Structural Basis for Hydration Dynamics in Radical Stabilization of Bilin Reductase Mutants<xref rid="fs1"></xref><xref rid="ac1"></xref>

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 IXR to phycocyanobilin;the common chromophore precursor for both classes of biliproteins. This interconversion occurs via semireduced 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-loadedAsp105Asn andHis88Glnmutants ofSynechocystis sp. PCC6803 PcyA in the initial oxidized and one-electron reduced radical states. 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.On thebasis of these structures and companion N-H long-rangeHMQCNMRanalyses 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, which prematurely binds and ejects from both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state. Linear 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 phycobiliproteins of cyanobacteria, red algae, and cryptomonads also function as light harvesting antennae to efficiently transfer light energy to photosynthetic reaction centers (6). 1 Bilins in nature are primarily generated from the enzymemediated degradation of heme. The oxygen-dependent interconversion of heme to biliverdin IXR (BV), catalyzed by the enzyme heme oxygenase, occurs widely among obligate aerobic and facultative anaerobic organisms (4, 7-9). Strongly productinhibited, heme oxygenase turnover is facilitated by BV reduction or removal by binding to bilin-binding proteins. In animals, BV is converted to bilirubin IXR by the NAD(P)H-dependent enzyme biliverdin reductase (10, 11). By contrast, in oxygenic photosynthetic organisms, BV is reduced by ferredoxin-dependent bilin reductases (FDBRs) to produce the phytobilins: phytochromobilin (PΦB), phycocyanobilin (PCB), and phycoerythrobilin (PEB) (3, 4). Originally identified in oxygenic photosynthetic organisms, FDBRs have been classified into five subfamilies that correspond to their unique substrate and/or product specificities (12). Owing to the recent discovery of PEB synthase (PebS), an FDBR encoded by a marine cyanophage, (13) and to the existence of novel bilins in cryptomonads whose biosynthesis is not yet understood (14), novel members of the FDBR family are expected to be discovered in photosynthetic organisms whose genomes have yet to be characterized. The best characterized FDBR is phycocyanobilin:ferredoxin oxidoreductase (PcyA, EC 1.3.7.5), which catalyzes the four-electron reduction of BV to PCB (Figure 1A, (15)). Like all known members of the FDBR family, PcyA possesses neither metal nor organic cofactors to mediate sequential one-electron, ferredoxin-mediated reduction of its bilin substrate. It was therefore hypothesized, and later shown for PcyA, that BV reduction proceeds through bilin radical intermediates that are regiospecifically protonated to yield its phytobilin product PCB (16). Using chemical modification, site-specific 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. and EY012347 to J.B.A. 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). *To whom correspondence should be addressed. E-mail: [email protected]. Phone: 530-754-6180. Fax: 530-752-8995. Abbreviations: FDBR, ferredoxin-dependent bilin reductase; PCB, phycocyanobilin; BV, biliverdin IXR; BV13, biliverdin 13; DHBV, 18,18-dihydrobiliverdin IXR; THBV, tetrahydrobiliverdin IXR; Fd, ferredoxin; FNR, ferredoxin:NADP oxidoreductase; PcyA, phycocyanobilin:ferredoxin oxidoreductase; rmsd, root-mean-square deviation. Article Biochemistry, Vol. 49, No. 29, 2010 6207 mutagenesis, and substrate analogue experiments, a mechanism for the PcyA-mediated conversion of BV to PCB via the twoelectron reduced intermediate 18,18-dihydrobiliverdin (DHBV) was subsequently proposed (17, 18). High-resolution structures of PcyA from the cyanobacteria Synechocystis sp. PCC6803 (19-21) and Nostoc sp. PCC7120 (18) reveal a central seven-stranded antiparallel β-sheet that is sandwiched on either side by fourR-helixes. The substrate-bound PcyA structure shows that BV inserts between the β-sheet and helices seven and eight (19). In substrate-free PcyA, the His88 imidazolium side chain forms a clear salt linkage with the carboxylate side chain of Asp105 (Synechocystis numbering), a linkage that is disrupted to accommodate BV binding. The catalytic importance of these residues is underscored by the observation that mutation of either His88 or Asp105 drastically inhibits catalytic turnover (17). Interestingly, both H88Q and D105N mutants of PcyA generate stable, catalytically stalled bilin radical complexes that fail to be further reduced in the presence of excess