STATIC AND TIME-RESOLVED ABSORPTION-SPECTROSCOPY OF THE BACTERIORHODOPSIN MUTANT TYR-185-]PHE - EVIDENCE FOR AN EQUILIBRIUM BETWEEN BR(570) AND AN O-LIKE SPECIES

被引:38
作者
SONAR, S
KREBS, MP
KHORANA, HG
ROTHSCHILD, KJ
机构
[1] BOSTON UNIV, DEPT PHYS, 590 COMMONWEALTH AVE, BOSTON, MA 02215 USA
[2] BOSTON UNIV, PROGRAM CELLULAR BIOPHYS, BOSTON, MA 02215 USA
[3] MIT, DEPT BIOL, CAMBRIDGE, MA 02139 USA
[4] MIT, DEPT CHEM, CAMBRIDGE, MA 02139 USA
关键词
D O I
10.1021/bi00060a019
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The light-dark adaptation, photocycle kinetics, and acid-induced blue formation of the bacteriorhodopsin (bR) mutant Tyr-185-->Phe (Y185F) expressed in Halobacterium halobium have been investigated by both static and time-resolved visible absorption spectroscopy. Evidence is presented that a pH-dependent equilibrium exists between a bR570-like form (bR570Y185F) and a red-shifted species in the light-adapted form of Y185F. In two related papers, we show that this species has vibrational features similar to the O intermediate. Key findings are that light adaptation causes formation of a purple species similar to bR570 and a second long-lived red-shifted species with a lambda(max) near 630 nm, well above the pH for the acid-induced blue transition. The concentration of the red-shifted species is pH- and salt-dependent, decreasing reversibly at high pH and high ionic strength. The dark-adapted state of Y185F also contains a small amount of the red-shifted species which is reversibly titratable. Dark adaptation is much slower than wild-type bR and causes a parallel decay of light-adapted bR and the red-shifted species. Time-resolved visible absorption spectroscopy reveals that the purple and the red-shifted species undergo separate photocycles. The purple species exhibits a relatively normal photocycle except for an increased rate of M formation kinetics. The red-shifted species has a photocycle involving a red-shifted K intermediate and a second longer lived intermediate possibly similar to N. The apparent absence of an O intermediate in the late photocycle of Y185F is attributed to cancellation by depletion bands due to the photoreacting red-shifted species. These results are discussed in terms of active-site interactions including the effect of eliminating a Tyr-185/Asp-212 hydrogen bond on the pK(a) of Asp-85 and Asp-212.
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页码:2263 / 2271
页数:9
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