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 sitedirected 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 protondonating residues surrounding the bilin substrate in the active sites. Phytochromobilin (P⌽B)2 is an open chain tetrapyrrole chromophore critical for light-sensing phytochromes to regulate growth and development of plants. The phytochromebound P⌽B absorbs light energy and proceeds with reversible structural rearrangement to alter the biochemical activities of phytochrome. P⌽B is covalently linked to apophytochrome through a thioether bond between a conserved cysteine residue on the apoprotein and the ethylidene group on the A-ring of P⌽B (1). The biosynthesis of P⌽B has been shown to reside in the plastids, where heme is first linearized by a heme oxygenase into the reaction intermediate biliverdin IX␣ (BV) and then subsequently reduced by a P⌽B synthase (2-5).In Arabidopsis, the HY2 (LONG HYPOCOTYL 2) gene encodes the P⌽B synthase (EC 1.3.7.4), which catalyzes the ferredoxin-dependent reaction of double bond reduction at the A-ring 2,3,3 1 ,3 2 -diene system of BV to yield 3Z/3E-P⌽B (Fig. 1A) (2). Mutations in HY2 have been shown to severely affect photomorphogenetic processes due to the loss of all functional phytochromes (6, 7). Many HY2-related proteins have been identified from various oxygenic photosynthetic organisms and collectively named ferredoxin-dependent bilin reductases (FDBRs) (8). FDBRs utilize reduced ferredoxin as the electron donor to reduce BV into different biliprotein chromophores with different double bond specificities. Phycocyanobilin:ferredoxin oxidoreductase (PcyA; EC 1.3.7.5) catalyzes two double bond reductions of BV on A-and D-rings to yield phycocyanobilin. 15,16-Dihydrobiliverdin:ferredoxin oxidoreductase (PebA; EC 1.3.7.2) and phycoerythrobilin (PEB):ferredoxin oxidoreductase (PebB; EC 1.3.7.3) work together to reduce the C-15-C-16 and A-ring double bond to produce PEB (8). A recently identified PEB synthase (PebS) can itself catalyze the two double bond reductions to yield PEB (9).PcyA is the most extensively studied FDBR enzyme. Previous biochemical analysis has identified a two-electron reduced intermediate, 181 ,18 2 -dihydrobiliverdin IX␣, present in the PcyA reaction, indicating that D-ring reduction precedes A-ring reduction (Fig. 1B) (10). Two organic radical intermediates have also been ...