Structural and spectroscopic characterisation of a heme peroxidase from sorghum
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A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV–visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A FeIII/FeII reduction potential of −266 mV vs NHE was determined. Stopped-flow experiments with H2O2 showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na+ ion) and proximal (assigned as a Ca2+) sides of the heme, which is consistent with the Ca2+-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions.
KeywordsHeme Porphyrin X-ray crystallography Sorghum Peroxidase Calcium
Sorghum (Sorghum bicolor) is an important worldwide cereal whose entire genome has recently been sequenced . After wheat, maize, barley and rice, Sorghum is one of the world’s most important cereals; it is the most extensively grown cereal grain in Nigeria and is a widely used raw material in the brewing industry. Interest in peroxidases from sorghum arises from their potential biotechnological applications. However, there is a relatively limited amount of information on sorghum peroxidases. A peroxidase was reported in crude sorghum grain extract in 1971 , but with very limited biochemical characterization. Later studies reported partial purification of a peroxidase from malting sorghum [3, 4]. The most detailed characterization so far of a peroxidase from sorghum was in 2006 .
There are some key features of sorghum peroxidase which are not yet established—in particular, there is no information on the reactivity of the enzyme with hydrogen peroxide, there is limited information on the Ca2+-dependency of the enzyme, and there is no structural information at all. In the present work, we report the purification of a sorghum peroxidase from a yellow sorghum grain (SK 5912) that is widely used in brewing. We have characterised the function and reactivity of the enzyme using spectroscopic, kinetic and electrochemical methods. We also report the crystal structure of this enzyme—the first for a sorghum peroxidase—and we compare this with the known features of other class III peroxidase enzymes.
Materials and methods
Protein isolation and purification
Since there is no recombinant expression system available for the enzyme, it was not possible to isolate the enzyme from E. coli in the normal ways. Instead, the enzyme was isolated directly from sorghum grains that Dr. Nnamchi provided from Nigeria.
Sorghum grains (Sorghum bicolor L. Moench variety SK 5912) grown in 2009 and purchased from the Institute for Agricultural Research of the Ahmadu Bello University Zaria, Nigeria, were used. The grains were manually sorted to remove broken kernels and foreign materials before being surface sterilized by immersion in 1 % (v/v) hypochlorite solution . After thoroughly rinsing with tap and distilled water, the grains were spread on a clean surface layered with soft absorbent paper and allowed to dry at room temperature overnight. Grains were crushed to a fine powder by violent bashing over an extended period in a mortar and pestle, and the protein extracted using a modified method of Nwanguma and Eze . Sorghum flour (0.2 g/mL) was incubated with 100 mM sodium phosphate buffer (pH 6.0) for 30 min at 4 °C and then centrifuged at the same temperature for 30 min at 5000 rpm using a SLC 6000 Sorvall Evolution centrifuge. The protein from the supernatant was precipitated using ammonium sulphate (to 30 %, 60 % and finally 90 % saturation) on ice and with continuous stirring; the solution was centrifuged (as described above) once each saturation point had been reached and assayed for peroxidase activity (see below). The precipitate after centrifugation was redissolved in 50 mM phosphate buffer pH 8.5 and dialysed against the same buffer in three 4-hour periods and used for further purification.
For purification, a 2.5 × 20 cm column was packed with DE-52 DEAE cellulose (Whatman International Ltd, Maidstone, England) and equilibrated with 50 mM phosphate buffer, pH 8.5. The dialysed crude enzyme from the 60–90 % ammonium sulphate cut, which showed the highest peroxidase activity, was loaded onto the column and the column washed with equilibration buffer at a flow rate of 150 mL/h. The peroxidase did not bind to the DE-52 resin and was eluted during this step. Fractions (10 mL) were collected and the absorbance of the fractions monitored at 280 nm and assayed for peroxidase activity (see below). Fractions containing peroxidase activity were pooled and concentrated by centrifugation at 4000 rpm using a 10 kDa molecular weight cut-off membrane in a SLC 6000 Sorvall Evolution Centrifuge. The concentrated protein was dialysed against 50 mM phosphate buffer, pH 6.0, and loaded onto a 2.5 × 15 cm CM-52 carboxymethyl cellulose (Whatman) column equilibrated with 50 mM phosphate buffer, pH 6.0 (equilibration buffer). The column was washed with equilibration buffer using a flow rate of 90 mL/h. The bound protein was eluted using a linear gradient of 0–0.6 M NaCl (200 mL, in equilibration buffer) at the same flow rate. Small (5.0 mL) fractions were collected and monitored for peroxidase activity. Fractions containing sorghum peroxidase were pooled and concentrated (ca. 5 mL) using a 10 kDa molecular weight cut-off membrane and loaded onto a Sephadex G-75 column that had been previously equilibrated with equilibration buffer. The column was run at a flow rate 0.5 mL/min and eluted fractions were assayed for peroxidase activity and protein purity assessed by SDS-PAGE. Heme concentrations were determined using the pyridine hemochromogen assay . Mass spectrometry using both MALDI-TOF and ESI (data not shown) revealed a molecular mass for the protein of 35,571 and 35,647 Da, respectively, although other peaks in the 35.5 kDa range were also present in both mass spectrometry experiments indicating that the enzyme as isolated comprises several different isoforms (probably differing in their glycosylation).
