Mechanistic studies of the phytochromobilin synthase HY2 from Arabidopsis

Phytochromobilin (PPhiB) is an open chain tetrapyrrole molecule that functions as the chromophore of light-sensing phytochromes in plants. Derived from heme, PPhiB is synthesized through an open chain tetrapyrrole intermediate, biliverdin IXalpha (BV), in the biosynthesis pathway. BV is subsequently reduced by the PPhiB synthase HY2 in plants. HY2 is a ferredoxin-dependent bilin reductase that catalyzes the reduction of the A-ring 2,3,3(1),3(2)-diene system to produce an ethylidene group for assembly with apophytochromes. In this study, we sought to determine the catalytic mechanism of HY2. Data from UV-visible and EPR spectroscopy showed that the HY2-catalyzed BV reaction proceeds via a transient radical intermediate. Site-directed mutagenesis showed several ionizable residues that are involved in the catalytic steps. Detailed analysis of these site-directed mutants highlighted a pair of aspartate residues central to proton donation and substrate positioning. A mechanistic prediction for the HY2 reaction is proposed. These results support the hypothesis that ferredoxin-dependent bilin reductases reduce BV through a radical mechanism, but their double bond specificity is decided by strategic placement of different proton-donating residues surrounding the bilin substrate in the active sites.