Aspartate 75 mutation in sensory rhodopsin II from Natronobacterium pharaonis does not influence the production of the K-like intermediate, but strongly affects its relaxation pathway

The early steps in the photocycle of the aspartate 75-mutated sensory rhodo psin II from Natrobacterium pharaonis (pSRII-D75N) were studied by time-res olved laser-induced optoacoustic spectroscopy combined with quantum yield d eterminations by flash photolysis with optical detection. Similar to the ca se of pSRII-WT, excitation of pSRII-D75N produces in subnanosecond time a K -like intermediate. Different to the case of K in pSRII-WT, in pSRII-D75N t here are two K states. K-E decays into K-L with a lifetime of 400 ns (indep endent of temperature in the range 6.5-52 degrees C) which is optically sil ent under the experimental conditions of our transient absorption experimen ts. This decay is concomitant with an expansion of 6.5 ml/mol of produced i ntermediate. This indicates a protein relaxation not affecting the chromoph ore absorption. For pSRII-D75N reconstituted into polar lipids from purple membrane, the mutation of Asp-75 by the neutral residue Asn affects neither the K-E production yield (Phi K-e 0.51 +/- 0.05) nor the energy stored by this intermediate (EEKE = 91 +/- 11 kJ/mol), nor the expansion upon its pro duction (Delta V-R,V-1 = 10 +/- 0.3 ml/mol). All these values are very simi lar to those previously determined for K with pSRII-WT in the same medium. The millisecond transient species is attributed to K-L with a lifetime corr esponding to that determined by electronic absorption spectroscopy for K-56 5. The determined energy content of the intermediates as well as the struct ural volume changes for the various steps afford the calculation of the fre e energy profile of the phototransformation during the pSRII-D75N photocycl e. These data offer insights regarding the photocycle in pSRII-WT. Detergen t solubilization of pSRII-D75N affects the sample properties to a larger ex tent than in the case of pSRII-WT.