Electric-Field Dependent Decays of Two Spectroscopically Different M-States of Photosensory Rhodopsin II from Natronobacterium pharaonis

Abstract

Sensory rhodopsin II (NpSRII) from Natronobacterium pharaonis was studied by resonance Raman (RR) spectroscopic techniques. Using gated 413-nm excitation, time-resolved RR measurements of the solubilized photoreceptor were carried out to probe the photocycle intermediates that are formed in the submillisecond time range. For the first time, two M-like intermediates were identified on the basis of their C=C stretching bands at 1568 and 1583 cm(-1), corresponding to the early M(L)(400) state with a lifetime of 30 micro s and the subsequent M(1)(400) state with a lifetime of 2 ms, respectively. The unusually high C=C stretching frequency of M(1)(400) has been attributed to an unprotonated retinal Schiff base in a largely hydrophobic environment, implying that the M(L)(400) --> M(1)(400) transition is associated with protein structural changes in the vicinity of the chromophore binding pocket. Time-resolved surface enhanced resonance Raman experiments of NpSRII electrostatically bound onto a rotating Ag electrode reveal that the photoreceptor runs through the photocycle also in the immobilized state. Surface enhanced resonance Raman spectra are very similar to the RR spectra of the solubilized protein, ruling out adsorption-induced structural changes in the retinal binding pocket. The photocycle kinetics, however, is sensitively affected by the electrode potential such that at 0.0 V (versus Ag/AgCl) the decay times of M(L)(400) and M(1)(400) are drastically slowed down. Upon decreasing the potential to -0.4 V, that corresponds to a decrease of the interfacial potential drop and thus of the electric field strength at the protein binding site, the photocycle kinetics becomes similar to that of NpSRII in solution. The electric-field dependence of the protein structural changes associated with the M-state transitions, which in the present spectroscopic work is revealed on a molecular level, appears to be related to the electric-field control of bacteriorhodopsin's photocycle, which has been shown to be of functional relevance.

Resolving voltage-dependent structural changes of a membrane photoreceptor by surface-enhanced IR difference spectroscopy

“…The presented SEIDA data complement those derived from surface-enhanced resonance Raman spectroscopy (SERRS) that reveal distinct structural changes of the retinal cofactor dependent on the applied potential (33). Thus, spectroscopic methods that combine structural and electrochemical sensitivity are highly relevant and form the basis of the biophysical scrutiny of the physiological function of membrane proteins and its regulation by the membrane potential.…”

Role of Arg-72 of pharaonis Phoborhodopsin (Sensory Rhodopsin II) on its Photochemistry

“…We should take into consideration the transition from M1 to M2, or the population of these two M-states being affected by the mutation. The spectroscopically separated two-M-states of the wild-type ppR was described in Chizhov et al (1998) and Rivas et al (2003). Further quantitative study on this respect should be necessary.…”

Time‐resolved resonance Raman spectroscopy of sensory rhodopsin II in the micro‐ and millisecond time range using gated cw excitation

“…3 The advantages of this approach are demonstrated using sensory rhodopsin II from Natronbacterium pharaonis (NpSRII), 4 for which due to its long recovery time RR spectroscopic studies have been restricted so far to quasistationary conditions. 5,6 NpSRII serves as photoreceptor in bacterial phototaxis. 7 The physiological function is linked to a reversible photoinduced reaction cycle of the retinal chromophore that is covalently bound to the apoprotein via a Schiff base linkage.…”