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Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective  in vivo  optogenetics is in many cases severely limited due to the strong absorption and scattering of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR). In this study, UV-Visible-NIR absorption and resonance Raman spectroscopy were used to study the double mutant, D212N/F234S, of green absorbing proteorhodopsin (GPR) regenerated with MMAR, a retinal analog containing a methylamino modified β-ionone ring. Four distinct subcomponent absorption bands with peak maxima near 560, 620, 710 and 780 nm are detected with the NIR bands dominant at pH <7.3, and the visible bands dominant at pH 9.5. FT-Raman using 1064-nm excitation reveal two strong ethylenic bands at 1482 and 1498 cm -1  corresponding to the NIR subcomponent absorption bands based on an extended linear correlation between λ max  and γ C = C . This spectrum exhibits two intense bands in the fingerprint and HOOP mode regions that are highly characteristic of the O 640  photointermediate from the light-adapted bacteriorhodopsin photocycle. In contrast, 532-nm excitation enhances the 560-nm component, which exhibits bands very similar to light-adapted bacteriorhodopsin and/or the acid-purple form of bacteriorhodopsin. Native GPR and its mutant D97N when regenerated with MMAR also exhibit similar absorption and Raman bands but with weaker contributions from the NIR absorbing components. Based on these results it is proposed that the NIR absorption in GPR-D212N/F234S with MMAR arises from an O-like chromophore, where the Schiff base counterion D97 is protonated and the MMAR adopts an all- trans  configuration with a non-planar geometry due to twists in the conjugated polyene segment. This configuration is characterized by extensive charge delocalization, most likely involving nitrogens atoms in the MMAR chromophore.

Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate