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J. Biol. Chem., Vol. 279, Issue 53, 55886-55894, December 31, 2004

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Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis^*

Jay M. Janz and David L. Farrens

From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E_a) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of 2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.

Received for publication, August 2, 2004 , and in revised form, October 8, 2004.

^* This work was supported by National Institutes of Health Grants EY12095 and DA14896 (to D. L. F.) and T32-EY07123-09 (to J. M. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Rockefeller University, 1230 York Ave., New York, NY 10021.

To whom correspondence should be addressed: Oregon Health and Science University, Mail Code L224, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-494-0583; Fax: 503-494-8393; E-mail: farrensd{at}ohsu.edu.

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