Properties of Asp212 → Asn bacteriorhodopsin suggest that ASp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base

R. Needleman, M. Chang, B. Ni, G. Varo, J. Fornes, S. H. White, J. K. Lanyi

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Abstract

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH > 7, the Asp212 → Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH < 3 and the blue form of the Asp85 → Glu protein at pH < 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pK(a) of the Schiff base. At pH < 7, the Asp212 → Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K ⇆ L1 ⇆ L2 → BR, but with the addition of an L1 ⇆ M reaction with unfavorable pK(a) for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.

Original languageEnglish
Pages (from-to)11478-11484
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number18
Publication statusPublished - Nov 5 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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