The effect of pH on the initial rate kinetics of the dimeric biliverdin-IXa reductase from the cyanobacterium Synechocystis PCC6803 Jerrard M. Hayes and Timothy J. Mantle School of Biochemistry and Immunology, Trinity College, Dublin, Ireland Introduction Cyanobacteria utilize linear tetrapyrroles as light-har- vesting pigments that are found covalently attached to phycobiliproteins in the ‘light pipes’ known as phyco- bilisomes [1]. Two genera, Synechococcus and Prochlo- rococcus, are suggested to be responsible for 25% of global photosynthesis [2] and, although some strains of Prochlorococcus do not express phycobiliproteins (e.g. MED4), others (e.g. SS120) express a phycourobilin- containing type-III phycoerythrin [3]. Linear tetrapyr- role metabolism in cyanobacteria is therefore a major physiological pathway. Cyanobacteria express ferre- doxin-dependent bilin reductases (PcyA, PebA and PebB) that synthesize phycocyanobilin and phycoery- throbilin from biliverdin-IXa [4]. These linear tetrapyr- Keywords biliverdin reductase; compulsory ordered mechanism; dimer; pH; Synechocystis Correspondence J. M. Hayes, School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland Fax: +353 677 2400 Tel: +353 895 1612 E-mail: jehayes@tcd.ie (Received 23 April 2009, revised 9 June 2009, accepted 11 June 2009) doi:10.1111/j.1742-4658.2009.07149.x Biliverdin-IXa reductase from Synechocystis PCC6803 (sBVR-A) is a stable dimer and this behaviour is observed under a range of conditions. This is in contrast to all other forms of BVR-A, which have been reported to behave as monomers, and places sBVR-A in the dihydrodiol dehydrogenase ⁄ N-ter- minally truncated glucose–fructose oxidoreductase structural family of dimers. The cyanobacterial enzyme obeys an ordered steady-state kinetic mechanism at pH 5, with NADPH being the first to bind and NADP + the last to dissociate. An analysis of the effect of pH on k cat with NADPH as cofactor reveals a pK of 5.4 that must be protonated for effective catalysis. Analysis of the effect of pH on k cat ⁄ K m NADPH identifies pK values of 5.1 and 6.1 in the free enzyme. Similar pK values are identified for biliverdin binding to the enzyme–NADPH complex. The lower pK values in the free enzyme (pK 5.1) and enzyme–NADPH complex (pK 4.9) are not evident when NADH is the cofactor, suggesting that this ionizable group may inter- act with the 2¢-phosphate of NADPH. His84 is implicated as a crucial resi- due for sBVR-A activity because the H84A mutant has less than 1% of the activity of the wild-type and exhibits small but significant changes in the protein CD spectrum. Binding of biliverdin to sBVR-A is conveniently monitored by following the induced CD spectrum for biliverdin. Binding of biliverdin to wild-type sBVR-A induces a P-type spectrum. The H84A mutant shows evidence for weak binding of biliverdin and appears to bind a variant of the P-configuration. Intriguingly, the Y102A mutant, which is catalytically active, binds biliverdin in the M-configuration. Abbreviations hBVR-B, human biliverdin-IXb reductase; HSA, human serum albumin; sBVR-A, biliverdin-IXa reductase from Synechocystis PCC6803; GST, glutathione S-transferase. 4414 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS roles are then incorporated into the phycobilisome complex. Intriguingly, some strains of cyanobacteria express biliverdin-IXa reductase (BVR-A), which catal- yses the pyridine nucleotide-dependent reduction of biliverdin-IXa to bilirubin-IXa. The first report of a cyanobacterial BVR-A was from Synechocystis PCC6803 (sBVR-A) [5] and BVR-A-like sequences are also clearly identifiable in Gleobacter, Anabena, Nostoc and Trichodesmium (E. Franklin & T. J. Mantle, unpublished results). Schluchter and Glazer [5] reported on the unusual acidic pH optimum for sBVR- A. They also describe features of a Synechocystis PCC6803 strain lacking sBVR-A, which they interpret as indicating that the reaction product, bilirubin-Ixa, plays a role in phycobiliprotein biosynthesis [5]. We have been intrigued that sBVR-A can potentially divert flux from phycobilin biosynthesis and also potentially reduce the phycobilins to the corresponding rubin, a reaction clearly catalysed in vitro [5], albeit with a rela- tively high K m for phycocyanobilin [5]. Questions on the possible function of BVR-A in cyanobacteria parallel a major re-evaluation of the function of BVR-A in mammals. Once considered to play a role solely in the elimination of excess haem, it is now implicated in the maintenance of a major anti- oxidant, bilirubin-IXa [6]. At high doses, biliverdin appears to tolerize the immune system of recipients undergoing organ transplantation in animal studies [7,8], although it is presently unclear whether this effect is caused by biliverdin-IXa or bilirubin-IXa. Because BVR-A is reponsible for the production of bil- irubin-IXa in neonates at birth, it is also a pharmaco- logical target for treating neonatal jaundice [9]. The rat [10,11] and human enzymes [12] have been crystal- lized; however, although there are complexes with NAD + [11] and NADP + [12], little is known about the biliverdin binding site. Although the mammalian enzymes have received the most attention, comparative studies on the salmon and Xenopus tropicalis