Biochemical Journal

Research article

Radical mechanism of cyanophage phycoerythrobilin synthase (PebS)

Andrea W. U. Busch, Edward J. Reijerse, Wolfgang Lubitz, Eckhard Hofmann, Nicole Frankenberg-Dinkel


PEB (phycoerythrobilin) is a pink-coloured open-chain tetrapyrrole molecule found in the cyanobacterial light-harvesting phycobilisome. Within the phycobilisome, PEB is covalently bound via thioether bonds to conserved cysteine residues of the phycobiliprotein subunits. In cyanobacteria, biosynthesis of PEB proceeds via two subsequent two-electron reductions catalysed by the FDBRs (ferredoxin-dependent bilin reductases) PebA and PebB starting from the open-chain tetrapyrrole biliverdin IXα. A new member of the FDBR family has been identified in the genome of a marine cyanophage. In contrast with the cyanobacterial enzymes, PebS (PEB synthase) from cyanophages combines both two-electron reductions for PEB synthesis. In the present study we show that PebS acts via a substrate radical mechanism and that two conserved aspartate residues at position 105 and 206 are critical for stereospecific substrate protonation and conversion. On the basis of the crystal structures of both PebS mutants and presented biochemical and biophysical data, a mechanism for biliverdin IXα conversion to PEB is postulated and discussed with respect to other FDBR family members.

  • bilin reductase
  • cyanophage
  • EPR
  • open-chain tetrapyrrole
  • phycoerythrobilin synthase
  • radical


Cyanobacteria form a large and diverse group of photoautotrophic bacteria that contribute significantly to the global primary production. They efficiently harvest light via their antenna complexes, the phycobilisomes [1]. One major chromophore incorporated into the phycobilisomes is PEB (phycoerythrobilin), a pink-coloured open-chain tetrapyrrole molecule (phycobilin). The biosynthesis of PEB starts with the oxidative cleavage of haem by haem oxygenase to yield BV (biliverdin IXα) [2]. Further reduction of BV is then catalysed by FDBRs [Fd (ferredoxin)-dependent bilin reductases] [3]. As the name implies, electrons are provided by the one-electron transferring redox protein Fd. FDBRs are a class of radical enzymes that do not possess any metal or organic co-factors [35]. They catalyse not only the biosynthesis of PEB, but also of the phycobilins PCB (phycocyanobilin) and PΦB (phytochromobilin), the chromophore of plant phytochromes (Figure 1) [3,6].

Figure 1 Bilin biosynthesis pathway

The substrate BV is reduced in two- and four-electron reduction steps by the FDBRs. Synthesis of the plant chromophore PΦB requires a two-electron reduction by HY2. In cyanobacteria, PebA reduces BV to 15,16-DHBV which is further reduced to PEB catalysed by PebB. Phage-derived PebS catalyses the four-electron reduction from BV to PEB with 15,16-DHBV as the intermediate. PcyA also performs a formal four-electron reduction to PCB with 181,182-DHBV as the intermediate.

