Induced-fitting and electrostatic potential change of PcyA upon substrate binding demonstrated by the crystal structure of the substrate-free form

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the sequential reduction of the vinyl group of the D-ring and the A-ring of biliverdin IXα (BV) using ferredoxin to produce phycocyanobilin, a pigment used for light-harvesting and light-sensing in red algae and cyanobacteria. We have determined the crystal structure of the substrate-free form of PcyA from Synechocystis sp. PCC 6803 at 2.5 Å resolution. Structural comparison of the substrate-free form and the PcyA–BV complex shows major changes around the entrance of the BV binding pocket; upon BV binding, two α-helices and nearby side-chains move to produce tight BV binding. Unexpectedly, these movements localize the positive charges around the BV binding site, which may contribute to the proper binding of ferredoxin to PcyA. In the substrate-free form, the side-chain of Asp105 was located at a site that would be underneath the BV A-ring in the PcyA–BV complex and hydrogen-bonded with His88. We propose that BV is protonated by a mechanism involving conformational changes of these two residues before reduction.