Resonance Raman studies of bovine metarhodopsin I and metarhodopsin II

Doukas, Apostolos G.; Aton, B.; Callender, Robert; Ebrey, Thomas G.

doi:10.1021/bi00605a028

Cited by 158 publications

(170 citation statements)

References 16 publications

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“…The "time scale" of the pulsed experiment is not dictated by the pulse duration but by the light intensity, because the light-dependent rates are so much larger than the light-independent rates. Doukas et al (16) have shown that metarhodopsin I does not exhibit these low wavenumber lines and looks like an all-trans chromophore. Therefore, Raman measurements of the conversion of bathorhodopsin lumirhodopsin and lumirhodopsin -metarhodopsin I should provide evidence concerning the origin of these unique bathorhodopsin lines.…”

Section: Resultsmentioning

confidence: 99%

“…Second, selective resonance enhancement permits the assignment of particular Raman lines to specific molecules in the photostationary steady state (5). These experiments have already been used to examine the conformation and interactions of the retinal chromophore in rhodopsin, isorhodopsin, and several of their photolytic intermediates (5,(12)(13)(14)(15)(16). One objective of this study was to obtain a spectrum of bathorhodopsin that would provide information on the strength of the C=N bond at the C-15 position and on the degree of protonation of the Schiff base nitrogen.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Resonance Raman studies of bathorhodopsin: Evidence for a protonated Schiff base linkage

Eyring

Mathies

1979

Proc. Natl. Acad. Sci. U.S.A.

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A dual beam pump/probe laser technique has been used with a 585nm probe wavelength to obtain maximal resonance enhancement of the Raman lines of bathorhodopsin in a photostationary steady-state mixture at -160'C. These studies show that bathorhodopsin has a protonated Schiff base vibration at 1657 cm-1 which shifts upon deuteration to 1625 cm-l. Within our experimental error (±2 cm-i) these frequencies are identical to those observed in rhodo sin and isorhodopsin. These effects show that the strength ofthe C=N bond and the degree of protonation of the Schiff base nitrogen are the same in bathorhodopsin, rhodopsin, and isorhodopsin. The implications of these results for the structure of the retinal chromophore in bathorhodopsin are discussed. The resonance Raman spectrum of pure bathorhodopsin has been generated by accurately subtracting the residual contributions of rhodopsin and isorhodopsin from spectra of the low temperature photostationary mixture. Bathorhodopsin is found to have lines at 853, 875, 920, 1006, 1166, 1210, 1278, 1323, 1536 Early studies (1-3) on the mechanism of visual excitation demonstrated that rhodopsin, the visual pigment in vertebrate rod cells, contains an 1 1-cis retinal chromophore that is isomerized to an all-trans conformation after photon absorption. This led logically to the conclusion that the primary photochemical event was a cis -trans photoisomerization about the 11,12 double bond. However, recent work has begun to question this simple model. Picosecond absorption measurements (4) demonstrated that the first photolytic intermediate, bathorhodopsin, is formed in less than 6 psec at room temperature. It was felt that this time was too short to permit the complete isomerization of the chromophore. Also, resonance Raman measurements by Oseroff and Callender (5) showed that bathorhodopsin has intense Raman lines at frequencies (856, 877, and 920 cm-') that are not found in the Raman spectrum of the all-trans protonated Schiff base derivative of retinal. They therefore concluded that the formation of bathorhodopsin might not involve a simple cis -trans isomerization about the 1 1-cis double bond. Subsequently, it has been proposed (6-8) that the initial photochemical event that leads to the formation of bathorhodopsin is a proton translocation. This hypothesis has been supported by recent picosecond absorption measurements (8) on the rate of formation of bathorhodopsin. This proton translocation might proceed to form a carbonium ion, exomethylene, or retroretinal structure, as depicted in Fig. 1. A common feature of these models is that the formation of bathorhodopsin is associated with the reduction of the bond order of the C=N double bond at the C-15 position and the increase of the H-N bond order on the Schiff base nitrogen. It must be noted, however, that the proton translocation hypothesis has been questioned by several authors (9-11) on the basis of the interconvertibility of rhodopsin, bathorhodopsin, and isorhodopsin (see Fig. 2). More detailed information about the ...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Resonance Raman studies of bathorhodopsin: Evidence for a protonated Schiff base linkage

