Covalent Bond between the Lys-255 Residue and the Main Chain Is Responsible for Stable Retinal Chromophore Binding and Sodium-Pumping Activity of Krokinobacter Rhodopsin 2.

Microbial rhodopsins are light-receptive proteins with various functions triggered by the photoisomerization of the retinal chromophore from the all- trans to 13- cis configuration. A retinal chromophore is covalently bound to a lysine residue in the middle of the seventh transmembrane helix via a protonated Schiff base. Bacteriorhodopsin (BR) variants lacking a covalent bond between the side chain of Lys-216 and the main chain formed purple pigments and exhibited a proton-pumping function. Therefore, the covalent bond linking the lysine residue and the protein backbone is not considered a prerequisite for microbial rhodopsin function. To further examine this hypothesis regarding the role of the covalent bond at the lysine side chain for rhodopsin functions, we investigated K255G and K255A variants of sodium-pumping rhodopsin, Krokinobacter rhodopsin 2 (KR2), with an alkylamine retinal Schiff base (prepared by mixing ethyl- or n -propylamine and retinal (EtSB or n PrSB)). The KR2 K255G variant incorporated n PrSB and EtSB as similarly to the BR variants, whereas the K255A variant did not incorporate these alkylamine Schiff bases. The absorption maximum of K255G + n PrSB was 524-516 nm, which was close to the 526 nm absorption maximum of the wild-type + all- trans retinal (ATR). However, the K255G + n PrSB did not exhibit any ion transport activity. Since the KR2 K255G variant easily released n PrSB during light illumination and did not form an O intermediate, we concluded that a covalent bond at Lys-255 is important for the stable binding of the retinal chromophore and formation of an O intermediate to achieve light-driven Na+ pump function in KR2.