Photochemical reaction cycle and proton transfers in neurospora rhodopsin

It was recently found that NOP-1, a membrane protein of Neurospora crassa, shows homology to haloarchaeal rhodopsins and binds retinal after heterolog ous expression in Pichia pastoris. We report on spectroscopic properties of the Neurospora rhodopsin (NR). The photocycle was studied with flash photo lysis and time-resolved Fourier-transform infrared spectroscopy in the pH r ange 5-8. Proton release and uptake during the photocycle were monitored wi th the pH-sensitive dye, pyranine. Kinetic and spectral analysis revealed s ix distinct states in the NR photocycle, and we describe their spectral pro perties and pH-dependent kinetics in the visible and infrared ranges. The p henotypes of the mutant NR proteins, D131E and E142Q, in which the homologu es of the key carboxylic acids of the light-driven proton pump bacteriorhod opsin, Asp-85 and Asp-96, were replaced, show that Glu-142 is not involved in reprotonation of the Schiff base but Asp-131 may be. This implies that, if the NR photocycle is associated with proton transport, it has a low effi ciency, similar to that of haloarchaeal sensory rhodopsin II. Fourier-trans form Raman spectroscopy revealed unexpected differences between NR and bact eriorhodopsin in the configuration of the retinal chromophore, which may co ntribute to the less effective reprotonation switch of NR.