Peroxygenases offer attractive means to address challenges in selective oxyfunctionalisation chemistry. Despite their attractiveness, the application of peroxygenases in synthetic chemistry remains challenging due to their facile inactivation by the stoichiometric oxidant (H2O2). Often atom inefficient peroxide generation systems are required, which show little potential for large scale implementation. Here we show that visible light-driven, catalytic water oxidation can be used for in situ generation of H2O2 from water, rendering the peroxygenase catalytically active. In this way the stereoselective oxyfunctionalisation of hydrocarbons can be achieved by simply using the catalytic system, water and visible light.

We show here that the amino acid residues contributing to the active sites of the vanadate containing haloperoxidases are conserved within three families of acid phosphatases; this suggests that the active sites of these enzymes are very similar. This is confirmed by activity measurements showing that apochloroperoxidase exhibits phosphatase activity. These observations not only reveal interesting evolutionary relationships between these groups of enzymes but may also have important implications for the research on acid phosphatases, especially glucose-6-phosphatase-the enzyme affected in von Gierke disease-of which the predicted membrane topology may have to be reconsidered.Haloperoxidases are enzymes catalyzing the two electron oxidation of a halide (X Ϫ ) to the corresponding hypohalous acid according to Eq. 1.HOX may further react with a broad range of nucleophilic acceptors to form a diversity of halogenated compounds. These haloperoxidases are named after the most electronegative halide they are able to oxidize, and thus a chloroperoxidase (CPO) is able to oxidize chloride, bromide, and iodide. Three classes of haloperoxidases have been identified. One of these consists of enzymes without a prosthetic group and as such have been detected in a number of bacteria (1, 2). The remaining two classes are the so-called heme-containing haloperoxidases-exemplified by the CPO from the fungus Caldariomyces fumago (3) or myeloperoxidase which is present in white blood cells (4)-and the vanadium-containing haloperoxidases that bind a vanadate ion (VO 4 3Ϫ ) as a prosthetic group. Enzymes representing these two classes not only differ in the nature of their prosthetic group but also in at least two other aspects: catalytic mechanism and stability. Hemecontaining peroxidases catalyze the formation of the hypohalous acid by a redox mechanism, whereas in vanadatecontaining peroxidases the transition metal does not change its redox state (5, 6) but may function as a Lewis acid. Vanadatecontaining haloperoxidases not only posses a very high stability (7, 8) but they also resist a high concentration of their substrate (H 2 O 2 ) (9) and their product (HOX) (7) that would readily inactivate the heme-containing peroxidases.Vanadate-containing peroxidases were first discovered in seaweeds about a decade ago (10, 11); at present they are being discovered not only in an increasing number of seaweeds (12, 13) but also in fungi (8) and in a lichen (14).The fungus Curvularia inaequalis has been shown to express a vanadium-containing CPO (V-CPO) (15), and recently the gene encoding this enzyme was cloned and sequenced (16). The V-CPO gene codes for a protein of 609 amino acids with a calculated molecular mass of 67,488 kDa. Database searches using the entire V-CPO sequence showed very little sequence similarity to other known proteins (16). The recent determination of the crystal structure of this enzyme at 2.1-Å resolution (17) revealed that the protein has an overall cylindrical shape and measures approximately 50 ϫ 80 Å. Th...

The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His 496 is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg 360 , Arg 490 , and Lys 353. When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.Haloperoxidases are enzymes catalyzing the two-electron oxidation of a halide (X Ϫ

The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11 A resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.

We report 51V solid-state NMR spectroscopy of the 67.5-kDa vanadium chloroperoxidase, at 14.1 T. We demonstrate that, despite the low concentration of vanadium sites in the protein (one per molecule, 1 mumol of vanadium spins in the entire sample), the spinning sideband manifold spanning the central and the satellite transitions is readily detectable. The quadrupolar and chemical shift anisotropy tensors have been determined by numerical simulations of the spinning sideband envelopes and the line shapes of the individual spinning sidebands corresponding to the central transition. The observed quadrupolar coupling constant C(Q) of 10.5 +/- 1.5 MHz and chemical shift anisotropy delta(sigma) of -520 +/- 13 ppm are sensitive reporters of the geometric and electronic structure of the vanadium center. Density functional theory calculations of the NMR spectroscopic observables for an extensive series of active site models indicate that the vanadate cofactor is most likely anionic with one axial hydroxo- group and an equatorial plane consisting of one hydroxo- and two oxo- groups. The work reported in this manuscript is the first example of 51V solid-state NMR spectroscopy applied to probe the vanadium center in a protein directly. This approach yields the detailed coordination environment of the metal unavailable from other experimental measurements and is expected to be generally applicable for studies of diamagnetic vanadium sites in metalloproteins.