reductant (17). The unusual stabilization of bilin radical intermediates indicates that both residues play critical roles in subsequent proton-coupled electron transfers mediated by PcyA. The present work was undertaken to determine the structural basis of the stability of the semireduced BV radical intermediates in the H88Q and D105N mutants and their inability to support full catalytic turnover. Owing to the large size of ferredoxin, our studies exploit the small chemical reductant sodium dithionite to generate the trapped bilin radical intermediates in situ for both mutants in the crystalline state (22).Herewe report the 1.3-1.5 Å resolution crystal structures of the BV-substrate loaded H88Q and D105N mutants of PcyA before and after reduction with sodium dithionite. Companion studies also address the protonation state of histidine residues using N-H HMQC NMR spectroscopy. In addition to shedding new insight into the structural basis of bilin reduction, these investigations provide compelling support for the importance of both residues and a water molecule that becomes associated with the bilin substrate during catalysis in proton-coupled electron transfer by PcyA. EXPERIMENTAL PROCEDURES Site-Directed Mutagenesis, Expression, and Purification of Synechocystis PcyA. Site-directed mutagenesis of Synechocystis PcyA cloned in the pTYB12 vector (New England BioLabs, Ipswich, MA) was performed using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). D105N and H88Q mutant clones, confirmed by DNA sequencing (Davis Sequencing, Davis, CA), were transformed into Escherichia coli strain BL21-DE3 (Stratagene, La Jolla, CA) for protein expression. After lactose induction at 12 C for 19 h, mutant proteins were purified according to the IMPACT-CN protein purification protocol (New England BioLabs, Beverly, FIGURE 1: PcyA reaction. (A) PcyA catalyzes the four-electron reduction of biliverdin (BV) to phycocyanobilin (PCB) via the intermediacy of 18,18-dihydrobiliverdin (15). Four proton-coupled-electron-transfers are mediated by stepwise one-electron transfers from four reduced ferredoxins (Fd). Pyrrole rings are labeledA-D,“P” represents the propionate groups, and the small numbers indicate carbonnumbering forBV. (B) Different tautomeric forms of Asp105 with the D-ring pyrrole for wild-type PcyA. The major “bidentate” neutral conformation observed as 65%occupancy (19) is depictedbothneutral lactamand lactim tautomers.Theminor “axial” ion-pair conformation is shownon the right.Dashed lines represent hydrogen bonds. The positive charge of the minor conformation on the B-ring also can be delocalized on other pyrrole rings and on His88. 6208 Biochemistry, Vol. 49, No. 29, 2010 Kohler et al. MA) with the following modifications. Cell lysis buffer consisted of 50 mMTris-HCl, pH 8.0, 0.5MNaCl, and 0.1% v/v Triton-X 100, and the column equilibration buffer consisted of 50 mM Tris, pH 8.0, 0.5MNaCl. On-column cleavagewas performed by addition of 50 mM 2-mercaptoethanol to column equilibration buffer (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl) followed by incubation at 4 C for 48 h. Purified protein was dialyzed in 20 mM Tris-HCl, pH 8.0 for 24 h followed by concentration using Amicon Ultra 10,000 MWCO centrifugal tubes (Millipore Corp, Billerica, MA) to approximately 10 mg/mL. Concentrated PcyA protein was stored at -80 C prior to use. Crystallization of Synechocystis PcyAMutants. Crystallization of BV substrate-loaded PcyAwas performed under green safelight using the hanging-drop method with a drop size of 4 μL on 24-well Linbro plates using BV-loaded PcyA D105N and H88Q mutants. BV loading was performed by adding a stoichiometric amount of BV to the enzyme 30 min prior to crystal setup. Crystallization drops contained 2 μL of reservoir buffer plus 2 μL of 10 mg/mL PcyA:BV solution, which were suspended over reservoir buffer. Crystals were grown at 293 K in the dark. Conditions that resulted in the best diffracting dithionite-treated “reduced” D105N PcyA crystals were 1.45-1.8 M ammonium sulfate, 0.15-0.4MNaCl, and 0.1MHEPES pH7.0. Conditions that resulted in the best diffracting dithionite-treated “reduced” H88Q PcyA crystals were 1.7-2.2 M ammonium sulfate, 0.26-0.32 MNaCl, and 0.1 M sodium cacodylate pH 7.0. These conditions produced long, rectangular crystals that were dark blue in their native state. Diffraction Data Collection of Synechocystis PcyA. Individual PcyA crystals were sequentially transferred into a cryoprotectant solution consisting of 30% (v/v) ethylene glycol in mother liquor and flash frozen in liquid nitrogen. For in