The reaction of hydroxylamine with bacteriorhodopsin studied with mutants that have altered photocycles: Selective reactivity of different photointermediates

Sriram Subramaniam, Thomas Marti, Susanne J. Rösselet, Kenneth J. Rothschild, H. Gobind Khorana

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

The reaction of the retinylidene Schiff base in bacteriorhodopsin (bR) to the water-soluble reagent hydroxylamine is enhanced by >2 orders of magnitude under illumination. We have used this reaction as a probe for changes in Schiff base reactivity during the photocycle of wild-type bR and mutants defective in proton transport. We report here that under illumination at pH 6, the D85N mutant has a 20-fold lower rate and the D212N mutant has a >4-fold higher rate for the light-dependent reaction with hydroxylamine compared with wild-type bR. In contrast, the reactivities of wild-type bR and the D96N and T46V mutants are similar. It has been previously shown that the D96N and T46V replacements have no significant effect on the kinetics of "M" formation but have dramatic effects on rate of the decay of M. We therefore conclude that the hydroxylamine reaction occurs before formation of the M intermediate. Most likely it occurs at the "L" stage of the cycle and reflects increased water accessibility to the Schiff base due to a light-driven change in protein conformation.

Original languageEnglish (US)
Pages (from-to)2583-2587
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number6
StatePublished - 1991
Externally publishedYes

Keywords

  • M intermediate
  • Membrane protein
  • Proton pump
  • Time-resolved spectroscopy
  • Water accessibility

ASJC Scopus subject areas

  • General
  • Genetics

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