Eyring

Mathies

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…It is tempting to speculate these latter residues act in catalysis as water activators or proton donors/acceptors because the hydrolysis reaction requires Schiff base protonation to occur efficiently. This point leads to an interesting question; how can Schiff base hydrolysis even occur in MII rhodopsin, since a protonated Schiff base is not detected for rhodopsin by vibrational spectroscopy (22,68,69)? There are two likely possibilities: (i) protonation is transient and followed by rapid hydrolysis, or (ii) a small population of MII rhodopsin may exist in the protonated form that is not resolved by the above mentioned techniques.…”

Section: Deuteruim Isotope Studies Support That a Tetrahedral Carbinomentioning

confidence: 99%

Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis

Janz¹,

Farrens

2004

Journal of Biological Chemistry

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Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E a ) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of ϳ2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.Rhodopsin, the dim light photoreceptor of rod cells, is arguably the best characterized member of the class A superfamily of GPCR (1-7). A transmembrane receptor, it has evolved into an efficient photoreceptor by covalently binding its chromophore, 11-cis-retinal, to lysine 296 via a protonated Schiff base linkage within the helical bundle (8, 9). Dim light vision begins when the 11-cis-retinal chromophore absorbs a photon and isomerizes to the all-trans-retinal form. This change in retinal initiates a series of photo-intermediates and conformational changes in the protein, resulting in the formation of metarhodopsin II (MII), 1 the active conformation of rhodopsin that is able to bind and activate the G-protein transducin. The MII photoproduct is in dynamic equilibrium with its predecessor MI, and this MI/MII pool is thought to decay through two processes (10). The MII product may be directly hydrolyzed and release all-trans-retinal from the binding pocket, or the MI pool may undergo an addition thermal isomerization along the chromophore CϭN double bond (all-trans 15-syn) giving rise to the MIII storage product (10). This MIII intermediate also decays to opsin and all-trans-retinal (albeit at a slower rate) either through the MI/MII pool or possibly direct retinal Schiff base hydrolysis of the MIII intermediate. Rhodopsin deactivation ultimately requires hydrolysis of the all-trans-retinal Schiff base linkage and release of retinal from the binding pocket. Recycling the receptor and returning it to a photosensitive conformational state completes the recovery process (11). The retinoid cycle accomplishes this task by converting the released all-trans-...

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“…Thus the v(C = ND) represents an almost 'pure' C = N vibration. The v(C = NH) bands are Table 1 Summary of peak positions (given in cm-') of the v(C = N) mode in H,O and D20, the y(C-N-D) and the v(C = C)' vibrations [25][26][27][28][29] 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700…”

Section: Spectra Of the Initial Statesmentioning

confidence: 99%

Sensory rhodopsin I photocycle intermediate SRI₃₈₀ contains 13‐cis retinal bound via an unprotonated Schiff base

et al. 1994

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Sensory rhodopsin I (SRI), the mutated derivative SRI-D76N and the complex of SRI with its transducer HtrI were overexpressed in Halobacterium salinarium and analyzed by resonance Raman spectroscopy. In the initial state SRI contains all-tram retinal bound via a protonated Schiff base as confirmed by retinal extraction which yields 95 f 3% all-trans retinal. The photocycle intermediate absorbing maximally at 380 mn (SRI& contains a Schiff base linkage between the protein and 13-cis retinal. Extraction of illuminated SRI yields up to 93% 13"cis retinal. Neither the mutation D76N nor HtrI changed the vibrational pattern of the chromophore.

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Resonance Raman studies of bovine metarhodopsin I and metarhodopsin II

Cited by 158 publications

References 16 publications

Resonance Raman studies of bathorhodopsin: Evidence for a protonated Schiff base linkage

Resonance Raman studies of bathorhodopsin: Evidence for a protonated Schiff base linkage

Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis

Sensory rhodopsin I photocycle intermediate SRI₃₈₀ contains 13‐cis retinal bound via an unprotonated Schiff base

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Resonance Raman studies of bovine metarhodopsin I and metarhodopsin II

Cited by 158 publications

References 16 publications

Resonance Raman studies of bathorhodopsin: Evidence for a protonated Schiff base linkage

Resonance Raman studies of bathorhodopsin: Evidence for a protonated Schiff base linkage

Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis

Sensory rhodopsin I photocycle intermediate SRI380 contains 13‐cis retinal bound via an unprotonated Schiff base

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Sensory rhodopsin I photocycle intermediate SRI₃₈₀ contains 13‐cis retinal bound via an unprotonated Schiff base