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

Abstract

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...

The Existence of an Isolated Hydronium Ion in the Interior of Proteins

“…Even in biliverdin‐bound D105N mutant, where His88 was confirmed to be singly protonated at Nϵ,13 the experimentally measured NMR chemical shift of 7.86 ppm for the 1 Hϵ of His88 was far from those observed for LBHB (typically 17–22 ppm;14 Supporting Information, Figure S4). It is likely that the NMR results13 do not directly support the interpretation by Unno et al 7. for the presence of H 3 O + and deprotonated Nϵ His88 .…”

“…His88 was doubly protonated in biliverdin‐free native PcyA; the protonation state of His88 could not be determined for biliverdin‐bound native PcyA 13. Even in biliverdin‐bound D105N mutant, where His88 was confirmed to be singly protonated at Nϵ,13 the experimentally measured NMR chemical shift of 7.86 ppm for the 1 Hϵ of His88 was far from those observed for LBHB (typically 17–22 ppm;14 Supporting Information, Figure S4). It is likely that the NMR results13 do not directly support the interpretation by Unno et al 7.…”

“…In support of the presence of H 3 O + , Unno et al. stated7 that “in nuclear magnetic resonance (NMR) studies by Kohler et al.,13 His88 was reported to be singly protonated in native PcyA”. However, according to the original report by Kohler et al.,13 it was the D105N mutant that had singly protonated His88, and not the native PcyA 13.…”

“…However, according to the original report by Kohler et al.,13 it was the D105N mutant that had singly protonated His88, and not the native PcyA 13. His88 was doubly protonated in biliverdin‐free native PcyA; the protonation state of His88 could not be determined for biliverdin‐bound native PcyA 13. Even in biliverdin‐bound D105N mutant, where His88 was confirmed to be singly protonated at Nϵ,13 the experimentally measured NMR chemical shift of 7.86 ppm for the 1 Hϵ of His88 was far from those observed for LBHB (typically 17–22 ppm;14 Supporting Information, Figure S4).…”

His74 conservation in the bilin reductase PcyA family reflects an important role in protein-substrate structure and dynamics

“…Crystal 81 structures reveal that these residues interact in the absence of sub-82 strate, but move apart upon BV binding to interact with the bilin 83 substrate [11,12,15]. In the substrate-free PcyA structure, the pro- BV [13]. Substitution of either residue with a non-proton-donating 89 amino acid prevents transfer of a second electron thereby stabiliz-90 ing the one-electron reduced BVHÅ radical [16].…”