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Biophys J, May 2000, p. 2581-2589, Vol. 78, No. 5

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

Aba Losi,* Ansgar A. Wegener,dagger Martin Engelhard,dagger Wolfgang Gärtner,* and Silvia E. Braslavsky*

 *Max-Planck-Institut für Strahlenchemie, Postfach 10-13-65, D-45413 Mülheim an der Ruhr, and  dagger Max-Planck-Institut für molekulare Physiologie, Rheinlanddamm 201, D-44139 Dortmund, Germany

The early steps in the photocycle of the aspartate 75-mutated sensory rhodopsin II from Natrobacterium pharaonis (pSRII-D75N) were studied by time-resolved laser-induced optoacoustic spectroscopy combined with quantum yield determinations by flash photolysis with optical detection. Similar to the case 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 there are two K states. KE decays into KL with a lifetime of 400 ns (independent of temperature in the range 6.5-52°C) which is optically silent under the experimental conditions of our transient absorption experiments. This decay is concomitant with an expansion of 6.5 ml/mol of produced intermediate. This indicates a protein relaxation not affecting the chromophore absorption. For pSRII-D75N reconstituted into polar lipids from purple membrane, the mutation of Asp-75 by the neutral residue Asn affects neither the KE production yield (Phi Ke 0.51 ± 0.05) nor the energy stored by this intermediate (EEKE = 91 ± 11 kJ/mol), nor the expansion upon its production (Delta VR,1 = 10 ± 0.3 ml/mol). All these values are very similar to those previously determined for K with pSRII-WT in the same medium. The millisecond transient species is attributed to KL with a lifetime corresponding to that determined by electronic absorption spectroscopy for K565. The determined energy content of the intermediates as well as the structural volume changes for the various steps afford the calculation of the free energy profile of the phototransformation during the pSRII-D75N photocycle. These data offer insights regarding the photocycle in pSRII-WT. Detergent solubilization of pSRII-D75N affects the sample properties to a larger extent than in the case of pSRII-WT.

Biophys J, May 2000, p. 2581-2589, Vol. 78, No. 5
© 2000 by the Biophysical Society   0006-3495/00/05/2581/09  $2.00


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