Evidence for a perturbation of arginine-82 in the bacteriorhodopsin photocycle from time-resolved infrared spectra

Arginine-82 (R82) of bacteriorhodopsin (bR) has long been recognized as an important residue due to its absolute conservation in the archaeal rhodopsi ns and the effects of R82 mutations on the photocycle and proton release. H owever, the nature of interactions between R82 and other residues of the pr otein has remained difficult to decipher. Recent NMR studies showed that th e two terminal nitrogens of R82 experience a highly perturbed asymmetric en vironment during the M state trapped at cryogenic temperatures [Petkova et al. (1999) Biochemistry 38, 1562-1572]. Although previous low-temperature F T-IR spectra of wild-type and mutant bR samples have demonstrated effects o f R82 on vibrations of other amino acid side chains, no bands in these spec tra were assignable to vibrations of R82 itself. We have now measured time- resolved FT-IR difference spectra of bR intermediates in the wild-type and R82A proteins, as well as in samples of the R82C mutant with and without th ioethylguanidinium attached via a disulfide linkage at the unique cysteine site. Several bands in the bR --> M difference spectrum are attributable to guanidino group vibrations of R82, based on their shift upon isotope subst itution of the thioethylguanidinium attached to R82C and on their disappear ance in the R82A spectrum. The frequencies and intensities of these IR band s support the NMR-based conclusion that there is a significant perturbation of R82 during the bR photocycle. However, the unusually low frequencies at tributable to R82 guandino group vibrations in M, similar to 1640 and simil ar to 1545 cm(-1), would require a reexamination of a previously discarded hypothesis, namely, that the perturbation of R82 involves a change in its i onization state.