Transducer-binding and transducer-mutations modulate photoactive-site-deprotonation in sensory rhodopsin I

Sensory rhodopsin I (SRI) is a seven-transmembrane helix retinylidene protein that mediates color-sensitive phototaxis responses through its bound transducer HtrI in the archaeon Halobacterium salinarum. Deprotonation of the Schiff base attachment site of the chromophore accompanies formation of the SRI signaling state, S-373. We measured the rate of laser flash-induced S373 formation in the presence and absence of HtrI, and the effects of mutations in SRI or HtrI on the kinetics of this process. In the absence of HtrI, deprotonation occurs rapidly (halftime 10 mu s) if the proton acceptor Asp76 is ionized (pK(a) = similar to 7), and only very slowly (halftime > 10 ms) when Asp76 is protonated. Transducer-binding, although it increases the pK(a) of Asp76 so that it is protonated throughout the range of pH studied, results in a first order, pH-independent rate of S373 formation of similar to 300 mu s. Therefore, the complexation of HtrI facilitates the proton-transfer reaction, increasing the rate similar to 50-fold at pH6. Arrhenius analysis shows that Htr-binding accelerates the reaction primarily by an entropic effect, suggesting HtrI constrains the SRI molecule in the complex. Function-perturbing mutations in SRI and HM also alter the rate of S373 formation and the lambda(max) of the parent state as assessed by laser flash-induced kinetic difference spectroscopy, and shifts to longer wavelength are correlated with slower deprotonation. The data indicate that HtrI affects electrostatic interactions of the protonated Schiff base and not only receives the signal from SRI but also optimizes the photochemical reaction process for SRI signaling.