Inverse Hydrogen-Bonding Change Between the Protonated Retinal Schiff Base and Water Molecules upon Photoisomerization in Heliorhodopsin 48C12

Heliorhodopsin (HeR) is a new class of the rhodopsin family discovered in 2018 through functional metagenomic analysis (named 48C12). Similar to typical microbial rhodopsins, HeR possesses seven transmembrane (TM) a-helices and an all-trans-retinal covalently bonded to the lysine residue on TM7 via a protonated Schiff base. Remarkably, the HeR membrane topology is inverted compared with that of typical microbial rhodopsins. The X-ray crystal structure of HeR 48C12 was elucidated after the first report on a HeR variant from Thermoplasmatales archaeon SG8-52-1, which revealed the watermediated hydrogen-bonding network connected to the Schiff base region in the cytoplasmic side. Herein, low-temperature lightinduced FTIR spectroscopic analyses of HeR 48C12 and N-15 isotopically labeled proteins were used to elucidate the structural changes during retinal photoisomerization. N-D stretching vibrations of the protonated retinal Schiff base (PRSB) at 2286 and 2302 cm(-1) in the dark state, and 2239 and 2252 cm(-1) in the K intermediate were observed. The frequency changes indicated that the hydrogen bond of PRSB strengthens upon photoisomerization in HeR. Moreover, O-D stretching vibration frequencies of the internal water molecules indicate that the hydrogen-bonding strength decreases concomitantly. Therefore, the PRSB hydrogen bond responds to photoisomerization in an opposite way to the hydrogen-bonding network involving water molecules. No frequency changes of the indole N-H or N-D stretching vibrations of tryptophan residues were observed upon photoisomerization, suggesting that all tryptophan residues in the HeR 48C12 maintained the hydrogen-bonding strengths in the K intermediate. These results provide insights into the molecular mechanism of the energy storage and propagation upon retinal photoisomerization in HeR.

Automated summary

Heliorhodopsin (HeR) is a new class of rhodopsin discovered in 2018, exhibiting unique structural features compared to typical microbial rhodopsins. Functional analysis using low-temperature light-induced FTIR spectroscopy revealed changes in hydrogen bonding and structural rearrangement during retinal photoisomerization in HeR 48C12. These findings provide insights into the molecular mechanisms underlying energy storage and propagation in HeR upon retinal photoisomerization.