Dispensability of the second Cys for phycoviolobilin formation by unusual ring D fixation in the cyanobacteriochrome

Abstract

Cyanobacteriochromes are linear tetrapyrrole-binding photoreceptors produced by cyanobacteria. Their chromophore-binding GAF domains are categorized into many lineages. Among them, the DXCF cyanobacteriochrome GAF domains have a "second Cys" within the DXCF motif in addition to a highly conserved "first Cys" stably ligated to C31 of the A-ring. It has been long known that the second Cys is crucial for two color-tuning events: isomerization activity (reduction of C4=C5 double bond) from the initially incorporated phycocyanobilin to phycoviolobilin and reversible ligation activity to the C10 of the chromophore. Comprehensive site-directed mutagenesis, however, revealed that the second Cys is dispensable for isomerization activity, in which three residues participate by fixing the C- and D-rings. Fixation of the chromophore on both sides of the C5 bridge is necessary, even though one side of the fixation site is far from this bridge, with the other side at C31 fixed by the first Cys.