enzymes are available [13,14]. In this respect, the enzyme from Synechocystis is of considerable interest because it exhibits a narrow acidic pH optimum compared to the broad range of pH that can support activity for the mammalian enzymes [5,14]. The cyanobacterial enzyme is also refractory to activation by inorganic phosphate when NADH is the cofactor [14]. In preliminary exper- iments, we observed that sBVR-A is not subject to the potent substrate inhibition observed with the mamma- lian enzymes and is therefore the first candidate, among all BVR-A forms studied to date, where a com- plete initial rate study can be completed in the absence of a biliverdin-binding protein as well as at the opti- mum, presumably physiological, pH. In preliminary gel filtration experiments, we have also shown that the Synechocystis enzyme behaves as a dimer and such studies are extended to include the light-scattering and analytical ultracentrifugation studies described here. We report a complete initial rate study, including the effect of pH on the kinetic parameters and site-directed mutagenesis studies, to gain an understanding of the function of sBVR-A in cyanobacteria and also to increase our knowledge of the mechanism of an enzyme closely related to a pharmacological target for neonatal jaundice. Results The expression vector pETBVR-A allowed us to rou- tinely prepare 20 mg of electrophoretically homoge- nous sBVR-A from 4 L of culture using Escherichia coli BL21 (DE3) cells. Using this approach, the enzyme has two additional residues at the N-terminus (Ser-Gly) but lacks the His-tag in the preparation reported earlier [5]. The enzyme was colourless; how- ever, the UV spectrum revealed significant absorbance at 260 nm. Analysis of the protein sample by HPLC revealed that, in addition to the protein, there was one major and two minor peaks that absorbed at 254 nm. The major peak, which eluted at 38 min, was identified as NADPH by its retention time, fluorescence emission spectrum and UV absorbance spectrum. We have not pursued the identity of the two minor peaks. All three compounds were released from the enzyme when it was bound to 2¢,5¢-ADP-sepharose and, under these conditions, the enzyme was eluted without contamina- tion. In preliminary experiments, we observed that sBVR-A eluted just before BSA on gel filtration in 25 mm sodium citrate pH 5 (the optimum pH for activity; see below) and, by comparison with the elu- tion volume of standard proteins, this is consistent with a molecular mass of 69 kDa at 20 ° C and 74 kDa at 4 °C (Table 1). Although the enzyme is less active at pH 7.5, gel filtration was carried out at this pH and at 20 °C as well as 4 °C and, under all these condi- tions, the molecular mass of the enzyme corresponds to that of the dimer (Table 1). This result is novel because all BVR-As described to date have been reported to behave as monomers [15–17]. To confirm that sBVR-A is a dimer, we examined the native molecular mass using light-scattering and analytical ultracentrifugation (both sedimentation velocity and sedimentation equilibrium) and the results obtained are provided in Table 1. These confirm the results of the gel filtration and are consistent with the dimeric nature of sBVR-A because several purified prepara- tions have been shown to run with a subunit molecular J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4415 mass of 34 kDa on SDS ⁄ PAGE. Prior to a detailed kinetic analysis, the stability of sBVR-A at a range of pH values was determined. Over the pH range 5–7, the enzyme did not lose any activity when pre-incubated for 180 min. The enzyme was unstable outside this range, being particularly unstable below pH 4.5. At pH 4, the half life was 30 s. At pH 8, the enzyme started to lose activity after 60 min and, at pH 9, retained 75% of the activity after 60 min of incuba- tion. The initial rate demonstrates a linear dependence on enzyme concentration (from 0.5–5 lg ⁄ mL) when assayed at pH 5 with NADPH or NADH as cofactor. All initial rate experiments were conducted within this range of enzyme concentration. In a preliminary set of experiments, there was no substrate inhibition up to a biliverdin concentration of 50 lm, in clear distinction to the mammalian enzymes. Initial rate experiments with NADPH or NADH as the variable substrate were carried out by working at various fixed concentrations of biliverdin-IXa and varying the concentration of NADPH from 5–100 lm or NADH from 50–1000 lm. The ‘fixed’ concentra- tions of biliverdin were also varied from 0.5–10 lm to yield the plot for NADPH as the variable substrate shown in Fig. 1A. The apparent V max values for NADPH as the variable substrate were then replotted against the biliverdin concentration (Fig. 1B) to yield the true V max and true K m values for biliverdin with NADPH as cofactor (Table 2). The initial rate mea- surements also yielded linear double-reciprocal plots (not shown) that intersected to the left of the recipro- cal initial rate axis, suggesting that the mechanism was sequential. However, these experiments could not iden- tify which of the substrates bound first or whether there was any particular order in their binding. A simi- lar pattern was obtained with NADH as the variable substrate (data not