FDBRs can be subdivided into two classes depending on the number of electrons transferred to the substrate. One class catalyses two-electron reductions, the other four-electron reductions. In plants, PΦB synthesis proceeds via a two-electron reduction at the A-ring position catalysed by PΦB synthase (HY2) [7]. In cyanobacteria, PEB synthesis requires two sequential two-electron reductions, 15,16-DHBV (15,16-dihydrobiliverdin):Fd oxidoreductase (PebA) reduces the substrate BV at the 15,16-methine bridge of the D-ring to the intermediate 15,16-DHBV, which is further reduced in a second two-electron reduction step to the final product PEB by PEB:Fd oxidoreductase (PebB) [8]. In contrast, PCB synthesis is catalysed by PCB:Fd oxidoreductase (PcyA) in a formal four-electron reduction combining 181,182-DHBV:Fd oxidoreductase activity and PCB:Fd oxidoreductase activity. Here, the D-ring exovinyl reduction yielding the intermediate 181,182-DHBV precedes the A-ring reduction yielding the final product PCB (Figure 1) [4]. These reduction steps were shown to proceed via bilin radical intermediates and similar results were obtained for the Arabidopsis thaliana FDBR HY2 [5,9,10]. A second FDBR able to catalyse a four-electron reduction was discovered in the genome of the Prochlorococcus-infecting cyanophage P-SSM2 [11]. First annotated as a PebA, the enzyme is able to catalyse the synthesis of PEB with BV as the substrate in a formal four-electron reduction with 15,16-DHBV as the intermediate. Due to its novel properties, it was named PebS (PEB synthase) [12]. PebS combines the activities of cyanobacterial PebA and PebB in one enzyme, which has so far not been found in cyanobacteria [12]. PebS is a single globular protein with an α-β-α sandwich fold and high structural similarity to PcyA, the best studied member of the FDBR family. The structure revealed amino acid residues likely to be involved in substrate conversion and possible proton transfer [13]. These residues comprise two aspartate residues at position 105 and 206 that are also highly conserved in the whole FDBR family [9]. The Asp105 residue has been suggested to be involved in initial protonation of the BV substrate to facilitate subsequent electron transfer from Fd [13]. In the present paper we provide evidence that PebS-catalysed PEB synthesis indeed proceeds via a radical mechanism and that both aspartate residues are important for stereospecific substrate protonation and conversion.



All chemicals were American Chemical Society grade or better unless specified otherwise. All assay components were purchased from Sigma. Glutathione–Sepharose™ 4FF, PreScission Protease and expression vector pGEX-6P-3 were obtained from GE Healthcare. Alternatively, Protino® Glutathione Agarose 4B from Macherey-Nagel was used. HPLC-grade acetone, acetonitrile, formic acid and spectroanalytical grade glycerol were obtained from J.T. Baker Inc. Sep-Pak Light cartridges were obtained from Waters. BV was obtained from Frontier Scientific, Carnforth, Lancashire, U.K.

Protein expression, purification and site-directed mutagenesis

PebS from cyanophage P-SSM2, Fd7002 (Fd from Synechococcus sp. PCC 7002) or FdP–SSM2 (Fd from cyanophage P-SSM2) were recombinantly expressed and purified as described previously [12]. Purified Fd was dialysed against 25 mM Tes/KOH (pH 8.0), 100 mM KCl and 15% glycerol and stored at −20 °C. The concentration was determined as described in [12]. All site-directed mutants of PebS were generated in pGEX_pebS_P-SSM2 [12] using the QuikChange® site-directed mutagenesis kit (Stratagene) using the following primers (shown is only the forward primer, the reverse primer is the complement, introduced base pair changes are underlined): D105N, 5′-CTTGTTTTGGTATGAACCTGATGAAGTTTAGTG-3′; D105E, 5′-CTTGTTTTGGTATGGAACTGATGAAGTTTAGTG-3′; D206N, 5′-CTTATATGACTGAACTTAATCCTGTTAGAG-3′; and D206E, 5′-CTTATATGACTGAACTTGAACCTGTTAGAGG-3′. PebS mutants were expressed and purified following the method used for the WT (wild-type) protein.

Preparation of bilins

Preparative production of chromophores was performed under anaerobic conditions as described earlier with the following modifications [5]. FNR (Fd:NADP+ oxidoreductase) from Synechococcus sp. PCC7002 and FdP–SSM2 or Fd7002 were used in varying concentrations. PebS concentrations ranged from 10–200 μM. The assay was carried out at 20 °C and substrate was added sequentially in excess amounts. The reaction was started with an NADPH-regenerating system with final concentrations of 2.2 mM glucose 6-phosphate, 27 μM NADP+ and 0.37 units/ml glucose-6-phosphate dehydrogenase. The reaction was stopped and the products purified according to [8]. 15,16-DHBV was prepared with PebA from Synechococcus sp. WH8020 [8] or respective PebS mutants unable to perform 15,16-DHBV reduction. 15,16-DHBV was then purified according to the previously published protocol [8]. Product formation was verified via HPLC.

