Abstract
Heliorhodopsin (HeR) is a family of microbial rhodopsin discovered in 2018, whose genes are found from archaea, bacteria, unicellular eukaryotes, and giant viruses. Viral heliorhodopsins are classified into VHeR1-4 based on their amino acid sequences, and we previously reported the proton transport activity for V2HeR3. In this study, we report molecular properties of V2HeR2. V2HeR2 contains all-trans retinal predominantly in the dark, and the protonated Schiff base is stabilized by a counterion. The photocycle is described by the sequentially-formed K, M, and O intermediates. The O intermediate with a long lifetime (15.8 sec) and negligible ion transport activity implicate the light sensor function for V2HeR2, which is also the case for many HeRs. FTIR spectroscopy revealed that the chromophore structure is a distorted 13-cis form in the K and O intermediates. Although these properties are common for other HeRs, FTIR spectroscopy gain unique structural factors in the active O intermediate. The 13-cis chromophore is highly distorted near the Schiff base, and the hydrogen bond of the Schiff base is weaker than the resting state. The long-lived O intermediate with the distorted 13-cis retinal and without hydrogen bond of the Schiff base is unique in V2HeR2, which is regulated by the surrounding protein moiety. Strengthened hydrogen bond in amide-I band in the O intermediate of V2HeR2 is opposite to the case in Thermoplasmatales archaeon HeR (TaHeR) and HeR 48C12. Unique protein structural changes in V2HeR2, TaHeR, and HeR 48C12 are possibly correlated to different interaction with their partner proteins.