NpR3784 is the prototype for a distinctive group of red/green cyanobacteriochromes using alternative Phe residues for photoproduct tuning

Cyanobacteriochromes (CBCRs) are photosensory proteins found in cyanobacteria and are distantly related to the widespread phytochromes. Whereas plant phytochromes exhibit responses to red and far-red light, CBCRs use the same photoisomerization of a linear tetrapyrrole (bilin) chromophore to respond to a wide range of colors. NpR6012g4 from Nostoc punctiforme and AnPixJ from Anabaena sp. PCC 7120 belong to a large subfamily of red/green CBCRs that exhibit a red-absorbing dark state similar to that of phytochrome but a green-absorbing photoproduct rather than a far-red-absorbing one. In these canonical red/green CBCRs, the photoproduct is blue-shifted relative to the orange absorption observed in the absence of native protein structure. This spectral tuning of the photoproduct requires a conserved Phe residue on the second beta strand of the CBCR GAF domain, consistent with a trapped-twist mechanism in which the bilin is sterically constrained in the photoproduct. N. punctiforme also produces NpR3784, a CBCR with a similar red/green photocycle to that of NpR6012g4. NpR3784 lacks both the beta 2 Phe and other residues characteristic of the canonical red/green CBCRs. In the current work, we identify NpR3784 homologs with red/green photocycles in other cyanobacteria. Spectral tuning in this NpR3784 group is accomplished by a different set of conserved Phe residues, including a characteristic Phe residue on beta 1. This set of Phe residues cannot be interchanged with the Phe residues found in canonical red/green CBCRs such as NpR6012g4. Our results provide new insights into the flexible protein-chromophore interactions used by CBCRs to generate their remarkable spectral diversity.