shown). Initial rate experiments with biliverdin-IXa as the variable substrate were carried out similarly to those described for NADPH but using various ‘fixed’ con- centrations of NADPH and varying the biliverdin-IXa concentration in the range 0.5–10 lm. The data were Table 1. Relative molecular mass of native sBVR-A. AUC, area under the curve. pH Temperature (°C) MW (kDa) Gel filtration 5 20 69 54 74 7.5 20 66 7.5 4 64 Light scattering 5 20 73.2 7.5 20 66.2 AUC velocity 5 11 71 5 21 80.1 7.5 11 80.4 7.5 21 80 AUC equilibrium 11 612 g 5 4 69.2 18 144 g 5 4 63.9 26 127 g 54 55 11 612 g 7 4 73.8 18 144 g 7 4 77.7 26 127 g 74 72 A B Fig. 1. Initial rate kinetics of sBVR-A with NADPH as the variable substrate. (A) The reaction was conducted in 100 m M sodium cit- rate buffer (pH 5) and the reaction was initiated by the addition of sBVR-A (5 lg). The concentrations of NADPH are indicated and the concentrations of biliverdin-IXa were 0.5 l M ( ), 1 lM ( ), 2 lM (.), 5 lM (r) and 10 lM (•). Each point represents the mean and the error bars represent the standard deviation of triplicate values. The curves are least squares fits to a rectangular hyperbola. (B) A replot of the apparent V max from (A) against the concentrations of biliverdin-IXa. The curve is a least squares fit to a rectangular hyper- bola and the error bars are the standard errors from the fits in (A). Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle 4416 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS fitted to a rectangular hyperbola (Fig. 2A). The true V max and K m for NADPH were calculated by replot- ting the apparent V max values (obtained from the fits in Fig. 2A) against the NADPH concentration (Fig. 2B) and the kinetic constants are shown in Table 2. The initial rate data with biliverdin-IXa as the variable substrate also yielded linear intersecting double-reciprocal plots that were consistent with a sequential mechanism (data not shown). Although these data sets indicate that the enzyme obeys a sequential mechanism, product inhibition patterns are required to distinguish between steady-state ordered, random sequential and Theorell–Chance mechanisms. NADP + inhibition with NADPH as the variable substrate was carried out at saturating (10 lm) levels of biliverdin. The inhibitory concentrations of NADP + were in the range 0–100 lm, whereas the concentration of NADPH varied in the range 5–100 lm. Curves were again fitted to the initial rate data set and used to yield double-reciprocal plots (Fig. 3A). The pattern of the double-reciprocal plots shows that NADP + exhibits competitive kinetics against NADPH. When the slope values of the double-reciprocal plots were replotted against the inhibitor concentration, a linear relation- ship was obtained (Fig. 3B) and was used to determine the inhibitor constant K is for NADP + , which is shown in Table 3. NADP + inhibition was also carried out with biliverdin as the variable substrate. These experi- ments were performed as described for NADPH but keeping the NADPH concentration constant at nonsat- urating (10 lm) and saturating (1 mm) levels of NADPH and varying the biliverdin concentration in the range 0.5–10 lm. A concentration of 1 mm was used for NADPH to ensure saturation. NADP + showed mixed inhibition against biliverdin at nonsatu- rating levels of NADPH. When the experiment was repeated at saturating levels of NADPH, no inhibition A B Fig. 2. Initial rate kinetics of sBVR-A with biliverdin-IXa as the vari- able substrate. (A) The reaction was conducted in 100 m M sodium citrate buffer (pH 5) and the reaction was initiated by the addition of sBVR-A (5 lg). The concentrations of biliverdin-IXa are indicated and the concentrations of NADPH were 5 l M ( ), 10 lM ( ), 20 lM (.), 50 l M (r) and 100 lM (•). Each point represents the mean and the error bars represent the standard deviation of triplicate values. The curves are least squares fits to a rectangular hyperbola. (B) A replot of the apparent V max from (A) against the concentrations of NADPH. The curve is a least squares fit to a rectangular hyperbola and the error bars are the standard errors from the fits in (A). Table 2. Kinetic parameters for wild-type and mutant forms of sBVR-A. sBVR-A Variable substrate V max (lmolÆmin )1 Æ mg )1 ) K m (lM) k cat (s )1 ) Wild-type NADPH 0.78 ± 0.06 10.78 ± 3.2 0.44 ± 0.034 Biliverdin 0.79 ± 0.07 2.32 ± 0.59 0.45 ± 0.02 NADH 0.29 ± 0.04 207 ± 66 0.17 ± 0.023 Biliverdin 0.24 ± 0.027 1.6 ± 0.55 0.15 ± 0.015 Y102A NADPH 0.33 ± 0.06 3.55 ± 3.2 0.18 ± 0.034 Biliverdin 0.33 ± 0.022 16.19 ± 1.62 0.18 ± 0.012 R185A NADPH 0.089 ± 0.006 4.54 ± 1.4 0.05 ± 0.034 Biliverdin 0.089 ± 0.01 3.22 ± 0.9 0.05 ± 0.006 H129A NADPH 0.68 ± 0.01 4.53 ± 0.32 0.39 ± 0.006 Biliverdin 0.68 ± 0.055 1.3 ± 0.34 0.39 ± 0.03 H126A NADPH 0.62 ± 0.1 23.3 ± 10 0.35 ± 0.06 Biliverdin 0.64 ± 0.05 4.67 ± 0.72 0.36 ± 0.03 H97A NADPH 0.58 ± 0.05 5.81 ± 2.3 0.33 ± 0.03 Biliverdin 0.58 ± 0.026 2.26 ± 0.28 0.33 ± 0.015 H84A NADPH Biliverdin 0.008 0.008 Unable to calculate E101A NADPH 0.27 ± 0.034 8.8 ± 4 0.15 ± 0.02 Biliverdin 0.25 ± 0.22 21.66 ± 24.55 0.14 ± 0.13 D285A NADPH 0.1 ± 0.006 1 ± 0.73 0.057 ± 0.01 Biliverdin 0.11 ± 0.013 1.60 ± 0.6 0.062 ± 0.007 J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4417 was observed (data not shown). The inhibition con- stants (K is from the slope replot and K ii from the inter- cept replot) for NADP + with biliverdin as the variable substrate are shown in Table 3. NAD + showed com- petitive kinetics against NADH and was a mixed inhibitor against biliverdin at nonsaturating (100 lm) levels of NADH (Table 3). Bilirubin inhibition with biliverdin as the variable substrate was conducted at saturating (100 lm) levels of NADPH and revealed that bilirubin is a mixed inhibitor against biliverdin at saturating levels of NADPH. Product inhibition experiments with bilirubin as an inhibitor were also conducted with NADPH as the variable substrate (5–100 lm) at nonsaturating (1 lm) and saturating (10 lm) levels of biliverdin. Ini- tial rate data for nonsaturating levels of biliverdin show that bilirubin exhibits mixed inhibition kinetics at nonsaturating levels of biliverdin and, when the experiment was repeated at saturating levels of biliver- din, the inhibition becomes uncompetitive (Fig. 4). Bilirubin exhibits mixed inhibition against NADH at nonsaturating levels of biliverdin and mixed inhibition against biliverdin at saturating levels of NADH (data not shown). Inhibition constants are shown in Table 3. These product inhibition patterns are entirely consis- tent with sBVR-A obeying a steady-state ordered mechanism at pH 5, with NADPH being the first to bind and NADP + the last to dissociate. Inorganic phosphate anion has been shown to be an activator of human BVR-A [14]. Increasing amounts of sodium phosphate (0–100 mm) were added to the sBVR-A assay using both NADH and NADPH as cofactor and at pH 5 and pH 7. The pH was moni- tored before and after the assay to ensure that it did not change significantly when adding increasing amounts of phosphate. The effect of ionic strength was found to be minimal. Inorganic phosphate was found to have no effect on sBVR-A activity with either cofac- tor at either pH. This is a major discriminating feature between the cyanobacterial enzyme and the vertebrate BVR-A family members. It is often the case that, when determining the effect of pH on the kinetic parameters of a two-substrate enzyme, one substrate is held at 10 · K m (91% saturat- ing) and the variation of initial rate with the concen- tration of the second substrate is then used to estimate k cat and the K m for the variable substrate. However, the assumption that a concentration that saturates at one pH will saturate at all the pH values under investi- gation is not without risk. All the initial rate parame- ters reported in the present study were determined in accordance with the classic analysis of Florini and Ves- tling [18] to calculate K m and V max . The effect of pH on k cat was investigated over the pH range 4.25–7.0 with both NADPH and NADH. The values measured for k cat are shown when the data set is described with NADPH or biliverdin as the variable substrate. The same k cat profile should be obtained (irrespective of which substrate is held as the variable) and this is clearly seen in Fig. 5A. Evidently, there is a pK at 5.4 for the ‘less acidic’ limb of the pH curve defining a side chain that must be protonated for catalysis to occur. There is no co-operativity for this protonation because the plot of log k cat versus pH gives a slope of approximately –1 (Fig. 5B). On the ‘more acidic’ limb A B Fig. 3. Product inhibition by NADP + with NADPH as the variable substrate. The reaction was conducted in 100 m M sodium citrate buffer (pH 5) and the reaction was initiated by the addition of sBVR-A (5 lg). Biliverdin-IXa was held constant (10 l M) at saturat- ing levels and the levels of NADPH are indicated. The concentra- tions of NADP + were 0 lM ( ), 10 lM ( ), 20 lM (.), 50 lM (r) and 100 l M (•). (A) The data are represented as a double-reciprocal plot and (B) a slope replot (apparent K m ⁄ V max from fits to a rectan- gular hyperbola) against the concentration of NADP + . Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle 4418 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS of this curve, there is a second pK (4.7) and proton- ation of this group reduces the k cat (but only by 50%). Great care has to be taken because the enzyme is highly unstable at the ‘more acidic’ pH values. Initial rates were obtained at pH 4 during the first few sec- onds of the reaction under conditions when at least 90% of the activity was retained; however, these are clearly not ideal conditions. With NADH as cofactor, the pK on the ‘less acidic’ limb is clearly not co-opera- tive (data not shown) and has a similar value (5.7) to that observed with NADPH (5.4). It is intriguing that the k cat on the ‘more acidic’ limb shows very little dependence on pH with NADH as the cofactor. The effect of pH on the k cat ⁄ K m values for NADPH and NADH was also analysed. The log plot for k cat ⁄ K m is shown for the NADPH (Fig. 6A) and bili- verdin (Fig. 6B) data sets and for the NADH (Fig. 6C) and biliverdin (Fig. 6D) data sets. With NADPH as Table 3. Initial rate kinetic parameters for product inhibition studies of sBVR-A. Inhibitor Variable substrate Fixed substrate Inhibition K is (lM) K ii (lM) NADP + NADPH Biliverdin 10 lM (saturating) Competitive 12.7 – NADP + Biliverdin NADPH 10 lM (nonsaturating) Mixed 