and hydrolysis in relation to visual pigment photolysis and regeneration: resonance raman spectroscopy of a tetrahedral carbinolamine intermediate and oxygen-18 labeling of retinal at the metarhodopsin stage in photoreceptor membranes , 1987 .

[35]  T. Sakmar,et al.  Rhodopsin: insights from recent structural studies. , 2002, Annual review of biophysics and biomolecular structure.

[36]  H. Khorana,et al.  Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[37]  R. Birge,et al.  The photobleaching sequence of a short-wavelength visual pigment. , 2001, Biochemistry.

[38]  T. Morizumi,et al.  Molecular properties of rod and cone visual pigments from purified chicken cone pigments to mouse rhodopsin in situ , 2005, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[39]  W A Rushton,et al.  Rod/cone rivalry in pigment regeneration , 1968, The Journal of physiology.

[40]  R. Mathies,et al.  Structural Observation of the Primary Isomerization in Vision with Femtosecond-Stimulated Raman , 2005, Science.

[41]  R. Birge,et al.  Nature of the primary photochemical events in rhodopsin and bacteriorhodopsin. , 1990, Biochimica et biophysica acta.

[42]  Stefan Haacke,et al.  Absorption of schiff-base retinal chromophores in vacuo. , 2005, Journal of the American Chemical Society.

[43]  Edward N Pugh,et al.  G proteins and phototransduction. , 2002, Annual review of physiology.

[44]  Oliver P. Ernst,et al.  Crystal structure of opsin in its G-protein-interacting conformation , 2008, Nature.

[45]  R. Birge,et al.  Serine 85 in transmembrane helix 2 of short-wavelength visual pigments interacts with the retinylidene Schiff base counterion. , 2001, Biochemistry.

[46]  H. Mcconnell.  Adventures in physical chemistry. , 2010, Annual review of biophysics.

[47]  Krzysztof Palczewski,et al.  G protein-coupled receptor rhodopsin. , 2006, Annual review of biochemistry.

[48]  K. Fahmy,et al.  Characterization of rhodopsin-transducin interaction: a mutant rhodopsin photoproduct with a protonated Schiff base activates transducin. , 1994, Biochemistry.

[49]  A. Terakita,et al.  Conserved proline residue at position 189 in cone visual pigments as a determinant of molecular properties different from rhodopsins. , 2002, Biochemistry.

[50]  Y. Shichida,et al.  Multiple functions of Schiff base counterion in rhodopsins , 2010, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[51]  D. Kojima,et al.  Single amino acid residue as a functional determinant of rod and cone visual pigments. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[52]  K. Yau,et al.  Breaking the Covalent Bond— A Pigment Property that Contributes to Desensitization in Cones , 2005, Neuron.

[53]  P. Mcnaughton,et al.  Response properties of cones from the retina of the tiger salamander. , 1991, The Journal of physiology.

[54]  D. Oprian,et al.  Effect of carboxylic acid side chains on the absorption maximum of visual pigments. , 1989, Science.

[55]  T. Sakmar,et al.  Movement of the retinylidene Schiff base counterion in rhodopsin by one helix turn reverses the pH dependence of the metarhodopsin I to metarhodopsin II transition. , 1993, The Journal of biological chemistry.

[56]  A. Oseroff,et al.  Resonance Raman spectroscopy of rhodopsin in retinal disk membranes. , 1974, Biochemistry.

[57]  D. Kliger,et al.  Proton transfer reactions linked to rhodopsin activation. , 1998, Biochemistry.

[58]  G. Wald.  The Molecular Basis of Visual Excitation , 1968, Nature.

[59]  K. Yau,et al.  Molecular Properties of Rhodopsin and Rod Function* , 2007, Journal of Biological Chemistry.

[60]  Tetsuji Okada,et al.  Photoisomerization mechanism of rhodopsin and 9-cis-rhodopsin revealed by x-ray crystallography. , 2007, Biophysical journal.

[61]  K. Nakatani,et al.  Physiological Properties of Rod Photoreceptor Cells in Green-sensitive Cone Pigment Knock-in Mice , 2007, The Journal of General Physiology.

[62]  V. Govardovskii,et al.  Late stages of visual pigment photolysis in situ: Cones vs. rods , 2006, Vision Research.

[63]  Livia S. Carvalho,et al.  Spectral Tuning of Shortwave‐sensitive Visual Pigments in Vertebrates † , 2007, Photochemistry and photobiology.

[64]  D. Farrens,et al.  Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2* , 2003, The Journal of Biological Chemistry.

[65]  K. Yau,et al.  Quantal noise from human red cone pigment , 2008, Nature Neuroscience.

[66]  R. Birge,et al.  Vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base and a counterion switch during photoactivation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[67]  B. Honig,et al.  Visual-pigment spectra: implications of the protonation of the retinal Schiff base. , 1976, Biochemistry.

[68]  R. Birge,et al.  Phototransduction by vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base following photobleaching. , 2002, Biochemistry.

[69]  B. Knox,et al.  Cloning and expression of a Xenopus short wavelength cone pigment. , 1998, Experimental eye research.

[70]  C. Blazynski,et al.  Pathways in the hydrolysis of vertebrate rhodopsin , 1984, Vision Research.