Spectroscopic analysis of bilin reductase activity

Anaerobic bilin reductase assays were performed utilizing an Agilent 8453 UV–visible spectrophotometer as described previously [5] with the following modifications. Assay conditions consisted of 25 mM Tes/KOH (pH 8.0), 100 mM KCl, 0.01 μM FNR from Synechococcus sp. PCC7002 and 1 μM FdP–SSM2, 10 μM BSA, 10 μM BV, 10 μM purified PebS WT or mutant, 50 units/ml glucose oxidase, 100 mM glucose and 5 μM catalase. To initiate catalysis, 100 μl of NADPH-regenerating system was added. The final concentration of NADPH-regenerating system contained 3.25 mM glucose 6-phosphate, 41 μM NADP+ and 0.55 unit/ml glucose-6 phosphate dehydrogenase. Reaction mixtures were incubated at 17 °C for 10 min (determined to be within the linear range of PebS activity). A spectrum was taken every 30 s for 10 min. Crude bilins were extracted with a Sep-Pak Light C18 cartridge and subsequently evaporated to dryness using a SpeedVac concentrator. HPLC analysis was performed as described previously [4]. The concentration of protein and bilin was determined as described previously [8].

Freeze-quench EPR measurements

For EPR measurements, the anaerobic assay described above was used with 4-fold increased concentrations of PebS, BV, FNR and Fd, and 15% spectro-analytical grade glycerol was included in the reaction mixtures. The total volume was 3 ml. At various times after addition of 200 μM NADPH, 200 μl aliquots were withdrawn from reaction mixtures, transferred to 4 mm quartz EPR tubes, and immediately frozen in liquid nitrogen. Continuous-wave EPR studies of PebS were performed using a Bruker Elexsys E500 CW X-band EPR spectrometer. EPR spectra were acquired in a standard TE102 resonator at 40 K using a microwave frequency of 9.43 GHz, a power of 20 μW and a field modulation amplitude of 10 G. The temperature of the sample was maintained using an Oxford ESR900 liquid helium flow cryostat and an Oxford ITC 503 temperature controller.

Crystallization of PebS mutants in complex with BV

Protein was concentrated to 7–12 mg/ml in 25 mM Tes/KOH (pH 8.0) and 100 mM KCl. Crystallization conditions were screened by the sitting drop vapour diffusion method using the Classic, Cryo, PEG and JSCG+ Suites (Qiagen), applying 200/100 nl and 100/100 nl mixtures of the protein solution/reservoir solution incubated at 18 °C in the dark. Initial hits were refined using the hanging drop technique. For the D105N mutant, final crystals diffracting to 2.2 Å (1 Å = 0.1 nm) were obtained using a reservoir containing 170 mM (NH4)2SO4 and 20% PEG [poly(ethylene glycol)] 3350. Crystals were briefly soaked in mother liquor supplemented with 10% PEG 400 before transfer into liquid N2. Crystals for the D206N mutant were grown using a reservoir containing 170 mM (NH4)2SO4, 25% PEG 8000 and 15% glycerol. Crystals were directly transferred into liquid N2 and diffracted to 1.85 Å.

Data collection and structure determination

Oscillation data of the D105N mutant were collected at 100 K at the ESRF (European Synchrotron Radiation Source, Grenoble, France) on beamline ID23.2. Data of D206N mutant crystals were collected at 100 K at the SLS (Swiss Light Source, Villigen, Switzerland) on beamline X10SA. All data were processed and scaled using the XDS package [14]. Data statistics are given in Supplementary Table S1 (at

Both structures were solved by molecular replacement in Molrep using the structure of PebS with bound BV (PDB code 2VGR) as the search model. The models were improved by iterating manual rebuilding in Coots [15] and refinement using Refmac [16] for the initial cycle, and phenix.refine [17] for the final cycles. Model and refinement statistics are given in Supplementary Table S1. Non-crystallographic symmetry restraints were used throughout refinement and were only released for variable parts of the structures.


