Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates

Abstract

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.

Conformational Changes Detected in a Sensory Rhodopsin II-Transducer Complex

“…There is a decreased intensity near 1657 (Ϫ) cm Ϫ1 and an increase of the 1633 (Ϫ) cm Ϫ1 band, which is likely to reflect the expected downshift of the CϭN Schiff base stretching vibration assigned at 1650 cm Ϫ1 and 1627 cm Ϫ1 in H 2 O and D 2 O, respectively (38). Additional changes near 1618 (ϩ) cm Ϫ1 and 1600 (Ϫ) cm Ϫ1 may reflect the D 2 O induced downshift of a tyrosine ring mode near 1620 cm Ϫ1 as previously observed in bacteriorhodopsin (48). from the receptor are evident in this time series including: (i) the increased negative intensity near 1694 cm Ϫ1 (Asn/Gln or amide I); (ii) reduction in negative intensity near 1664 cm Ϫ1 (amide I); and (iii) reduction in intensity of negative band at 1545 cm Ϫ1 (ethylenic stretch of chromophore) along with the appearance of a shoulder at 1555 cm Ϫ1 , which might reflect the presence of the N intermediate.…”

Proton Translocation in Proteins

“…There is clearly an ample number of these residues for the formation of a membrane-spanning HBC, particularly when one recalls that a single HBC may be composed of residues from more than one transmembrane helix in the folded protein. Spectroscopic data have suggested that during the proton pumping photocycle of BR, protonation/ deprotonation events occur at several potential HBC residues, tyrosine and aspartate (15); environmental change also occurs about tryptophan residues (16). Most recently, Khorana and co-workers have expressed the www.annualreviews.org/aronline Annual Reviews gene for BR in E. coli and have begun systematic site-directed mutagenesis studies to identify the key amino acid residues required for proton pumping.…”

Trapping the M sub 1 and M sub 2 substrates of bacteriorhodopsin for electron diffraction studies

“…This is a reversible first-order reaction (Bray & White, 1966). The rate equations are, .100 95 Roepe et al, 1987;Gerwert et al, 1989;Bousche et al, 1991;Braiman et al, 1991;Pfefferle et al, 1991). (Bell, 1972).…”

“…The Schiff base is deprotonated (Lewis et al, 1974). Infrared spectroscopy has provided information concerning the protonation states of carboxyi and tyrosine sidechains at distinct points in the photocycle (Engelhard et al, 1985;Rothschild et al, 1986;Dollinger et al, 1986;Roepe et al, 1987). FTIR spectroscopy demonstrates that both…”