Abstract
Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced by absorption of green light, leads to active metarhodopsin II, in which the Schiff base is intact but deprotonated. The subsequent metabolic retinoid cycle starts with Schiff base hydrolysis and release of photolyzed all-trans-retinal from the active site and ends with the uptake of fresh 11-cis-retinal. To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UV-visible spectroscopy. A light-induced shortcut of the retinoid cycle, as it occurs in other retinal proteins, is not observed. The predominantly formed illumination product contains all-trans-retinal, although the spectra reflect Schiff base reprotonation and protein deactivation. By its kinetics of formation and decay, its low temperature photointermediates, and its interaction with transducin, this illumination product is identified as metarhodopsin III. This species is known to bind all-trans-retinal via a reprotonated Schiff base and forms normally in parallel to retinal release. We find that its generation by light absorption is only achieved when starting from active metarhodopsin II and is not found with any of its precursors, including metarhodopsin I. Based on the finding of others that metarhodopsin III binds retinal in all-trans-C(15)-syn configuration, we can now conclude that light-induced formation of metarhodopsin III operates by Schiff base isomerization ("second switch"). Our reaction model assumes steric hindrance of the retinal polyene chain in the active conformation, thus preventing central double bond isomerization.
Highlights
Living cells react to stimuli, which are realized in physical or chemical signals and are often detected by specialized membrane receptor proteins
To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UVvisible spectroscopy
When the Meta II sample is subsequently illuminated with blue light, the absorption maximum is shifted to the 475 nm region
Summary
Preparation of Samples—Rod outer segments were prepared from fresh, dark-adapted retinae of cattle eyes [23]. Rhodopsin disc membranes were subsequently purified by repetitive washes with a low ionic strength buffer [24]. Opsin membranes were prepared from rhodopsin membranes by bleaching, extraction of retinals with hydroxylamine, and repetitive washes with fatty acid-free bovine serum albumin. Excess retinal was subsequently removed by repetitive washes with 2% fatty acid-free bovine serum albumin. PH was adjusted with diluted NaOH or HCl. Deuterated samples were prepared by repetitive washes of the rhodopsin membranes with BTP buffer prepared in D2O. FTIR Measurements—The sample (200 M rhodopsin membrane suspension) was centrifuged for 30 min at 100,000 ϫ g and 4 °C to obtain a 2.4 mM rhodopsin pellet [25]. The supernatant was removed, and the pellet was transferred to a temperature-controlled (Ϯ0.5 °C) transmission cuvette with two BaF2 windows and a 3-m polytetrafluorethylene spacer. An OLIS RSM-16 spectrometer was used for data aquisi-
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