Static and time-resolved absorption spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: evidence for an equilibrium between bR570 and an O-like species.
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 (bRY185F570) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Sonar
S
S
Physics Department, Boston University, Massachusetts 02215.
Krebs
M P
MP
Khorana
H G
HG
Rothschild
K J
KJ
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D003624:Darkness; D019617:Halobacterium salinarum / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008027:Light; D009154:Mutation; D009994:Osmolar Concentration; D010649:Phenylalanine / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry*
D014443:Tyrosine; D010649:Phenylalanine; D001436:Bacteriorhodopsins