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Abstract

Phytochromes are a diverse family of bilin-binding photoreceptors that regulate a wide range of physiological processes. Their pho-tochemical properties make them attractive for applications in optogenetics and superresolution microscopy. Phytochromes un-dergo reversible photoconversion triggered by the Z reversible arrow E photo-isomerization about the double bond in the bilin chromophore. However, it is not fully understood at the molecular level how the protein framework facilitates the complex photoisomerization dynamics. We have studied a single-domain bilin-binding photo-receptor All2699g1 (Nostoc sp. PCC 7120) that exhibits photocon-version between the red light-absorbing (Pr) and far red-absorbing (Pfr) states just like canonical phytochromes. We present the crys-tal structure and examine the photoisomerization mechanism of the Pr form as well as the formation of the primary photoproduct Lumi-R using time-resolved spectroscopy and hybrid quantum me-chanics/molecular mechanics simulations. We show that the un-usually long excited state lifetime (broad lifetime distribution centered at similar to 300 picoseconds) is due to the interactions between the isomerizing pyrrole ring D and an adjacent conserved Tyr142. The decay kinetics shows a strongly distributed character which is imposed by the nonexponential protein dynamics. Our findings offer a mechanistic insight into how the quantum efficiency of the bilin photoisomerization is tuned by the protein environ-ment, thereby providing a structural framework for engineer-ing bil in-based optical agents for imaging and optogenetics applications.