ABSTRACT: Attractant and repellent signaling conformers of the dual-signaling phototaxis receptor sensory rhodopsin I and its transducer subunit (SRI-HtrI) have recently been distinguished experimentally by the opposite connection of their retinylidene protonated Schiff bases to the outwardly located periplasmic side and inwardly located cytoplasmic side. Here we show that the pKa of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by ∼1.5 units from that of the inwardly connected conformer. The pK a difference enables quantitative determination of the relative amounts of the two conformers in wild-type cells and behavioral mutants prior to photoexcitation, comparison of their absorption spectra, and determination of their relative signaling efficiency. We have shown that the onephoton excitation of the SRI-HtrI attractant conformer causes a Schiff base connectivity switch from inwardly connected to outwardly connected states in the attractant signaling photoreaction. Conversely, a second near-UV photon drives the complex back to the inwardly connected conformer in the repellent signaling photoreaction. The results suggest a model of the color-discriminating dual-signaling mechanism in which phototaxis responses (his-kinase modulation) result from the photointerconversion of the two oppositely connected SRI-HtrI conformers by one-photon and two-photon activation. Furthermore, we find that the related repellent phototaxis SRII-HtrII receptor complex has an outwardly connecte
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