HIS(166) IS CRITICAL FOR ACTIVE-SITE PROTON-TRANSFER AND PHOTOTAXIS SIGNALING BY SENSORY RHODOPSIN-I

Photoinduced deprotonation of the retinylidene Schiff base in the sens ory rhodopsin I transducer (SRI-Htrl) complex results in formation of the phototaxis signaling state S-373. Here we report identification of a residue, His(166), critical to this process, as well as to reproton ation of the Schiff base during the recovery phase of the SRI photocyc le. Each of the residue substitutions A, D, G, L, S, V, or Y at positi on 166 reduces the flash yield of S-373, to values ranging from 2% of wild type for H166Y to 23% for H166V. The yield of S-373 is restored t o wild-type levels in Htrl-free H166L by alkaline deprotonation of Asp (76), a Schiff base proton acceptor normally not ionized in the SRI-Ht rl complex, showing that proton transfer from the Schiff base in H166L occurs when an acceptor is made available. The flash yield and rate o f decay of S-373 of the mutants are pH dependent, even when complexed with Htrl, which confers pH insensitivity to wild-type SRI, suggesting that partial disruption of the complex has occurred. The rates of S-3 73 reprotonation at neutral pH are also prolonged in all H166X mutants , with half-times from 5 s to 160 s (wild type, 1 s). All mutations of His(166) tested disrupt phototaxis signaling. No response (H166D, H16 6L), dramatically reduced responses (H166V), or inverted responses to orange light (H166A, H166G, H166S, and H166Y) or to both orange and ne ar-UV light (H166Y) are observed. Our conclusions are that His(166) 1) plays a role in the pathways of proton transfer both to and from the Schiff base in the SRI-Htrl complex, either as a structurally importan t residue or possibly as a participant in proton transfers; 2) is invo lved in the modulation of SRI photoreaction kinetics by Htrl; and 3) i s important in phototaxis signaling. Consistent with the involvement o f the His imidazole moiety, the addition of 10 mM imidazole to membran e suspensions containing H166A receptors accelerates S-373 decay 10-fo ld at neutral pH, and a negligible effect is seen on wild-type SRI.