Abstract
Proton transfer reactions play important functional roles in many proteins, such as enzymes and transporters, which is also the case in rhodopsins. In fact, functional expression of rhodopsins accompanies intramolecular proton transfer reactions in many cases. One of the exceptional cases can be seen in the protonated form of marine bacterial TAT rhodopsin, which isomerizes the retinal by light but returns to the original state within 10(-5) s. Thus, light energy is converted into heat without any function. In contrast, the T82D mutant of TAT rhodopsin conducts the light-induced deprotonation of the Schiff base at high pH. In this article, we report the structural analysis of T82D by means of difference Fourier transform infrared (FTIR) spectroscopy. In the light-induced difference FTIR spectra at 77 K, we observed little hydrogen out-of-plane vibrations for T82D as well as the wild-type (WT), suggesting that the planar chromophore structure itself is not the origin of the reversion from the K intermediate in WT TAT rhodopsin. Upon relaxation of the K intermediate, T82D forms the following intermediate, such as M, whereas K of WT returns to the original state. Present FTIR analysis revealed the proton transfer from the Schiff base to D82 in T82D upon formation of the M intermediate. It is accompanied by the second proton transfer from E54 to the Schiff base, forming the N intermediate, particularly in membranes. The equilibrium between the M and N intermediates corresponds to the protonation equilibrium between E54 and the Schiff base. We also found that Ca(2+) binding takes place in T82D as well as WT but with 6 times lower affinity. An altered hydrogen-bonding network would be the origin of low affinity in T82D, where deprotonation of E54 is involved in the Ca(2+) binding.