Origins of Deuterium Kinetic Isotope Effects on the Proton Transfers of the Bacteriorhodopsin Photocycle ² Leonid S. Brown, ‡ Richard Needleman, § and Janos K. Lanyi* ,‡ Department of Physiology and Biophysics, UniVersity of California, IrVine, California 92697, and Department of Biochemistry, Wayne State UniVersity, Detroit, Michigan 48201 ReceiVed September 21, 1999; ReVised Manuscript ReceiVed NoVember 10, 1999 ABSTRACT: Deuterium kinetic isotope effects (KIE) were measured, and proton inventory plots were constructed, for the rates of reactions in the photocycles of wild-type bacteriorhodopsin and several site- specific mutants. Consistent with earlier reports from many groups, very large KIEs were observed for the third (and largest) rise component for the M state and for the decay of the O state, processes both linked to proton transfers in the extracellular region. The proton inventory plots (ratio of reaction rates in mixtures of H 2 O and D 2 O to that in H 2 O vs mole fraction of D 2 O) were approximately linear for the first and second M rise components and for M decay, as well as for O decay, indicating that the rates of these reactions are limited by simple proton transfer. Uniquely, the third rise component of M (and in the D96N mutant also a fourth rise component) exhibited a strongly curved proton inventory plot, suggesting that its rate, which largely accounts for the rate of deprotonation of the retinal Schiff base, depends on a complex multiproton process. This curvature is observed also in the E194Q, E204Q, and Y57F mutants but not in the R82A mutant. From these findings, and from the locations of bound water in the extracellular region in the crystal structure of the protein [Luecke, Schobert, Richter, Cartailler, and Lanyi (1999) J. Mol. Biol. 291, 899-911], we suspect that the effects of deuterium substitution on the formation of the M state originate from cooperative rearrangements of the extensively hydrogen-bonded water molecules 401, 402, and 406 near Asp-85 and Arg-82. The reaction cycle (“photocycle”) of the light-driven transport of protons in bacteriorhodopsin includes direct proton transfers between neighboring donors and acceptors within the protein, protonation/deprotonation reactions de- pendent on long-range interactions between protonatable groups, and proton release and uptake at the aqueous membrane interfaces (1-4). In the L to M 1 reaction of the photocycle the changed pK a ’s of donor and acceptor result in the development of a protonation equilibrium between the Schiff base of the photoisomerized 13-cis,15-anti-retinal and Asp-85. Protonation of Asp-85 causes release of a proton to the extracellular surface, and the coupling of this release to the pK a of Asp-85 (5-7), as well as what may be a protein conformation change in the kinetic step described as the M 1 to M 2 reaction (8, 9), shifts the protonation equilibrium toward complete deprotonation of the Schiff base. In the M 2 to N reaction the Schiff base is reprotonated by Asp-96, and during the N to O reaction Asp-96 is protonated from the cytoplasmic surface. In the final step, the BR state recovers through proton transfer from Asp-85 to the extracellular proton release site (10, 11). The spectral changes during the photocycle are more complex than expected from this simple sequence, and this complexity has been explained by various schemes, including some with equilibration reactions (12-15) and others with branched unidirectional reactions or multiple bacteriorhodop- sin populations (16-18). Although the observable relaxation times are complex functions of the actual rate constants of the reactions and dependent on the kinetic model, in a linear sequence they do reflect the distinct molecular steps in the transport cycle listed above, i.e., the shifts of protonation equilibria, the retinal isomerization, etc. With this in mind, it had been noted in many earlier reports that the magnitudes of deuterium kinetic isotope effects (KIE) 1 were quite dif- ferent for the different relaxations. Unusually large (5-10×) KIEs were observed for the component of the formation of M with greatest amplitude (19-22), assigned in one scheme to an “M 1 to M 2 ” reaction (8, 12) and in another to the L to M 1 reaction (22), and for the decay of the O state (10, 19, 22, 23). All other relaxations of the photocycle, including the decay of the M state, exhibited smaller (1-3×) KIEs. On this basis, it was suggested (22) that the proton transfers in the extracellular region occur by “ice-like” conduction, while those in the cytoplasmic region utilize conduction more like that in liquid water. Recently, the structure of bacteriorhodopsin was deter- mined by X-ray diffraction to resolutions high enough to ² This work was funded partly by grants from the National Institutes of Health (GM 29498 to J.K.L.), the Department of Energy (DEFG03- 86ER13525 to J.K.L.), and the U.S. Army Research Office (DAAL03- 92-G-0406 to R.N.). * Correspondence should be addressed to this author. E-mail: jlanyi@orion.oac.uci.edu. ‡ University of California. § Wayne State University. 1 Abbreviation: KIE, kinetic isotope effect (in this report it refers to the ratio of relaxation rates in 100% H2O and 100% D2O). 938 Biochemistry 2000, 39, 938-945 10.1021/bi9921900 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/08/2000