The reaction of hydroxylamine with bacteriorhodopsin studied with mutants that have altered photocycles: Selective reactivity of different photointermediates
- Massachusetts Institute of Technology, Cambridge (USA)
The reaction of the retinylidene Schiff base in bacteriorhodopsin (bR) to the water-soluble reagent hydroxylamine is enhanced by greater than 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 greater than 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.
- OSTI ID:
- 5948266
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 88:6; ISSN PNASA; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINES
CELL CONSTITUENTS
CHEMICAL REACTIONS
CYTOPLASM
EXTRACELLULAR SPACE
HYDROXYLAMINE
IMINES
MUTANTS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOTOCHEMICAL REACTIONS
PHOTOSYNTHETIC BACTERIA
PIGMENTS
PROTEIN STRUCTURE
PROTEINS
RHODOPSIN
SCHIFF BASES
SPACE
SPECTROPHOTOMETRY