Anaerobic bilin reduction

In order to get a deeper insight into the PebS reaction mechanism, the anaerobic assay system described for other FDBRs was employed. In contrast with an aerobic assay, this system allows the detection of possible bilin radical intermediates during the enzymatic reduction of BV and 15,16-DHBV by PebS and mutants. The PebS reaction was started by the addition of reducing equivalents and resulted in a fast increase of absorbance at ~460 nm and ~740–760 nm. Concomitantly, a decrease of BV absorption at ~680 nm was observed (Figure 2A, WT). Absorption at the indicated wavelengths is probably due to the formation of bilin radical intermediates as shown for other FDBRs [5,9,10]. Although the absorption of the putative radical intermediate(s) decreased during the course of the reaction, formation of the product PEB was observed at ~540 nm. Furthermore, the semi-reduced intermediate 15,16-DHBV becomes temporarily visible at ~600 nm but disappears as the reaction proceeds (Figure 2A). The final product of the PebS reaction is 3Z-PEB as confirmed by HPLC analysis (Figure 2B).

Figure 2 Absorption spectra under anaerobic conditions (A) and HPLC analyses (B) of PebS_WT and mutants

PebS–BV complex (10 μM) was used and absorption spectra were monitored for 10 min every 30 s. The reaction was started by addition of an NADPH-regenerating system. The reaction was stopped and the products analysed via HPLC at 380 nm (solid line) and 560 nm (dashed line). (A) Time course of substrate conversion. BV absorption due to reduction decreases at 680 nm, 15,16-DHBV formation results in an absorption increase at 605 nm, PEB increases with an absorption at 540 nm. Absorption of ~460 nm, ~740 nm and ~760 nm increases, with further decrease for PebS_WT and PebS_D206N/E and PebS_D105E indicated by double arrows, and is stable in the case of PebS_D105N. (B) HPLC analyses were conducted with C18 reverse-phase columns from Phenomenex. The Ultracarb 5 μ column was used for all mutants and WT, except for PebS_D105E and PebS_D206E, where the Luna 5 μ column was used. Retention times for the two columns slightly differ. The reaction products DHBV and PEB were detected at 560 nm. BV was detected at 380 nm. Products were confirmed by retention times of bilin standards and whole spectrum analysis of elution peaks. Asterisks indicate non-specific degradation products.

Asp105 and Asp206 are critical protonating residues

On the basis of the PebS crystal structure, the two conserved amino acid residues Asp105 and Asp206 were proposed to be crucial for PebS function, as their carboxy groups are in hydrogen bonding distance to the substrate [13], and both residues are conserved in many members of the FDBR family. We therefore generated PebS mutants in which the carboxy group was changed to the corresponding amide (D105N and D206N), or in which the aspartate residue was replaced by the more extended glutamate residue (D105E and D206E). In our anaerobic assay, incubation of D105N with BV and an excess of electron equivalents resulted in an absorption increase at ~460 nm and ~740–760 nm similar to WT, but with a very slow decay. These results suggest the formation of bilin radical intermediates and their stabilization/accumulation by the mutant (Figure 2A). Subsequent HPLC analyses revealed that this mutant is unable to convert the substrate BV (Figure 2B). Interestingly, the D105E mutant fully retained the ability to catalyse the first reduction at the 15,16-methine bridge, but could not catalyse the second reduction at the A-ring 2,3,31,32-diene system, thereby yielding 15,16-DHBV as the final product (Figure 2).

Investigation of PebS_D206N with BV as the substrate showed a decrease of BV absorption at ~680 nm with subsequent increase and decrease of a possible bilin radical absorption at ~460 nm and ~740–760 nm. HPLC analyses of this mutant demonstrated that only the first reduction step was performed. The final product of this reaction with a maximum absorption at ~605 nm is 15,16-DHBV (Figure 2). The same product was obtained when Asp206 was substituted by a glutamic acid residue (results not shown). However, in this mutant the reaction can be pushed further to produce PEB by a 10-fold increase in FNR concentration (Figure 2).

