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The cyanobacterial enzyme phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes the twostep four-electron reduction of biliverdin IXα to phycocyanobilin, the precursor of biliprotein chromophores found in phycobilisomes. It is known that catalysis proceeds via paramagnetic radical intermediates, but the structure of these intermediates and the transfer pathways for the four protons involved are not known. In this study, high-field electron paramagnetic resonance (EPR) spectroscopy of frozen solutions and single crystals of the one-electron reduced protein-substrate complex of two PcyA mutants D105N from the cyanobacteria Synechocystis sp. PCC6803 and Nostoc sp. PCC7120 are examined. Detailed analysis of Synechocystis D105N mutant spectra at 130 GHz and 406 GHz reveals a biliverdin radical with a very narrow g tensor with principal values 2.00359(5), 2.00341(5) and 2.00218(5). Using density-functional theory (DFT) computations to explore the possible protonation states of the biliverdin radical, it is shown that this g tensor is consistent with a biliverdin radical where the carbonyl oxygen atoms on both the A and the D pyrrole rings are protonated. This experimentally confirms the reaction mechanism recently proposed (Tu et al, Biochemistry 2007, 46, 1484.

Heme-derived linear tetrapyrroles (phytobilins) in phycobiliproteins and phytochromes perform critical light-harvesting and light-sensing roles in oxygenic photosynthetic organisms. A key enzyme in their biogenesis, phycocyanobilin:ferredoxin oxidoreductase (PcyA), catalyzes the overall four-electron reduction of biliverdin IXα to phycocyanobilin -the common chromophore precursor for both classes of biliproteins. This interconversion occurs via semi-reduced bilin radical intermediates that are profoundly stabilized by selected mutations of two critical catalytic residues, Asp105 and His88. To understand the structural basis for this stabilization and to gain insight into the overall catalytic mechanism, we report the high-resolution crystal structures of substrate-loaded Asp105Asn and His88Gln mutants of Synechocystis sp. PCC 6803 PcyA in the initial oxidized and one-electron reduced radical state. Unlike wild-type PcyA, both mutants possess a bilin-interacting axial water molecule that is ejected from the active site upon formation of the enzyme-bound neutral radical complex. Structural studies of both mutants also show that the side chain of Glu76 is unfavorably located for D-ring vinyl reduction. Based on these structures and companion 15 N-1 H long-range HMQC NMR analyses to assess the protonation state of histidine residues, we propose a new mechanistic scheme for PcyA-mediated reduction of both vinyl groups of biliverdin wherein an axial water molecule, that prematurely binds and ejects from both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state. KeywordsPhycocyanobilin; Biliverdin; Ferredoxin; Radical; Oxidoreductase † This work was supported in part by National Science Foundation Grant MCB -0843625 to A.J.F. and J.C.L., and by NIH grant GM73789 to R.D.B. A National Institutes of Health training grant T32-GM007377 supported D.D.G. # Protein coordinates have been deposited in the Protein Data Bank (IDs 3nb8, 3nb9, 3f0l, and 3f0m for the H88Q oxidized, H88Q radical, D105N oxidized, and D105N radical complexes respectively). * Corresponding Author: Prof. Andrew J. Fisher, Depts. of Chemistry, and Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, CA 95616, fisher@chem.ucdavis.edu, NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptLinear tetrapyrroles (bilins) perform important roles in the biology of bacteria, algae, plants, and animals. Bilins (aka. bile pigments) were first identified in animals where they are formed during metabolic breakdown of heme to recycle iron (1,2). In photosynthetic organisms, bilins covalently associated with proteins to carry out light-sensing functions vital to optimizing photosynthesis and light capture (3,4). As prosthetic groups of phytochromes, photoreceptors widely distributed in photosynthetic and some nonphotosynthetic organisms, bilins are activated by light to regulate complex signaling cascades during photomorphogenesis (5). The bilin prosthetic groups of p...