Absorbance changes following the photolysis of mildly sonicated membrane suspensions of bovine rhodopsin are monitored using multichannel detection at 15, 20, 25, 30, and 35 degrees C. Difference spectra collected with microsecond time resolution are analyzed by singular value decomposition and multiexponential fitting. Several kinetic schemes are tested using methods that compare the observed rates and associated spectral amplitudes to the eigenvalues and eigenvectors of kinetic matrices. The time evolution of the spectra is more complex than can be accounted for by the traditional lumi-->metarhodopsin I<-->metarhodopsin II scheme. Above 25 degrees C, the formation of metarhodopsin II is achieved without a large transient accumulation of metarhodopsin I. Within the framework of first-order kinetics, the observations are explained by simple kinetic schemes that lead to the formation of a deprotonated Schiff's base species temporally distinct from metarhodopsin II directly upon the decay of lumirhodopsin.

We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho). Rho 3 MII difference spectra were obtained at 10°C for unlabeled recombinant Rho and isotope-labeled L-[ring-2 H 4 ]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[ring-2 H 4 ]Tyr and isolated using a His 6 tag. A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibrational modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR 3 M transition in bacteriorhodopsin, although the sign of the bands is reversed. In bacteriorhodopsin, these bands were assigned to Tyr-185, which along with Pro-186 in the Fhelix, may form a hinge that facilitates ␣-helix movement.Elucidation of the mechanism of photoactivation of rhodopsin (Rho), 1 the light receptor in vision, remains an important problem in biology (1). Rhodopsin is an integral membrane protein found in the disc photoreceptor membranes of rod outer segments (2, 3) with a core structure consisting of seven transmembrane ␣-helices (4 -7). Upon light absorption, the rhodopsin chromophore, 11-cis-retinal, rapidly isomerizes to an alltrans configuration (8, 9) followed by a series of thermal transitions (Batho 3 Lumi 3 Meta I 3 Meta II) (10, 11). Signal transduction occurs upon formation of the Meta II intermediate, which binds and activates the G-protein transducin (12, 13). Because rhodopsin is a G-protein-coupled receptor, elucidation of its molecular mechanism is likely to be of general importance for the vast suprafamily of G-protein-coupled receptors, which include the ␤-adrenergic receptor (14, 15) and olfactory receptors (16).Thus far, bR is the only IMP with a seven-helix transmembrane motif whose structure has been elucidated at atomic resolution (17)(18)(19). This structure confirmed key features of an earlier "spectroscopic" model based in part on site-directed mutagenesis and FTIR difference spectroscopy (20 -23). For example, a retinal binding pocket was predicted on the basis of FTIR, UV-visible spectroscopy, and site-directed mutagenesis (20 -24), which is formed in part from several residues on the F-helix (helix 6 in rhodopsin), including two tryptophans (Trp-182, Trp-189), a proline (Pro-186), and a tyrosine (Tyr-185). These residues, along with several others from the C-helix (Trp-86, Thr-89, and Asp-85) are in close proximity to the retinal chromophore and act to constrain its structure in an all-trans configuration. In addition, these residues are in a good position to couple retinal isomerization to protein changes involved in proton transport, including a change in the structure and orientation of the F-helix.A comparable combination of Trp, Pro, and Tyr residues (WXPY) is fully conserved in helix 6 of all ...

Absorption changes following photolysis of bovine rhodopsin in mildly sonicated membrane suspensions are monitored at 25 degrees C. Difference spectra collected at 17 times between 1 microsecond and 75 ms following excitation are analyzed globally using singular value decomposition and non-linear least-squares fitting techniques. The results are not consistent with the simple scheme: Lumirhodopsin-->Metarhodopsin I<-->Metarhodopsin II, but indicate that an intermediate with a deprotonated Schiff's base is formed nearly simultaneously with metarhodopsin I upon the decay of Lumirhodopsin.

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A complete resonance Raman excitation profile of the heme charge-transfer band known as band III is presented. The data obtained throughout the near-infrared region show preresonance with the Q-band, but the data also clearly show the enhancement of a number of modes in the spectral region of band III. Only nontotally symmetric modes are observed to have resonance enhancement in the band III region. The observed resonance enhancements in modes of B(1g) symmetry are compared with the enhancements of those same modes in the excitation profiles of the Q-band of deoxy myoglobin, also presented here for this first time. The Q-band data agree well with the theory of vibronic coupling in metalloporphyrins (Shelnutt, J. A. J. Chem. Phys. 1981, 74, 6644-6657). The strong vibronic coupling of the Q-band of the deoxy form of hemes is discussed in terms of the enhancement of modes with both B(1g) and A(2g) symmetry. The comparison between the Q-band and band III reveals that, consistent with the theory, only modes of B(1g) symmetry are enhanced in the vicinity of band III. These results show that band III is vibronically coupled to the Soret band. The coupling of band III to modes with strong rhombic distortion of the heme macrocycle calls into question the hypothesis that the axial iron out-of-plane displacement is primarily responsible for the structure-dynamics correlations observed in myoglobin.