Histidine protonation controls structural heterogeneity in the cyanobacteriochrome AnPixJg2

Cyanobacteriochromes are compact and spectrally diverse photoreceptor proteins that bind a linear tetrapyrrole as a chromophore. They show photochromicity by having two stable states that can be interconverted by the photoisomerization of the chromophore. These photochemical properties make them an attractive target for biotechnological applications. However, their application is impeded by structural heterogeneity that reduces the yield of the photoconversion. The heterogeneity can originate either from the chromophore structure or the protein environment. Here, we study the origin of the heterogeneity in AnPixJg2, a representative member of the red/green cyanobacteriochrome family, that has a red absorbing parental state and a green absorbing photoproduct state. Using molecular dynamics simulations and umbrella sampling we have identified the protonation state of a conserved histidine residue as a trigger for structural heterogeneity. When the histidine is in a neutral form, the chromophore structure is homogenous, while in a positively charged form, the chromophore is heterogeneous with two different conformations. We have identified a correlation between the protonation of the histidine and the structural heterogeneity of the chromophore by detailed characterization of the interactions in the protein binding site. Our findings reconcile seemingly contradicting spectroscopic studies that attribute the heterogeneity to different sources. Furthermore, we predict that circular dichroism can be used as a diagnostic tool to distinguish different substates.