Catalytic turnover was also tested with the intermediate of the reaction, 15,16-DHBV. As expected, none of the asparagine residue mutants was able to convert 15,16-DHBV, whereas the WT protein catalysed the formation of PEB (indicated by an absorption increase at ~540 nm; results not shown). Interestingly, UV–visible spectra of the catalytic turnover of these mutants with 15,16-DHBV as the substrate showed no indication of absorbance that could point to the formation of a 15,16-DHBV radical intermediate (results not shown). In this regard, it has to be noted that externally applied 15,16-DHBV to D206N revealed a 60 nm spectral shift to the shorter wavelength of the UV–visible spectrum as compared with the enzymatically produced and enzyme bound 15,16-DHBV (see Supplementary Figure S1 at

Bilin radical intermediates during PebS reaction

Since the performed anaerobic assays suggested the formation of bilin radical intermediates and their accumulation in the D105N mutant, additional anaerobic bilin reductase assays were performed using an excess of NADPH as the reducing agent and 40 μM of the PebS–BV complex. The reactions were monitored via UV–visible spectroscopy (see Supplementary Figure S2 at and EPR spectroscopy (Figure 3). For the latter, aliquots were taken at different time points and flash frozen for further EPR measurements. Paramagnetic species could be detected for PebS_WT and the mutants D105N and D206N, indicated by an isotropic EPR signal at g~2 with a first derivative peak to peak linewidth of approx. 15 G (Figure 3). The PebS_WT protein incubated with BV showed a fast increase and decay of the detectable radical with the strongest signal being observed after 0.5 min. The PebS_D105N mutant generated a relatively stable paramagnetic intermediate as shown by an increasing EPR signal with no significant decrease (Figure 3, middle panel). The PebS_D206N mutant showed a similar behaviour of radical signal increase and decrease in the course of the reaction as observed for the WT reaction. At the end of the reaction at 20 min, a weak signal was still detectable either due to weak radical stabilization or an uncompleted reaction (Figure 3, right-hand panel).

Figure 3 EPR measurement of bilin radicals occurring during reactions of PebS with BV

EPR spectroscopic measurement of BV complexes of PebS_WT, PebS_D105N and PebS_D206N mutant. Samples were withdrawn before addition of NADPH and 0.5 min, 2.5 min, 4.5 min and 20 min after the reaction was started and were immediately frozen in liquid nitrogen. All spectra are on the same scale and were recorded at 40 K, 20 μW power and 10 Gauss field modulation (see text for further details).

Absorption changes at the long and short wavelength (~460 nm, ~740–760 nm), which do not correspond to the substrate (BV) or product (DHBV, PEB) absorption maxima followed similar kinetics as the EPR signal (Figure 4). For comparison, the time point with the highest EPR intensity has been assigned as 100%. Since the EPR linewidth of the radical signal was invariant over the course of the experiment, the EPR signal intensities were taken as the peak to peak amplitudes of the first derivative signals as presented in Figure 3. Relative EPR signal intensity and absorbance at 760 nm were normalized to the amplitude of the signal at 0.5 min for the PebS_WT reaction and at 2.5 min for the reaction of the two mutants. As shown in Figure 4, the EPR intensity followed very similar kinetics as the absorbance change at 760 nm. Therefore, absorption at this wavelength can most probably be attributed to the formation of bilin radical intermediates. These results confirm a radical mechanism of BV reduction catalysed by PebS. Unfortunately, similar experiments employing 15,16-DHBV as a substrate to detect paramagnetic species of the second reduction catalysed by PebS were technically not feasable due to the high amount of 15,16-DHBV required for the analysis.

Figure 4 Correlation of EPR signal and absorption at 760 nm

EPR signal intensity (black bar) was calculated from the first derivative peak to peak amplitude and compared with the absorption change at 760 nm (white bar) during the reaction with BV. The time point of strongest EPR signal was set to 100%. Relative absorbance at 760 nm and EPR signal intensity were normalized to the time point at 0.5 min (PebS_WT) and 2.5 min (PebS_D105N and PebS_D206N).

