ACS DIVISION OF BIOLOGICAL CHEMISTRY

March 23rd, 2015 by Ogren, J. I., Yi, A., Mamaev, S., Li, H., Spudich, J. L., Rothschild, K. J.

Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel which exhibits properties useful for optogenetic applications such as a slow light-inactivation and a red-shifted visible absorption maximum compared to the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR-difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR-difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon formation of the long-lived conducting P2380 state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp299 (homolog to Asp212 in BR) becomes protonated and Asp169 (homolog to Asp85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1→P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu169 to Asp299 during the primary phototransition and from the Schiff base to Glu169 during P2380 formation. The unusual charge neutrality of both Schiff base counterions in the P2380 conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs.