Ultraviolet-visible transient spectroscopy of bacteriorhodopsin mutants: Evidence for two forms of tyrosine-185 → phenylalanine

Mireia Duñach, Stuart Berkowitz, Thomas Marti, Yi Wu He, Sriram Subramaniam, H. Gobind Khorana, Kenneth J. Rothschild

Research output: Contribution to journalArticlepeer-review


The photocycle kinetics of the bacteriorhodopsin mutant Tyr-185 → Phe has been investigated by UV-visible transient spectroscopy. Flash-induced spectral changes were measured from 100 ns to 500 ms using a gated optical multichannel analyzer on protein samples that were reconstituted in vesicles with Halobacterium halobium lipids. Tyr-185 → Phe exhibits a pH-dependent absorbance spectrum reflecting contributions from two different species. At pH 6, the dominant photocycling species has a λmax near 610 nm although the absorption maximum of light-adapted Tyr-185 → Phe is at 581 nm. This red-shifted species does not form any M-like intermediate and undergoes a photocycle similar to that observed for deionized blue membrane. At pH 8, the dominant photoactive form exhibits a λmax near 550 nm. This purple species, which is blue shifted 20 nm relative to wild-type bacteriorhodopsin, exhibits a photocycle similar to the wild type. However, M formation occurs in 8 μs, approximately three times faster than wild-type bacteriorhodopsin at pH 8. In addition, an unusually long lived intermediate absorbing at 610 nm is observed at high pH. In the UV region, a broad band near 300-310 nm is absent in the mutant relative to wild type, consistent with earlier measurements made at low temperature which suggest that Tyr-185 undergoes a change in protonation. Steady-state proton pumping action spectra indicate that the 550 nm species does transport protons but that the blue species is inactive. These results are discussed in terms of a model that hypothesizes that Tyr-185 is located close to the bacteriorhodopsin chromophore and stabilizes the interaction of helices F and G through formation of a polarizable bond with Asp-212.

Original languageEnglish (US)
Pages (from-to)16978-16984
Number of pages7
JournalJournal of Biological Chemistry
Issue number28
StatePublished - Oct 5 1990
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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