Structure of the PebS mutants D105N and D206N

In addition to the biochemical evidence that both aspartate residues are critical for catalysis, the respective asparagine mutants were crystallized to obtain structural information to help elucidate the underlying catalytic mechanism. X-ray data of PebS_D105N and PebS_D206N with bound BV have been collected. The structure of PebS_D105N (PDB code 2X9I) was refined at 2.2 Å resolution and PebS_D206N (PDB code 2X9J) at 1.85 Å. Both mutants show the identical overall fold as observed for PebS_WT [RMSD (root mean square deviation) ~0.27 Å for approx. 190 Cα atoms for all observed domains] (see Supplementary Figure S3 at

In all PebS structures, the substrate BV is bound in a pocket parallel to the central β-sheet with the propionate side chains facing the solvent. As already observed for the WT structures, the amino acid residues located on the proximal β-sheet side define the rigid part of the active site. In contrast, residues on the distal side are more flexible (Figure 5 and Supplementary Figure S4 at Accordingly, residue Asp105 and the respective Asn105 mutant superimpose perfectly (Figure 5A and Supplementary Figures S4A and S4B). Asp206, on the other hand, has been found in three different conformational modes in our PebS_WT structures. In one conformation, Asp206 faces the solvent (‘out’). In the second dominant conformation, Asp206 co-ordinates the substrate indirectly via a central pyrrole water (‘in’). In the third conformation observed in the WT enzyme, Asp206 directly co-ordinates the pyrrole nitrogens and effectively displaces the pyrrole water (‘deep’). Using this nomenclature, both copies of the D206N mutant in the asymmetric unit are in the ‘in’ conformation. In the D105N structure, we find four copies in the asymmetric unit, two of which have Asp206 in the ‘deep’ conformation and two in the ‘in’ conformation (see Supplementary Table S2 at In one of the latter we did not find electron density to support the placement of the pyrrole water. In both PebS mutants, BV is found exclusively in the porphyrin-like planar form.

Figure 5 Superposition of the active site of the WT, the D105N (A) and D206N (B) mutant with bound substrate BV

The WT is shown in grey and the mutants in red (D105N) and blue (D206N). Only the residues at position 88, 105 and 206 are shown. The hydrogen bonding network is only shown for the mutants.


PebS is a phage-derived FDBR involved in the biosynthesis of PEB, one of the main chromophores in cyanobacterial light-harvesting complexes. So far, PebS activity is solely found in the enzyme derived from the marine cyanophage P-SSM2 infecting low-light-adapted Prochlorococus strains [11]. Putative PebS orthologues have been identified in sequenced cyanophage genomes and in metagenome-derived sequences of phage origin only [12,18]. In a first two-electron reduction step, PebS regiospecifically reduces the 15,16-methine bridge of BV forming the intermediate 15,16-DHBV. 15,16-DHBV is further reduced to PEB at the A-ring 2,3,31,32-diene structure in a second two-electron reduction step. Both reduction steps generate two new chiral C-atoms at position C-2 and C-16, both of which are in the R configuration [19]. This second reduction step also formally resembles the second reduction catalysed by PcyA and the reaction catalysed by HY2 as they all target the same structure at the tetrapyrrole's A-ring but use different substrates [15,16-DHBV (PebS), 181,182-DHBV (PcyA) and BV (HY2)].

The sole product of the PebS reaction is 3Z-PEB

Improvement of our assay work-up conditions enabled us to ultimately prove that the sole product of the PebS reaction is the 3Z-isomer of PEB (Figure 1). Although we previously had indications from UV–visible spectroscopy that this is the case [13], HPLC analyses now clearly show that 3Z-PEB is the final product (Figure 2B). Therefore, the occurrence of the 3E-isomer is simply due to experimental artefacts such as assay work-up conditions at slightly higher temperatures [3]. Further studies from our laboratory furthermore suggest similar results for PcyA, where the 3Z-isomer of PCB is the sole product (A.W.U. Busch and N. Frankenberg-Dinkel, unpublished results). Earlier results from phytochromobilin synthase from oats and other FDBRs from Cyanidium caldariorum also confirmed the production of the 3Z-isomer [2022]. Therefore, we propose that all FDBRs only produce the 3Z-isomer.

