Vanadium K-edge XAS studies on the native and peroxo-forms of vanadium chloroperoxidase from Curvularia inaequalis

doi:10.1016/j.jinorgbio.2010.02.007

Journal of Inorganic Biochemistry

Volume 104, Issue 6, June 2010, Pages 657-664

https://doi.org/10.1016/j.jinorgbio.2010.02.007 Get rights and content

Abstract

Vanadium K-edge X-ray Absorption Spectra have been recorded for the native and peroxo-forms of vanadium chloroperoxidase from Curvularia inaequalis at pH 6.0. The Extended X-ray Absorption Fine Structure (EXAFS) regions provide a refinement of previously reported crystallographic data; one short V = O bond (1.54 Å) is present in both forms. For the native enzyme, the vanadium is coordinated to two other oxygen atoms at 1.69 Å, another oxygen atom at 1.93 Å and the nitrogen of an imidazole group at 2.02 Å. In the peroxo-form, the vanadium is coordinated to two other oxygen atoms at 1.67 Å, another oxygen atom at 1.88 Å and the nitrogen of an imidazole group at 1.93 Å. When combined with the available crystallographic and kinetic data, a likely interpretation of the EXAFS distances is a side-on bound peroxide involving V–O bonds of 1.67 and 1.88 Å; thus, the latter oxygen would be ‘activated’ for transfer. The shorter V–N bond observed in the peroxo-form is in line with the previously reported stronger binding of the cofactor in this form of the enzyme. Reduction of the enzyme with dithionite has a clear influence on the spectrum, showing a change from vanadium(V) to vanadium(IV).

Graphical Abstract

Vanadium K-edge X-ray Absorption Spectra have been recorded for the native and peroxo-forms of vanadium chloroperoxidase from Curvularia inaequalis at pH 6.0. The EXAFS regions provide a refinement of previously reported crystallographic data. The presented spectra are the first on a peroxo-intermediate of a vanadium haloperoxidase.

Introduction

Vanadium haloperoxidases (VHPOs) are a class of enzymes that catalyse the oxidation of halides by hydrogen peroxide to the corresponding hypohalous acid (Eq. (1)) [1], [2]. $\begin{matrix} X^{-} + H_{2} O_{2} + H^{+} \to HOX + H_{2} O & (X = Cl, Br, or I) \end{matrix}$

These VHPOs are named after the most electronegative halide that they are able to oxidize. Thus, a vanadium chloroperoxidase (VCPO) is able to oxidize chloride, bromide and iodide. Physiologically, these enzymes are produced on the surface of organisms and appear to be engaged in chemical aggression. For example, in case of nutrient depletion, the fungus C. inaequalis produces a VCPO on the surface of its growing hyphen tip, that produces HOCl to facilitate its entry into a plant cell by causing cell wall degradation [3], e.g. via reaction with lignin [4]. The vanadium bromoperoxidases (VBPOs), that are produced in and on the surface of seaweeds, are thought to play a role in anti-fouling by either the direct biocidal activity of HOBr or by bromination of bacterial signalling molecules, thus preventing biofilm formation [5]. VHPOs contain vanadium and this is directly involved in the catalytic action; the corresponding apo-enzymes are inactive [6], [7] and activity can be restored by the addition of vanadate. For VCPO from C. inaequalis, direct observation of the interaction of the substrates with the vanadate cofactor was reported; the nature of a UV–VIS absorption of the bound cofactor is changed upon addition of substrates [2]. This UV–VIS study confirmed that the enzyme first reacts with H₂O₂ to form a peroxo-intermediate, which then reacts with the halide to complete the catalytic cycle. Pre-steady state UV–VIS stopped-flow studies showed that the rate constants of individual steps in the catalytic mechanism that are in line with the kinetic constants obtained from steady state studies [1], [8].

For the VCPO from C. inaequalis, the crystal structures of both the native enzyme and the peroxo-intermediate are available (at 2.03 and 2.24 Å resolution, respectively) [9]; the structures of these vanadium centres are shown in Fig. 1. The information obtained from these crystallographic studies, in conjunction with kinetic, mutagenesis [8], [10], [11], and computational studies [12], [13], and several spectroscopic studies [2], [14], have led to proposals for the catalytic cycle of this and related enzymes. However, given that the X-ray data were obtained with a limited resolution and the crystals were obtained from a solution of the enzyme at pH 8.0, whereas the enzyme shows maximum activity at pH 5–6 [11], [15], further studies are necessary to clarify the structure of the catalytic centre of this enzyme and improve the understanding of the catalytic mechanism.