All absorbance spectra and equilibrium ligand binding experiments were measured in 100 mM sodium phosphate buffer, pH 6.0, at 25.0 °C using a Jasco V630 UV–VIS spectrophotometer. Ferrous sorghum peroxidase was generated by addition of sodium dithionite to the ferric enzyme and the ferrous-CO derivative was produced by direct bubbling of CO gas through the dithionite-reduced sample. Equilibrium binding constants, K D, were determined according to published procedures  and involved addition of small volumes (0.5–2.0 μL) of ligand (from an appropriate stock solution) to the protein (~2–4 μM) until no further spectral change occurred.
Steady state activities
Steady state kinetic assays were performed in 50 mM sodium acetate buffer (pH 5.5) at 25 °C. The initial rate of oxidation of guaiacol was monitored at 470 nm in the absence and presence of calcium chloride (0.5 mM) (ε 470 = 22.6 mM−1 cm−1). All reactions contained 10 nM sorghum peroxidase and were initiated by the addition of 100 μM H2O2. Values for K m and k cat parameters were determined by fitting the data to the Michaelis–Menten equation using the GraphPad Prism 6 software package.
Transient state kinetics
Transient state kinetic measurements were performed using an SX.18MV stopped-flow spectrophotometer (Applied Photophysics, UK) fitted with a Neslab RTE-200 circulating water bath (25.0 ± 0.1 °C). Multiple wavelength absorption studies were carried using a photodiode array detector in 50 mM sodium acetate buffer, pH 5.5. 1.7 μM Sorghum peroxidase was rapidly mixed with 50 μM H2O2 and the spectral changes monitored over an appropriate time period; the experiment was repeated in the presence of 1 mM calcium chloride. Spectral deconvolution was performed by global analysis and numerical integration methods using Pro-Kineticist software.
Aliquots of sorghum peroxidase (sodium phosphate buffer, pH 6) were frozen in Wilmad SQ EPR tubes (Wilmad LabGlass, Vineland, NJ, USA) in methanol kept on dry ice. The EPR spectra of the frozen samples were recorded at 10 K on a Bruker EMX spectrometer (X-band) equipped with a spherical high-quality ER 4122 SP9703 resonator and an Oxford Instruments liquid helium system.
Reduction potential determination
Determination of the ferric/ferrous reduction potential (50 mM potassium phosphate buffer, pH 7.0) was using the xanthine/xanthine oxidase method, as described previously . The method allows the determination of the reduction potentials from equilibrium concentrations in the presence of a suitable dye and without the need for measuring a potential; it thus avoids some of the difficulties associated with other electrochemical methods for the determinations of reduction potentials in proteins, because equilibria are achieved rapidly and there is no interference from surface contamination of electrodes . Potassium phosphate buffer was made anaerobic by addition of glucose (5 mM), glucose oxidase (50 µg/ml) and catalase (5 µg/ml). A dye of known potential, in this case phenosafranin (E o ′ = −252 mV ), is used and the assay mixture also included xanthine (300 µM), xanthine oxidase (50 nM), benzyl viologen (0.2 µM), and enzyme (20 µM). Changes in absorbance corresponding to the reduction of heme were measured at the isosbestic point of phenosafranin (407 nm). Reduction of the dye was measured at 520 nm, where the change caused by heme reduction was negligible. Using this method, linear Nernst plots for one-electron reduction of heme [25 mV ln(D ox /D red)], and the two-electron reduction of dye [12.5 mV ln(D ox /D red)], where E ox , E red and D ox , D red are the concentrations of oxidized (ox) and reduced (red) forms of enzyme (E) and dye (D), respectively, produced the expected slope (slope = 1) across a wide range of potentials, and the intercept gives a reliable value for ∆E o ′ versus NHE (normal hydrogen electrode) with an error margin of ±2 mV (according to [10, 12]).
The sorghum peroxidase crystals were grown using the sitting drop vapour diffusion technique at 291 K. The crystallisation drops were set up by mixing 2 µl enzyme (7 mg/ml) in 100 mM sodium phosphate pH 6.0 with 2 µl reservoir solution (4 M sodium formate). The crystals were prepared for X-ray diffraction by brief immersion in the reservoir solution containing 10 % glycerol for cryo-protection followed by flash-freezing in liquid nitrogen.