PebS acts via a radical mechanism

Since one common feature of all FDBRs is the lack of any metal or organic cofactors, the presence of bilin radical intermediates was proposed [3]. Radical species could already be confirmed for PcyA [5] and HY2 [9]. In the present study, we show that PebS also acts via bilin radical intermediates which makes them a specific feature of this family of enzymes [5,9]. Employing the anaerobic assay system in combination with UV–visible spectroscopy, these radical species can be observed by their distinct absorption bands in the long and short wavelength range. During the course of the PebS reaction, the observed signal emerges from different radical species that cannot be distinguished here since the reaction probably proceeds via a concerted proton-coupled electron transfer.

Asp105 and Asp206 are both proton donating residues in the PebS reaction

A strong accumulation of radical intermediate was observed for the mutant D105N. Since this mutant is unable to catalyse the conversion of BV, it is catalytically stalled in one of the protonations of the first reduction. The observed radical is therefore most likely one of the two radicals occurring in the first two-electron reduction of BV to 15,16-DHBV. This finding is in agreement with studies on PcyA where the homologous amino acid exchange leads to a loss of activity and also to BV radical stabilization [23]. In contrast, D206N is still able to convert BV to the intermediate 15,16-DHBV and therefore is only essential for A-ring reduction. This is consistent with observations for plant PΦB synthase (HY2) which catalyses the two-electron reduction of the BV A-ring to PΦB. For HY2, the Asp206 homologue Asp256 has been found to be essential for BV reduction as well [9]. Although PcyA also contains an aspartate residue at this position, its mutation to asparagine only has a minimal effect on activity [23]. Therefore, it is not surprising that this residue is found in the ‘out’ position in the PcyA crystal structure [24]. This conserved residue seems to play a special role in both A-ring reductions of HY2 and PebS but not in PcyA. In this regard, PcyA is the only member of the FDBR family where this amino acid residue is not strictly conserved (see Supplementary Figure S5 at Alternative residues are found in PcyA of Prochlorococcus marinus MED4 (glutamic acid) and the cyanophage P-SSM4 (lysine). Both enzymes were shown to be catalytically active and produce PCB [12,25]. Another fundamental difference between the PcyA and PebS structures is the existence of a continuous proton uptake channel (proton relay) leading to a conserved His88 residue in PcyA. This channel plays a central role in the PcyA reaction scheme and has been proposed to be important for reprotonation of Asp105 [10]. Therefore, although we find certain structural features to be conserved within the whole FDBR family, no common catalytic mechanism can be proposed.

Proposed catalytic mechanism of PebS

On the basis of our biochemical, biophysical and structural data we propose the following catalytic mechanism for PebS (Figure 6A). The bound bilins will always be present as a mixture of tautomers (lactim–lactam) regarding the location of the protons. We envisage the first event of the reaction being a protonation of a mono-lactim BV (in the ACδ configuration; using the nomenclature in [26]) through the pyrrole water (H2O). The resulting monohydrobiliverdin cation will take up an electron from Fd and generate a neutral monohydrobiliverdin radical (ACDδ). Residue Asp105 will be important for the next concerted proton-electron transfer and also for directing the proton stereospecifically to C-16 to generate the R configuration at this carbon atom. We envisage that the latter might be possible through a Asp105-mediated stereospecific tautomerization (indicated by a black asterisk in Figure 6A) to yield 15,16-DHBV. We expect that PebA will work in a similar fashion with Asp105 being the critical proton-donating residue. This scheme would also suggest that a D105N mutant accumulates a neutral monohydrobiliverdin radical. The nature of this radical will be further investigated by future high-field EPR measurements.