26 51 NADPH 1000 l M (saturating) No inhibition – – NAD + NADH Biliverdin 10 lM (saturating) Competitive 613 NAD + Biliverdin NADH 100 lM (nonsaturating) Mixed 2615 1771 NADH 1000 l M (saturating) No inhibition – – Bilirubin NADPH Biliverdin 1 l M (nonsaturating) Mixed 13 28 Biliverdin 10 l M (saturating) Uncompetitive – 17.5 Bilirubin NADH Biliverdin 1 l M (nonsaturating) Mixed 5.2 9.6 Bilirubin Biliverdin NADPH 100 l M (saturating) Mixed 13 28 Bilirubin Biliverdin NADH 1000 l M (saturating) Mixed 16 42 Fig. 4. Product inhibition by bilirubin-IXa with NADPH as the vari- able substrate at saturating levels of biliverdin. (A) The reaction was conducted in 100 m M sodium citrate buffer (pH 5) and the reaction was initiated by the addition of sBVR-A (5 lg). Biliverdin- IXa was held constant at saturating levels (10 l M) and the concen- trations of NADPH are indicated. The concentrations of bilirubin-IXa were 0 l M ( ), 1 lM ( ), 2 lM (.), 5 lM (r) and 10 lM (•). The data are represented as a double-reciprocal plot. A B Fig. 5. Effect of pH on k cat with NADPH as cofactor: 25 mM sodium citrate (pK values of 3.13, 4.76 and 6.4) was used as buffer over the entire pH range studied. (A) Values for k cat were obtained with NADPH as the variable substrate ( •) and with biliverdin-IXa as the variable substrate ( ). (B) The log ⁄ log plot for (A) is shown. J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4419 cofactor, the k cat ⁄ K m data reveal two pK values of 5.1 and 6.1 with NADPH as the variable substrate and 4.9 and 5.6 with biliverdin as the variable substrate. This is consistent with two ionizing groups in the free enzyme with pK values of 5.1 and 6.1 that define NADPH binding. Interestingly, with NADH (Fig. 6C) and biliverdin (Fig. 6D) as the variable substrates, there is only a single pK (i.e. 5.3 for NADH binding to the free enzyme and 5.5 for biliverdin binding to the enzyme–NADH complex). The only difference between NADPH and NADH is the 2¢-phosphate on NADPH and it is tempting to suggest that there may be a disso- ciable group with a pK of 5.1 in the free enzyme (4.9 in the enzyme–NADH complex) that is not involved in binding NADH. This pK may be associated with an ionizing residue that is involved in binding the 2¢-phos- phate of NADPH. The effect of pH on the initial rate kinetics is consis- tent with two ionizing groups in the enzyme active site involved in binding NADPH, which may be perturbed slightly in the enzyme–NADPH complex but which are both required for binding biliverdin. In the ternary com- plex, a group with a pK of 5.4 must be protonated for efficient catalysis with NADPH (in the case of the ter- nary complex with NADH as cofactor, this pK is 5.7). The nature of the second pK in the ternary complex with NADPH (4.7) is unclear. There is no analogous pK in the binding of NADH and it is not readily appar- ent in the ternary complex with NADH as cofactor. To identify the ionizing residues, we have attempted to crystallize sBVR-A, so far without success. We have therefore built a model using the rat enzyme as a tem- plate and this is shown in Fig. 7. In this model, we have highlighted residues from the sBVR-A model that are candidates for the ionizing residues. These include four His (84, 97, 126 and 129) one Glu (101), one Asp (285) and one Tyr (102) residue. All were mutated to Ala residues and the sequences confirmed. The gluta- thione S-transferase (GST) fusions were purified, the GST domain cleaved and removed by affinity chroma- tography and the mutant sBVR-As analysed in terms of CD spectra, induced CD spectra for biliverdin and initial rate kinetic parameters. The kinetic parameters of all the mutants are shown in Table 2. This clearly rules out His97, His126 and His129, which have k cat and K m values that are very close to those displayed by the wild-type enzyme. In addition. these three His to Ala mutants show CD spectra and induced CD spectra for biliverdin that are very close to those exhibited by the wild-type enzyme (Fig. 8A). However, a clear candidate for a key active site residue is His84. The specific activity of the H84A mutant is 1% of the wild-type and is so low that we were unable to A B C D Fig. 6. log k cat ⁄ K m versus pH for NADPH and NADH as the variable substrates. (A) Log k cat ⁄ K m with NADPH as the variable substrate. (B) Log k cat ⁄ K m with biliverdin as the variable substrate and NADPH as cofactor. (C) Log k cat ⁄ K m with NADH as cofactor. (D) Log k cat ⁄ K m with biliverdin as the variable substrate and NADH as cofactor. Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle 4420 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS determine the kinetic parameters with confidence. Examination of the CD spectrum of H84A protein reveals that it is close to, but not identical with, that of the native enzyme (data not shown). We suggest that H84A may be the residue responsible for proton- ating the pyrollic nitrogen prior to hydride transfer (see Discussion); however, we cannot discount a mod- est global structural change having some role in the decreased catalytic activity. It should be noted that, in this respect, the H84A mutant was isolated with bound nucleotide and is clearly able to bind biliverdin-IXa (Fig. 8B), suggesting that both substrates are able to bind to the H84A mutant. The binding of biliverdin to wild-type sBVR-A stabi- lizes the helical P-configuration (Fig. 8A) of the linear tetrapyrrole, also known as the ‘lock washer’ [19] and, by this criteria, biliverdin can be seen to bind weakly to the H84A mutant (Fig. 8B), albeit with a broaden- ing of the positive ellipticity into a peak at 400 nm and a significant shoulder at 325 nm. Two of the mutants (R185A and D285A) have k cat values that are only 10% of wild-type (Table 2). The D285A mutant has modest changes in the K m values and the induced CD spectrum for biliverdin is similar to wild-type. The R185A mutant also shows similar K m values to the wild-type; however, the positive ellipticity of the induced CD for biliverdin shows a sharp peak at 400 nm (the wild-type shows a broad peak centred at 390 nm) and a clear minor peak at 325 nm (Fig. 8C). In the case of the E101A mutant, there is a significant increase in the K m for biliverdin (nine-fold) and the k cat is reduced to a third of that of the wild-type (Table 2). The induced CD spectrum for biliverdin bound to the E101A mutant shows a considerably reduced amplitude, with the positive ellipticity split into two peaks at 325 nm and 400 nm (Fig. 8D). In this case, the trough is centered at 580 nm (compared to 700 nm in the wild-type). Intriguingly the Y102A mutant exhibits CD behaviour that reflects the M-con- figuration (Fig. 8E). The ability of this mutant to stabilize the opposite enantiomer is associated with a modest (50%) drop in the k cat and a seven-fold increase in the K m for biliverdin. Discussion All mammalian forms of BVR-A are reported to behave as monomers. These include the enzymes from pig spleen and rat liver [15], human liver [16] and ox kidney [17]. We have artificially created a dimer of rat BVR-A by using fused GST domains as sites for dimerization [20]. The Synechocystis enzyme is there- fore the first natural dimer reported for BVR-A. We were careful to use a range of techniques to measure the native molecular mass of sBVR-A and to conduct these experiments under a range of conditions, includ- ing temperature, pH and the presence or absence of phosphate, because this has such a pronounced activat- ing effect on the mammalian enzymes with NADH as cofactor [14]. Under all of these conditions, the native enzyme exhibits a molecular mass of 66–80 kDa and, because the molecular mass is 34 kDa as measured by SDS ⁄ PAGE, we conclude that sBVR-A is a stable dimer. In light of the recent reports on the structures of monkey dihydrodiol dehydrogenase [21] and the N-terminally truncated dimeric form of glucose–fruc- tose oxidoreductase [22], we propose that sBVR-A joins this small family of pyridine nucleotide-depen- dent oxidoreductases that dimerize via the C-terminal b-sheet domain. It is intriguing that we purify sBVR-A with bound pyridine nucleotide because this is also a feature of the glucose–fructose oxidoreductase enzyme. In addition to its unique quaternary structure, sBVR-A also exhibits a sharp pH optimum, which we reproducibly measured as pH 5. This behaviour is in contrast to that displayed by the mammalian BVR-A monomers, which show activity over a broad range of pH values in the range 5–9 [14]. The cyanobacterial BVR-A is not subject to the potent substrate inhibition observed with the mammalian forms and this has Fig. 7. A model for sBVR-A. sBVR-A model (green) superimposed on the rat BVR-A crystal structure (grey). The amino acid residues that mutated and their positions within the sBVR-A model are shown. The numbers indicated represent the amino acid residues of sBVR-A. J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4421 allowed us to complete a full initial rate study on the Synechocystis enzyme and to rigorously establish that it obeys an ordered steady-state mechanism. The effect of pH on the initial rate parameters has allowed us to identify two ionizing groups in the free enzyme that are required in the unprotonated (pK 5.1) and proton- ated forms (pK 6.1), respectively, for binding NADPH. The protonation state of the lower pK does not affect the binding of NADH. This is consistent with an ioniz- ing residue, pK 5.1, in the free enzyme which, in the deprotonated state, may promote interaction with the 2¢-phosphate group of NADPH but plays no signifi- cant role in binding NADH. In the case of human BVR-A, we have suggested that the protonation state of Glu75, may effect the interaction with Arg44, which is a key residue involved in binding the 2¢-phosphate of NADPH [14]. Further work is required to identify possible analogous candidates in sBVR-A. There is clearly a pK of 5.4 in the ternary complex that is required to be protonated for efficient catalysis with NADPH and which, with NADH as cofactor, exhibits apK of 5.7. Our mutagenesis studies tentatively iden- tify this residue as His84. We have recently discussed the possibility that a His residue may be responsible for supplying a proton to the pyrrole nitrogen atom of biliverdin-IXb prior to hydride transfer in the case of human biliverdin-IXb reductase (hBVR-B). Although structurally distinct to BVR-A, BVR-B is a good model for mechanistic studies on the reduction of the linear tetrapyrrole ‘C10’ position by