Structural Basis for Hydration Dynamics in Radical Stabilization of Bilin Reductase Mutants

摘要

Heme-derived linear tetrapyrroles (phytobilins) in phycobiliproteins and phytochromes performncritical light-harvesting and light-sensing roles in oxygenic photosynthetic organisms. A key enzyme in theirnbiogenesis, phycocyanobilin:ferredoxin oxidoreductase (PcyA), catalyzes the overall four-electron reductionnof biliverdin IXR to phycocyanobilin the common chromophore precursor for both classes of biliproteins. Thisninterconversion occurs via semireduced bilin radical intermediates that are profoundly stabilized by selectednmutations of two critical catalytic residues, Asp105 and His88. To understand the structural basis for thisnstabilization and to gain insight into the overall catalytic mechanism, we report the high-resolution crystalnstructures of substrate-loadedAsp105Asn andHis88Glnmutants of Synechocystis sp. PCC 6803 PcyA in the initialnoxidized and one-electron reduced radical states. Unlike wild-type PcyA, both mutants possess a bilin-interactingnaxial water molecule that is ejected from the active site upon formation of the enzyme-bound neutral radicalncomplex. Structural studies of both mutants also show that the side chain of Glu76 is unfavorably located fornD-ring vinyl reduction.On the basis of these structures and companion 15nN-1nHlong-rangeHMQCNMRanalysesnto assess the protonation state of histidine residues, we propose a new mechanistic scheme for PcyA-mediatednreduction of both vinyl groups of biliverdin wherein an axial water molecule, which prematurely binds and ejectsnfrom both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state.