Tyrosine protonation changes in bacteriorhodopsin. A Fourier transform infrared study of BR548 and its primary photoproduct.

P D Roepe,P L Ahl,J Herzfeld,J Lugtenburg,K J Rothschild

doi:10.1016/s0021-9258(18)60686-6

P D Roepe, P L Ahl + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)60686-6

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Abstract

The structural alterations which occur in bacteriorhodopsin (bR) during dark adaptation (BR570----BR548) and the primary phototransition of the dark photocycle (BR548----KD610) have been investigated by Fourier transform infrared and UV difference spectroscopy. Possible contributions of tyrosine to the Fourier transform infrared difference spectra of these transitions were assigned by incorporating ring per-deuterated tyrosine into bR. Based on these data and UV difference measurements, we conclude that a stable tyrosinate exists in BR570 at physiological temperature and that it protonates during formation of BR548. A tyrosinate protonation has also been observed at low temperature during the primary phototransition of BR570 to the red-shifted photoproduct K630 (1). However, we now find that no tyrosine protonation change occurs during the primary phototransition of BR548 to the red-shifted intermediate KD610. Through analysis of bR containing isotopically labeled retinals, it was also determined that the chromophore of KD610 exits in a 13-trans, 15-cis configuration. On the basis of this evidence and previous studies on the structure of the chromophore in BR570, BR548, and K630, it appears that only the 13-trans,15-trans configuration of the protonated chromophore leads to a stable tyrosinate group. It is proposed that a tyrosinate residue is stabilized due to its interaction with the Schiff base positive charge in the BR570 chromophore. Isomerization of the chromophore about either the C13 = C14 or C = N bond disrupts this interaction causing a protonation of the tyrosinate.

Highlights

Tyrosine Protonation Changes in BacteriorhodopsinOn the basis of this evidence and occurs upon light absorption by BR548does not result in the previous studies on the structureof the chromophore formation of a blue-shifted ‘“”-like intermediate or result in in BR570, BR548, and &30, it appears that only the 13- proton pumping as found for the photocycle of the lighttrans,l&trans configuration of the protonated chro- adapted BR570 species
Rhodopsin during dark adaptation(BRs70+BRs4a) Photon absorption by the light-adapted state of purple memand the primary phototransitoiof nthe darkphotocycle brane (BR570) results in the initiation of a photochemical (BR648+&D10) have been investigatebdy Fourier trans- cycle which is characterized by several distinct intermediates form infrared andUV difference spectroscopy
The formation ble contributionsof tyrosine to the Fourier transform of the early photointermediate K630 involves a C13=C14trans infrared difference spectra of these transitions were assigned by incorporating ring per-deuterated tyrosine into bR. Based on these data andUV difference measurements, we conclude that a stable tyrosinate exists in BR670 at physiological temperature and thatit protonates during formation ofBR648

Summary

Tyrosine Protonation Changes in Bacteriorhodopsin

On the basis of this evidence and occurs upon light absorption by BR548does not result in the previous studies on the structureof the chromophore formation of a blue-shifted ‘“”-like intermediate or result in in BR570, BR548, and &30, it appears that only the 13- proton pumping as found for the photocycle of the lighttrans,l&trans configuration of the protonated chro- adapted BR570 species. We have used FTIR spectroscopy in combination with isotopic labeling of both the retinal chromophore and bR tyrosine residues in order to study the molecular changes occurring in these moieties during both dark adap-. The abbreviations used are: bR, bacteriorhodopsin; bR-[*H4]Tyr, analogy tothe K photoproduct oBf R67, This name avoids bR containingring per-deuterated tyrosines; FTIR, Fourier transform potential confusion which might arise from designations which imply infrared HOOP, hydrogen-out-of-plane; RPSB,retinal-protonated a cis or trans structure of the intermediate, which is under investi-. Samples were illuminated using a 1-kilowatt HgXe lamp in combination with a monochromator and either a 540- or 640-nm narrow band interference filter (Ditric Optics, Hudson, MA)

RESULTS

FTIR of Bacteriorhodopsin

DISCUSSION