hydride. BVR-B is a ‘non-Ixa’ biliverdin reductase [23] and is unable to accommodate biliverdin-IXa in a productive orienta- tion, although we have shown that it clearly binds, albeit rotated by 90° [24], when compared with the bil- iverdin isomers that are substrates (i.e. the IXb,IXd and IXc isomers). Mutagenesis studies on BVR-B have indicated that a solvent hydroxonium ion may be the source of the proton and this was found to be consis- tent with quantum mechanical ⁄ molecular mechanical calculations [25]. However, our studies with BVR-B as a model have demonstrated that there is a requirement for proton transfer to the pyrrole nitrogen atom prior to hydride transfer in the hBVR-B reaction co-ordinate [25] and we suggest that His84 is a good candidate for this function in sBVR-A. The second ‘more acidic’ pK in the k cat data set (pK 4.7) is also prominent with NADPH but less so with NADH. We have taken advantage of the induced CD spectra of biliverdin when enantiomeric forms are stabilized by binding to proteins, including serum albumins [26] and, as reported in the present study, sBVR-A. In solution, these chiral forms are clearly in equilibrium so that no CD spectrum is seen. Bilirubin adopts two enantiomeric ‘ridge tile’ configurations [19,27], whereas A BC D E Fig. 8. Induced CD spectra of biliverdin-IXa bound to sBVR-A and various mutants. sBVR-A and the mutants indicated (all at 29 lM) were incubated with biliverdin-IXa (30 l M) and NADP + (100 lM). (A) Wild-type, (B) H84A, (C) D285A, (D) E101A and (E) Y102A. Effect of pH on the dimeric Synechocystis BVR-A J. M. Hayes and T. J. Mantle 4422 FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS biliverdin is suggested to oscillate between two helical ‘lock washer’ configurations, and one of these, the P- configuration, is clearly stabilized in a biliverdin–myo- globin complex [28]. Although it is tempting to suggest that trapping oscillations between two helical forms is the phenomenon responsible for the P(lus) and M(inus) spectra of biliverdin when bound to human serum albumin (HAS) and BSA respectively [26], this remains to be confirmed. A recent X-ray structure of HSA with bilirubin bound [29] shows a ZZE configu- ration (not a ridge tile), whereas, in solution, HSA stabilizes a P-type induced CD spectrum so that, until we have CD spectra of the appropriate crystals, abso- lute assignments will not be possible. The wild-type and most mutants that we have studied show P-behav- iour [by convention the sign of the longer wavelength defines (P)lus or (M)inus]. However the Y102A mutant appears to stabilize the inverted chiral M-form. This mutant exhibits catalytic activity (k cat is approximately 50% of wild-type), albeit with a seven-fold increase in the K m BILIVERDIN . Because the K m NADPH is very simi- lar to wild-type, this suggests that hydride transfer from the C4 of the nicotinamide ring can be accom- plished relatively efficiently, even with variable configu- rations of biliverdin bound at the active site. The Y102A mutant therefore accomodates a variant config- uration of biliverdin to the wild-type enzyme but retains the ability to catalyse the transfer of hydride from both pyridine nucleotides. As discussed previ- ously [30], the most likely model for the biliverdin binding site can accommodate a number of conforma- tions of biliverdin, including the various locked iso- mers that have been shown to bind productively in hBVR-A [30] and the two helical P- and M-conformers described in the present study. The description of a functional BVR-A in some cyanobacteria introduces an important issue with regard to the subcellular localization of both PcyA and sBVR-A. These two enzymes potentially compete for substrate and different subcellular localizations would provide a way out of this hypothetical dilema. The opti- mum pH for activity for sBVR-A at acid pH values is consistent with the hypothesis that sBVR-A may be localized in the lumen of the thylakoid [5], which is reported to maintain a pH in the range 5.5–5 [31]. As a result of the low abundance of this protein, we have not been able to confirm this using immunogold labelling (L. Weaver, J. M. Hayes & T. J. Mantle, unpublished results). The enzymes responsible for the synthesis of the light-harvesting pigments phycocyanobilin and phy- coerythrobilin (PcyA, PebA and PebB) are all ferre- doxin-dependent and their reaction product is destined for incorporation into the phycobilisomes that decorate the cytosolic side of the thylakoid membrane. The bilin reductase PcyA exhibits a pH optimum of 7.5 [32], whereas PebA and PebB are assayed at pH 7.5 [4], con- sistent with a distinct subcellular localization to sBVR- A and most likely the cytosol, which has been reported to maintain a pH in the range 6.8–7.2 [31]. Further work is required to resolve this important question. Experimental procedures The protein coding DNA for sBVR-A was amplified from Synechocystis PCC6803 genomic DNA using forward (5¢- CGCGGATCCCATGTCTGAAAATTTTG-3¢) and reverse (5¢-CGCCTCGAGCTAATTTTCAACTATATC-3¢) primers containing BamH1 and Xho1 sites, respectively, to allow directional cloning into a modified pET41a expression vec- tor (Novagen, Madison, WI, USA). The GST-sBVR-A fusion protein expressed from pETBVR-A in