Circular dichroism spectroscopy reveals multiple phytochrome photoproducts in equilibrium.

Phytochromes are a widespread family of red/far-red photoreceptors including master regulators of plant growth and development. Phytochromes use 15,16-photoisomerization of linear tetrapyrrole (bilin) chromophores to toggle between a 15Z red-absorbing dark-adapted state (P) and a 15E far-red-absorbing photoproduct (P). The bilin is bound within a conserved, N-terminal PAS-GAF-PHY photosensor tridomain and is covalently attached to a conserved Cys residue, but the mechanism(s) permitting detection of far-red light are not well understood. Plant and cyanobacterial phytochromes exhibit complex P CD spectra that are also not well explained. In this work, we use the model cyanobacterial phytochrome Cph1 from Synechocystis sp. PCC 6803 to examine the basis for this complex CD spectrum. We employ truncations with and without the PHY domain (N514 and N322) as well as a panel of variants with point substitutions in N514. We identify two classes of photoconversion: type 1 produces P, whereas type 2 produces a blue-shifted alternative photoproduct (P) with a distinct CD spectrum and with properties similar to those of the previously observed Meta-R intermediate. Both type 1 and type 2 variants exhibit efficient photoisomerization, indicating that type 2 variants are specifically deficient in spectral tuning of the 15E photoproduct. Subtle differences within type 1 variants can be ascribed to the presence of varying amounts of P. We show that P formation can proceed at pH 6 in Type 2 cases, whereas even wild-type N514 is unable to form P at pH 9. We, thus, demonstrate that the photoproduct of Cph1 contains two 15E species in pH-dependent equilibrium, shedding new light on the P state.