Properties of Asp 212 + Asn Bacteriorhodopsin Suggest That Asp 212 and Aspa 6 Both Participate in a Counterion and Proton Acceptor Complex near the Schiff

Abstract

The gene coding for bacteriorhodopsin was modified in vitro to replace Aspz1' 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 Aspzlz 4 Asn chromophore was blue (absorption maximum at 586 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 Asp'' + 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 AspsB. However, an important difference is that only Asps5 affects the pK, of the Schiff base. At pH 7, the Aspz1' 4 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 c-, L1 c-, Lz + BR, but with the addition of an LI M reaction with unfavorable pK, 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 Asp", AspZ", and at least one other protonable residue participate in it.

Cite this paper

@inproceedings{Needleman2001PropertiesOA, title={Properties of Asp 212 + Asn Bacteriorhodopsin Suggest That Asp 212 and Aspa 6 Both Participate in a Counterion and Proton Acceptor Complex near the Schiff}, author={Richard B. Needleman and Man Chang and Baofu Ni and FornesBII and Stephen H White and Janos and L{\'a}nyi}, year={2001} }