Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome

Abstract

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state (15ZPb) and orange-absorbing 15E state (15EPo). pH titration analysis revealed that 15ZPb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy, 15EPo harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state (15ZPg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in 15ZPb and bilin protonation in 15EPo, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated 15ZPg and protonated 15EPr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed 15ZPg formation as an intermediate in the 15EPr-to-15ZPg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP-to-15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blue-absorbing CBCR subfamilies.