The Photobleaching Sequence of a Short-Wavelength Visual Pigment

• Published in: Biochemistry (Easton) 2001-07, Vol.40 (26), p.7832-7844
• Main Authors: Kusnetzow, Anakarin, Dukkipati, Abhiram, Babu, Kunnel R, Singh, Deepak, Vought, Bryan W, Knox, Barry E, Birge, Robert R
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Cattle; COS Cells; Freezing; Photochemistry; Protein Binding; Protons; Retinal Cone Photoreceptor Cells - chemistry; Rhodopsin - chemistry; Rhodopsin - metabolism; Rod Opsins - chemistry; Rod Opsins - metabolism; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Xenopus laevis
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010387y

The photobleaching pathway of a short-wavelength cone opsin purified in delipidated form (λmax = 425 nm) is reported. The batho intermediate of the violet cone opsin generated at 45 K has an absorption maximum at 450 nm. The batho intermediate thermally decays to the lumi intermediate (λmax = 435 nm) at 200 K. The lumi intermediate decays to the meta I (λmax = 420 nm) and meta II (λmax = 388 nm) intermediates at 258 and 263 K, respectively. The meta II intermediate decays to free retinal and opsin at >270 K. At 45, 75, and 140 K, the photochemical excitation of the violet cone opsin at 425 nm generates the batho intermediate at high concentrations under moderate illumination. The batho intermediate spectra, generated via decomposing the photostationary state spectra at 45 and 140 K, are identical and have properties typical of batho intermediates of other visual pigments. Extended illumination of the violet cone opsin at 75 K, however, generates a red-shifted photostationary state (relative to both the dark and the batho intermediates) that has as absorption maximum at ∼470 nm, and thermally reverts to form the normal batho intermediate when warmed to 140 K. We conclude that this red-shifted photostationary state is a metastable state, characterized by a higher-energy protein conformation that allows relaxation of the all-trans chromophore into a more planar conformation. FTIR spectroscopy of violet cone opsin indicates conclusively that the chromophore is protonated. A similar transformation of the rhodopsin binding site generates a model for the VCOP binding site that predicts roughly 75% of the observed blue shift of the violet cone pigment relative to rhodopsin. MNDO-PSDCI calculations indicate that secondary interactions involving the binding site residues are as important as the first-order chromophore protein interactions in mediating the wavelength maximum.