Herein, we report vanadium K-edge XAS studies, both XANES and EXAFS, of C. inaequalis VCPO for the native enzyme and its peroxo-derivative. The results obtained have been interpreted in light of the crystallographic and XAS information available for this and other VHPOs, together with kinetic data, and have provided new information concerning the nature of the vanadium centre in the catalytic cycle of this enzyme. The results are also compared with the computational and solid-state ⁵¹V-NMR data on this enzyme. In an attempt to explain the long known substrate inhibition at lower pH values a spectrum was also recorded of the native enzyme in the presence of a high concentration of chloride. The spectrum of dithionite-reduced enzyme was also recorded and compared to the previously reported spectrum of reduced vanadium bromoperoxidase from Ascophyllum nodosum [16].

Section snippets

Enzyme preparation

VCPO from C. inaequalis was recombinantly produced in Saccharomyces cerevisiae, as described previously [8]. The enzyme was dialysed against H₂O, concentrated, freeze-dried, then dissolved in 100 mM (N-morpholino)ethanesulfonic acid (MES) pH 6.0, glycerol (20%) and 200 mM Na₂SO₄. The sample of native enzyme investigated had a concentration of 1.3 mM (88 mg/mL). The sample of the native enzyme (1.2 mM, 81 mg/mL) plus H₂O₂ (5.0 mM) was prepared by the addition of 10 μL of a 150 mM solution of H₂O₂ to 290

Overall XAS parameters of all samples

Fig. 2 shows the profiles of the pre-edge, edge, and X-ray absorption near edge structure (XANES) regions of the vanadium K-edge XAS recorded for the five samples investigated. For each of these samples, the position, height and width of the pre-edge peak and the energy of vanadium K-edge are listed in Table 1.

As expected, the observed position of the vanadium K-edge correlates with the oxidation state of the metal centre. Thus, the samples of Na₃VO₄, native VCPO, native VCPO + H₂O₂, and native

Native VCPO

The long V–O bond of 1.95 Å is attributed to oxygen O(4) (Fig. 1, Fig. 6) that was placed 1.93 Å from the vanadium in the crystallographic study [9] (see Table 3). This oxygen was suggested to be present as a hydroxide ion or a water molecule [9] and this seems to be a sensible proposal given the V–O distance. The X-ray crystallographic studies placed the other three oxygen atoms bound to the vanadium at the same distance 1.65 [9] or 1.60 [27] Å. However, these three atoms are resolved in this

Conclusions

The vanadium K-edge XAS studies reported herein have provide clear evidence for the presence of V(V) in the native and peroxo-forms of the VCPO of C. inaequalis. The associated EXAFS information provides accurate values for the length of the V–O and V–N bonds involving the vanadium centre of the two forms of the enzyme at a catalytically relevant pH, 6.0. The EXAFS data obtained for the peroxo-form of this enzyme are the first such data to be reported for this important catalytic intermediate

Abbreviations

XAS

X-ray Absorption Spectroscopy

EXAFS

Extended X-ray Absorption Fine Structure

XANES

X-ray Absorption Near Edge Structure

UV–VIS

Ultraviolet–Visible

QM/MM

Quantum Mechanics/Molecular Mechanics

SS-MAS

Solid State Magic Angle Spinning

VCPO

Vanadium Chloroperoxidase

VBPO

Vanadium Bromoperoxidase

VHPO

Vanadium Haloperoxidase

MES

2-(N-morpholino)ethanesulfonic acid

Acknowledgements

We are grateful to Daresbury Laboratory for provision of beamtime and would like to thank the station scientist Dr. Fred Mosselmans for his assistance during the EXAFS data collection. Mr. Henk L. Dekker is kindly thanked for his help during purification and concentration of the enzyme. This work was supported by the Netherlands Organization for Scientific Research (NWO), the Netherlands Technology Foundation (STW), the Dutch National Research School Combination (NRSC-Catalysis), and DSM

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Vanadium K-edge XAS studies on the native and peroxo-forms of vanadium chloroperoxidase from Curvularia inaequalis

Abstract

Graphical Abstract

Introduction

Section snippets

Enzyme preparation

Overall XAS parameters of all samples

Native VCPO

Conclusions

Abbreviations

Acknowledgements

J. Biol. Chem.

Biochim. Biophys. Acta

Inorg. Chim. Acta

Phytochemistry

J. Biol. Chem.

J. Biol. Chem.

Inorg. Chim. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

FEBS Lett.

Polyhedron

J. Mol. Biol.

Struct. THEOCHEM

J. Biol. Chem.

FEBS Lett.

Biochemistry

Proc. Natl. Acad. Sci.

Appl. Environ. Microbiol.

Biol. Chem.

Theory Comput.

Inorg. Chem.