Data collection and refinement statistics
Unit cell (Å)
58.62, 58.62, 208.41
Data collection statistics*
Wilson B value (Å2)
No. of reflections
No. of reflections for Rfree
R/R free (%)
RMSD from standard bond length/angles (Å/°)
Average B value (Å2)
26 (2395 atoms)
38 (284 atoms)
Chain B (Ligands)
45 (21 atoms)
Chain C (sugar molecules)
40 (107 atoms)
Ramachandran statistics (%)
Results and discussion
We have purified pure cationic sorghum peroxidase from the Nigerian Sorghum grain variety SK 5912. The purification profile, using cation exchange chromatography, revealed a single basic peroxidase, similar to that previously reported for a related sorghum peroxidase . SK 5912 sorghum peroxidase shows a high degree of sequence identity to other class III plant peroxidases, Figure S1.
The potentiometric reduction of SK 5912 sorghum peroxidase (data not shown) yields a value for the Fe3+/Fe2+ reduction potential, E o = −266 ± 5 mV. Values for the Fe3+/Fe2+ reduction potentials vary considerably across peroxidases (from around −30 to −320 mV), as summarised in a recent review ; the sorghum potential measured in this work falls in the range of those that have been measured for other peroxidases.
The class III heme peroxidases typically contain two calcium binding sites. In the sorghum structure there are also two metal ions, and at the same locations as found in HRP and PNP, Fig. 5a, d; in the sorghum structure, these are assigned as Na+ (on the distal side) and Ca2+ (on the proximal side) binding sites based on the ligand coordination geometries. The proximal metal site is seven coordinate, Fig. 5d (bottom panel), with an average distance of 2.40 Å to its ligands T212 (O and OG1), D257 (OD2), T260 (Oand OG1), A263 (O) and D265 (OD1). It was refined as a Ca2+ based on the bond distance to the ligands and the coordination number. This proximal Ca2+ site is indirectly connected to the proximal histidine residue (His211) through the adjacent residue Thr 212. The second (distal) metal binding site is observed to be six-coordinate in the sorghum structure, Fig. 5d, and with longer bond lengths to the ligands [D82 (O and OD1), V85 (O), G87 (O), D89 (OD1) and S91 (OG); average distance 2.44 Å], which is consistent with the presence of a Na+ ion; this is different from the HRP and PNP structures, which show seven coordinate Ca2+ in the same position. This site was, thus refined as sodium, as also assigned in the BP1 structure,3 but the presence of sodium at this site instead of the more typical calcium might merely be a reflection of the crystallization conditions (in sodium formate). The presence of Ca2+ and Na+ ions at these sites is consistent with the Ca2+-dependence of the steady state and pre-steady state kinetics above. It is probably the case that the catalytic activity of the sorghum enzyme is dependent on metal ion incorporation at these positions, or even that the activity is dependent on replacement of the distal sodium site with calcium (which would account for the calcium activation). Calcium incorporation at the distal metal site might even be accompanied by structural changes that shorten the distance of the distal histidine from the heme iron. Previous work would be consistent with this interpretation, as Smith and co-workers observe  that Ca2+ is required for formation of fully folded HRP during recombinant expression of HRP in E. coli, and they  and others  have noted that calcium depletion is likely to affect catalytic performance in HRP. BP1 is similarly activated by calcium . The structural and kinetic information presented in this work for the sorghum enzyme are consistent with these studies on the other class III enzymes, and our data also align nicely with the earlier suggestion  that the activity of sorghum enzymes is calcium dependent.
Reducing the temperature at which the reaction was carried out (to 10 °C), or decreasing the concentration of Ca2+ (to 100 μM), did not slow down the rate at which Compound I was formed, so that ferric enzyme was never observed as the first isolated spectrum in the presence of Ca2+.
There is another region of density, close to the δ-heme edge, which we have assigned as glycerol (probably binding to the enzyme during the purification process), Fig. 6b.
Refining the distal metal ion as Ca2+ gave a negative peak in Fo-Fc map. The B factor of the Ca2+ also doubled (~30 Å2) when the metal was refined as Ca2+, which was also twice the B factor of its surrounding ligands. This was not the case when metal was refined at Na+, as the B factors of the metal and the ligands in this case were lower and similar in magnitude to one another.
We acknowledge Louise Fairall for help with the protein crystallography work, Diamond Light Source staff at beam line I03 (BAG MX6388), BBSRC (grants BB/K008153/1 (to JA), BB/L00458/1 (to ER) and BB/K015656/1 (to PM/ER), EPSRC (studentship to GP), and TEFUND for a travel scholarship (to CIN).
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