Figure 6 PebS-catalysed reduction of BV

(A) Proposed mechanism. Black stars indicate stereospecific reductions. See text for details. (B) Illustration of the used nomenclature of protonation sites in the substrate BV. The pyrrole nitrogens are denoted A, B, C and D, whereas the two carbonyl-oxygens on the A- and D-ring are denoted α and δ. Nomenclature was adapted from [26].

Asp206 has been found to be flexible (ranging from an ‘out’ to a deep ‘in’ conformation in direct contact with the BV) and to be essential for the second reduction step of PebS. Including this information in our mechanism we propose an initial protonation of a 15,16-DHBV monolactim (αCD) from Asp206 to generate a trihydrobiliverdin cation, concurrent electron transfer from Fd will result in a neutral radical (αBCD). Next would be the stereospecific protonation of C-2. Again, Asp105 could play a crucial role in this step. The still deprotonated Asp105 could catalyse a stereospecific tautomerization, yielding a neutral lactam radical (BCD). The final step could be a concerted proton-electron transfer followed by a final tautomerization. The exact nature of which is still unknown but could possibly again involve Asp206 and water molecules. We would envisage that the homologous aspartic acid residues Asp107 and Asp231 of PebB from Synechococcus sp. WH8020 are also crucial for the PebB reaction. However, the initial protonation state of Asp107 in PebB is currently unknown and is the subject of current investigation in our laboratory.

One has to note that due to the reduced extent of the conjugated π-system, the reaction intermediate 15,16-DHBV will adopt a different conformation in the active site than the BV found in our ‘ground state’ structures. In addition, the D105E mutant, which retains the catalytically critical carboxy group, can still catalyse the first, but not the second, reduction. The longer side chain changes the geometry of the active site, thereby hindering the productive positioning of the reaction intermediate 15,16-DHBV. Clearly more work is needed to clarify the structural changes involved in these intermediates, both by analysing the two glutamate mutants, and the accumulated radicals of PebS.

In summary, the following points would be fundamental for the proposed mechanism: (i) electron transfer from Fd is directly coupled to proton transfer from water or the protein; (ii) tautomerization of the different protonation states of the substrate occurs constantly; (iii) stereospecific reduction in both steps is enforced by Asp105; and (iv) flexibility of Asp206 facilitates water release and re-protonation.


Andrea Busch and Nicole Frankenberg-Dinkel designed the study; Andrea Busch performed all the experiments; Andrea Busch and Edward Reijerse performed the EPR experiment; Andrea Busch, Edward Reijerse and Wolfgang Lubitz analysed the EPR data; Eckhard Hofmann solved the crystal structures; Andrea Busch, Eckhard Hofmann and Nicole Frankenberg-Dinkel analysed all biochemical data; Andrea Busch, Eckhard Hofmann and Nicole Frankenberg-Dinkel wrote the paper.


This work was financially supported by the SFB 480 (Teilprojekt C6 and C8) from the Deutsche Forschungsgemeinschaft (to N.F.D. and E.H.). A.B. received a PhD fellowship from the Ruhr-University Bochum Research School.


We thank the beamline staff at the ESRF and the SLS and colleagues from the Max-Planck Institute for Physiology (Dortmund, Germany) for help during data collection. Thanks are due to Dr Shih-Long Tu for his help in setting up the anaerobic assay system in our laboratory and to Dr Jessica Wiethaus for helpful discussion.


  • The structural co-ordinates reported will appear in the PDB under accession code 2X9I for PebS_D105N and 2X9J for PebS_D206N.

Abbreviations: BV, biliverdin IXα; DVBV, dihydrobiliverdin; Fd, ferredoxin; Fd7002, Fd from Synechococcus sp. PCC 7002; FdP–SSM2, Fd from cyanophage P-SSM2; FDBR, ferredoxin-dependent bilin reductase; FNR, ferredoxin:NADP+ oxidoreductase; PCB, phycocyanobilin; PEB, phycoerythrobilin; PebS, PEB synthase; PEG, poly(ethylene glycol); PΦB, phytochromobilin; WT, wild-type


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