CIRCULAR-DICHROISM SPECTROSCOPY OF THE RETINAL CHROMOPHORE DURING THEPHOTOCYCLE OF BACTERIORHODOPSIN AND ITS D96N MUTANT DERIVATIVE

Low-temperature UV-visible and circular dichroism (CD) spectroscopy we re employed to study the photocycle of native (wild-type, WT) bacterio rhodopsin (bR) and its mutant derivative D96N. For both bR forms, irra diation at -80 degrees C yields the M(412) photocycle intermediate (la mbda(max) = 412 nm). The CD spectrum matches the UV-visible absorbance , including the (vibrational) fine structure. Conversion of the M form into subsequent photocycle intermediates and the re-formation of the bR parent state are accomplished on stepwise temperature elevation. Fo r WT bR, the CD spectrum of the M intermediate appears to be comprised of the 412 nm band and a long-wavelength component (with a broad maxi mum around 560 nm). This interpretation is complicated by the fact tha t, at -80 degrees C, and increasing with increasing temperature, the l ong-wavelength CD absorption becomes negative, whereas no qualitative change of the CD band around 410 nm is observed. This new intensity in terferes with the CD band of M(412) in this wavelength region. Further warming of the sample leads to the formation of another intermediate with a positive CD spectrum around 550 nm which, under our experimenta l conditions (low temperature, high pH), is expected to be the interme diate N-550. The UV-visible absorption of N-550 has a similar waveleng th, but lower extinction coefficient, than that of bR(568), whereas it s CD spectrum has not yet been reported. The same experimental apparat us was employed for the D96N mutant. The UV-visible changes with incre asing temperature are governed by the retarded M decay, as reported pr eviously (Butt et al., EMBO J., 8 (1989) 1657). The ''temperature-reso lved'' CD spectra of this mutant also reflect the retarded conversion of the M form into the bR parent state through the N form. The nearly unaffected UV-visible and CD spectra in the UV range and the concomita nt changes in the long-wavelength CD signals are interpreted as the ob servation of an M --> M' conversion, which has been proposed by Varo a nd Lanyi (Biochemistry, 30 (1991) 5008). Sodium azide addition to D96N , which accelerates the photocycle kinetics, induces a shift of the ab sorption band to shorter wavelengths (lambda(max) = 490 nm) which rese mbles the behaviour of bR at high pH (pH > 9.0).