E. coli BL21 (DE3) cells was purified on glutathione-sepharose (Chroma- trin Ltd, Dublin, Ireland) cleaved with thrombin (Sigma– Aldrich, St Louis, MO, USA) and the GST fragment removed by affinity chromatography on glutathione-sepha- rose. Prior to HPLC analysis, the purified protein was incubated in 6 m urea at 95 °C for 2 min, centrifuged at 16 000 g for 2 min and immediately loaded onto a Supelco Discovery C18 reversed phase HPLC column (Supelco, Bellefonte, PA, USA) (25 · 4 mm) at a flow rate of 1mLÆmin )1 . The HPLC column was equilibrated in 100 mm potassium phosphate (pH 6) and elution was achieved using a linear gradient of 0–40% methanol. Size-exclusion chromatography was conducted using 1 · 100 cm Sephacryl 200 HR (Sigma–Aldrich) columns equilibrated at pH 5 (25 mm sodium citrate, 100 mm NaCl) and pH 7.5 (25 mm Tris ⁄ HCl, 100 mm NaCl) at both 4 °C and 20 °C. The calibration proteins used [b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa)] were individually applied to the column and their elution volumes used to construct a standard curve of log molecular mass versus elution volume. Light scattering was performed on sBVR-A at pH 5 and pH 7.5 at 20 °C. Protein samples (0.25 mgÆmL )1 ) were clar- ified using a 0.22 lm filter and applied to an S-200 Super- dex HR gel-filtration column connected to an AKTA FPLC system (Amersham Biosciences, Little Chalfont, UK). The column was run at 20 °C and a flow rate of 0.5 mgÆmL )1 in the desired equilibration buffer (25 mm sodium citrate, pH 5, 100 m m NaCl or 25 mm Tris ⁄ HCl, pH 7.5, 100 mm NaCl). The gel-filtration column was con- nected online to a miniDawn Tristar light-scattering detec- tor (Wyatt Technology, Santa Barbara, CA, USA) and an Optilab rEX Rayleigh interference detector (Wyatt Tech- nology). The weight-average molar mass of sBVR-A was calculated using the software astra (Wyatt Technology). J. M. Hayes and T. J. Mantle Effect of pH on the dimeric Synechocystis BVR-A FEBS Journal 276 (2009) 4414–4425 ª 2009 The Authors Journal compilation ª 2009 FEBS 4423 [...]... reciprocal of the apparent Vmax was plotted against the concentration of inhibitory product and the inhibition constant for the intercept effect (Kii) was determined from the intersection on the inhibitor concentration axis For pH studies, determination of pK values has been described previously [35,36] Acknowledgements We thank Tatsiana Rakovich and Kieran CrosbieStaunton for initial work on the pH kinetics. .. at 460 nm as a result of the appearance of bilirubin-IXa using a Hexios spectrophotometer (Thermo Spectronic, Cambridge, UK) with online chart recorder (Kipp & Zonen, Hilperton, UK) The typical reaction mixture contained 1–5 lg purified sBVR-A, 100 mm sodium citrate buffer (pH 5) and various concentrations of the substrate biliverdin-IXa and cofactor NAD(P)H (Calbiochem) The reaction was performed at... For initial rate and product inhibition studies, data sets were converted to double-reciprocal plots To determine inhibition constants involving slope changes, the apparent Km ⁄ Vmax was replotted against the concentration of inhibitory product The straight line intercepted the x-axis at –Kis to give the inhibition constant for the slope effect For inhibition constants involving intercept effects, the. .. initiated by the addition of enzyme or NAD(P)H The extinction coefficient for bilirubin under these conditions is 35.75 mm)1Æcm)1 For initial rate kinetics, data points (in triplicate) were fitted to the Michaelis–Menten equation using a least squares fitting routine and the computer software prism (GraphPad Software Inc., San Diego, CA, USA) or wincurve fit, 4424 version 1.3 (Kevin Raner Software, Victoria,... kinetic and physical properties of biliverdin reductase Biochem Soc Trans 9, 275–278 Florini JR & Vestling CS (1957) Graphical determination of the dissociation constants for two-substrate enzyme systems Biochim Biophys Acta 25, 575–578 Trull FR, Ibars O & Lightner DA (1992) Conformation inversion of bilirubin formed by reduction of the biliverdin-human serum albumin complex: evidence from circular... 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Structure of monkey dimeric dihydrodiol dehydrogenase in complex with isoascorbic acid Acta Crystallogr D Biol Crystallogr 64, 532–542 Lott JS, Halbig D, Baker HM, Hardman MJ, Sprenger GA & Baker EN (2000) Crystal structure of a truncated mutant of glucose-fructose oxidoreductase shows that an N-terminal arm controls tetramer formation J Mol Biol 304, 575–584 Effect of pH on the dimeric Synechocystis. .. ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms Plant Cell 13, 965–978 5 Schluchter WM & Glazer AN (1997) Characterization of cyanobacterial biliverdin reductase Conversion of biliverdin to bilirubin is important for normal phycobiliprotein biosynthesis J Biol Chem 272, 13562–13569 6 Baranano DE, Rao M, Ferris CD & Snyder SH (2002) Biliverdin reductase: a major physiologic cytoprotectant . The effect of pH on the initial rate kinetics of the dimeric biliverdin-IXa reductase from the cyanobacterium Synechocystis PCC6803 Jerrard. two-substrate enzyme, one substrate is held at 10 · K m (91% saturat- ing) and the variation of initial rate with the concen- tration of the second substrate is then used