situ reduction, substrate-bound H88Q and D105N PcyA crystals were soaked with 100 mM sodium dithionite in reservoir buffer solutions and allowed to sit in the dark until a color change from blue to green occurred, typically within 10 min. X-ray diffraction data from untreated “oxidized” and dithionite-treated “reduced” BV substrate-bound H88Q and D105N PcyA crystals were collected on beamline 7-1 at the Stanford Synchrotron Radiation Lightsource (SSRL). Diffraction data were indexed and integrated using Mosflm and scaled with the CCP4 program SCALA. Oxidized H88Q and D105N data sets were resolved to 1.3 Å resolution with a completeness of 90.1% and 97.6%, respectively. Both mutants crystallized in space group P21212 with unit cell parameters of: a = 70.8 Å, b = 95.6 Å, c=42.7 Å. Oxidized H88Q and D105N crystals had a calculated Matthews coefficient VM (23) of 2.3 Å /Da (∼45% solvent content) and 2.1 Å/Da (∼42% solvent content), respectively, assuming one monomer per crystallographic asymmetric unit (ASU). Synchrotron X-ray radiation has been shown to reduce cofactors in enzymes (24-26). To monitor the potential reduction of the radical intermediate, microspectrometry was used to record changes in the UV/vis spectrum of the crystal during exposure to the X-ray beam. Spectral changes in the visible spectrum correlates well with reduction state of BV bound to PcyA in solution (16). To limit the reduction potential from X-ray radiation, our radical structure data sets resulted from the merging of diffraction data collected in 12 increments from five reduced H88Q crystals and 10 reduced D105N crystals. Each 12 increment had less than 60 s of X-ray radiation exposure (or radiation dose of 0.064 MGy). Reduced H88Q and D105N data sets were resolved to 1.5 Å resolution with a completeness of 80.6% and 95.2%, respectively. Diffraction data on the initial oxidized mutants were collected from a single crystal of each mutant. The oxidized crystals were exposed in the X-ray beam for a total time of 8 min (radiation dose of 0.51MGy) to collect a complete data set. The X-ray radiation dose absorbed by crystals was calculated using the program RADDOSE (27). Mutant Synechocystis PcyA Structure Determination, Model Building, and Structure Refinement. Structures were solved by molecular replacement using the wild-type Synechocystis PcyA structure (PDB ID: 2d1e) (19). Model building was performed using the molecular graphics program COOT (28). Maximum-likelihood coordinate and B-factor refinement was carried out with the program REFMAC (29) using 95% of the collected data to the respective maximum resolution (5% of the data was set aside for Rfree cross validation). Model quality was checked using the programPROCHECK (30), and the results are summarized in Table 1. EPRSpectroscopy. EPR spectroscopy was performed at the CalEPR facility using a laboratory-constructed pulsed EPR spectrometer operating at 130 GHz (D-band) with the oscillating magnetic field of the cylindrical microwave resonator perpendicular to the appliedmagnetic field. TheD-band spectrometer was described previously in a 130GHz EPR spectroscopic characterization of the D105Nmutant of PcyA (22). A single crystal of the H88Q mutant of PcyA was soaked in sodium dithionite solution and mounted inside of a 0.5 mm ID quartz capillary with the long crystal axis parallel to the capillary axis and flash frozen in liquid nitrogen. NMR Spectroscopy. N-labeled PcyA samples, obtained from cells grown on N-(NH4)2SO4 supplemented minimal media (31), were buffer-exchanged into 95% H2O/5% D2O solution containing 10 mM phosphate (pH 7.0) to a final protein concentration of ∼0.5 mM. All NMR experiments were performed at 25 C on a Bruker Avance III 600 MHz spectrometer equipped with a four-channel interface and triple-resonance cryoprobe (TCI) with pulsed field gradients. Two-dimensional N-H long-range HMQC (LR-HMQC) NMR experiments were performed to correlate histidine ring nitrogen-15 resonances (Nδ1 andNε2) with carbon-attached ring protons, Hδ2 andHε1. A dephasing delay of 45 ms was chosen to select the desired twobond correlations (JNH = 11.35 Hz) in the histidine ring and to suppress signals from one-bond JNH amide couplings (32). LRHMQC spectra of uniformly N-labeled PcyA and mutants (H88Q and D105N) were performed with H and N carrier frequencies at 4.70 and 210 ppm, respectively. The N dimension had a sweep width of 110 ppm with 128 complex points, and the H dimension had a sweep width of 12 ppm with 2048 complex points. Decoupling of N was accomplished with the GARP sequence (33) using a 1.39 kHz field.