29th Annual Meeting of the American Society of Photobiology

Downtown Marriot

Chicago, Il.

Effect of medium and anion substitution on the early thermodynamics in Natronobacterium pharaonis halorhodopsin photocycle

Abstract-
The enthalpy (H) and structural volume changes (V) associated with the formation and decay of the early, red-shifted intermediate, K₆₀₀, in the photocycle of Natronobacterium pharaonis halorhodopsin (pHR) (1) were obtained from Laser-Induced Optoacoustic Spectroscopy (LIOAS). An expansion, 12 ml/mol V_K 19 ml/mol, accompanies the formation of K₆₀₀, the exact value depending on the medium and on the anion present in the cavity (Cl^-, NO₃^-, Br^-, I^-). A smaller expansion, 1 ml/mol V_KL 10 ml/mol, accompanies the decay of _K into the blue-shifted intermediate L₅₂₀. V_KL is negative (-6.8 mL/mol) only in the case of azide-loaded pHR, which transports protons in the opposite direction than the anion pumping pHR. This points to a key role of the conformational changes occurring in L₅₂₀ in determining the direction and sign of charge translocation. The negative linear correlation between H and V for chloride-loaded pHR observed upon mild variations in the medium is attributed to enthalpy-entropy compensation effects and allows the calculation of the constant free energy variation for these steps, G_K = (120 20) kJ/mol and G_KL = -(2 2) kJ/mol. Different to other systems (2), S and V are negatively correlated in these first steps of pHR photocycle, probably as a consequence of the necessity of the anion to remain hydrogen bound. Thus, the environment around the anion becomes bigger and at the same time more rigid during each of the two steps of the process. The photoisomerization quantum yield for the all-trans 13-cis isomerization was 0.65, as measured by laser flash photolysis, by detecting the L₅₂₀ concentration and the parent state bleaching in the microsecond time range, and taking into account the distribution of isomers in the parent state. (1) G. Varo, L.S. Brown, J. Sasaki, H. Kandori, A. Maeda, R. Needleman, J.K. Lanyi (1995) Biochemistry 44, 14490 (2) A. Losi, A.A. Wegener, M. Engelhardt, S.E. Braslavsky (2001) J. Am. Chem. Soc 123, 1766

Keywords: retinal proteins, enthalpy-entropy compensation, structural volume change, quantum yield