Vanadium Compounds - American Chemical Society

Chapter 15

Reactivity of Vanadium Bromoperoxidase

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on August 27, 2015 | http://pubs.acs.org Publication Date: December 10, 1998 | doi: 10.1021/bk-1998-0711.ch015

Alison Butler, Richard A. Tschirret-Guth, and Matthew T. Simpson Department of Chemistry, University of California, Santa Barbara, CA 93106-9510

Vanadium haloperoxidases catalyze the halogenation of organic substrates (i.e., chloride, bromide or iodide) or the halide-assisted (i.e., chloride, bromide or iodide) disproportionation of hydrogen peroxide forming dioxygen and water. Competitive kinetic studies and fluorescent quenching studies have been used to probe binding of organic substrates to vanadium bromoperoxidase (V-BrPO). Certain organic substrates have been found to bind to V-BrPO such as the indole derivatives presented herein, among other substrates. When organic substrates bind to V -BrPO, a freely diffusible oxidized bromine intermediate is not released from the enzyme active site. The pH dependence and the selectivity of V-BrPO is also examined.

Haloperoxidases catalyze the oxidation of a halide (e.g., chloride, bromide or iodide) by hydrogen peroxide, which can result in halogenation of organic substrates, i.e., R-H in Equation 1, 1

B r + H 0 + R-H -> Br-H + 2 H 0 2

2

2

Εφι 1

the production of dioxygen through subsequent oxidation of a second equivalent of hydrogen peroxide, or the production of hypohalous acid (i.e., HOC1), depending on the identity of the haloperoxidase. The three types of haloperoxidases which have been identified to date include the vanadium haloperoxidases (1-4), the FeHeme halo peroxidases (5,6) and the nonmetallo haloperoxidases (7). Vanadium bromoperoxidase, which is the focus of this article, has been isolated primarily from marine algae (for recent reviews see references 8,9). Because of the abundance of halogenated marine

202

©1998 American Chemical Society In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

203 natural products, and because V-BrPO can catalyze halogenation reactions, the physiological role of V-BrPO is thought to be in the biosynthesis of the halogenated marine natural products. These natural products range from relatively simple volatile halogenated hydrocarbons (e.g., CHBrç, C P ^ B ^ C H B r C l (10-12)) to more complex compounds, including halogenated indoles and terpenes (13; Figure 1). 2


Scope We are interested in the reactivity of V-BrPO towards organic substrates that are likely precursors of the halogenated natural products, including substituted indole derivatives. In this article we will review the results of competitive kinetic studies which address the mechanism of halogenation and then examine new results on the pH dependence of the competitive kinetic studies as well as fluorescent quenching studies which can probe binding of organic substrates to V-BrPO. The Vanadium Site Recently, the crystal structure of V-C1PO isolated from C. inaequalis as well as the peroxide-bound V-C1PO derivative were reported by Messerschmidt and Wever (14,15 and see related articles in this volume). The x-ray structure of V-BrPO (A. nodosum) has not yet been reported (16,17), however, the sequence alignment of V-BrPO (A. nodosum) and V-C1PO (C. inaequalis) (1,14,15) shows good overlap, particularly in the active site region. Thus the structure of V-BrPO is expected to be very similar to V C1PO. The main structural motif of V-C1PO is α-helical. Vanadium is coordinated at the top of one of two four-helix bundles in a broad channel which is lined on one side with predominantly polar residues including an ion-pair between Arg-360 and Asp-292 and several main chain carbonyl oxygens (Figure 2). The other side of the channel is hydrophobic, containing Pro-47, Pro-211, Try-350, Phe-393, Pro-395, Pro-396, and Phe397. The vanadium site is remarkably simple, comprised essentially of vanadate coordination to a single amino acid residue, His 496 (V-N 1.96 Â), in a pentagonal bipyramidal geometry (Figure 3A) ( 14,15). The "vanadate" site (i.e., three equatorial oxygens at a V - 0 distance of 1.65 Â and one apical V - 0 at 1.93 Â, interpreted as V OH) is stabilized by multiple hydrogen bonding between the vanadate oxygen atoms and certain positively charged protein residues (see Figure 3A). His 404 (C. inaequalis) is hydrogen bonded to the apical hydroxide ligand; this residue is important for peroxide binding and catalysis (18) and is referred to as the acid-base histidine. The x-ray structure of the peroxide form of V-C1PO (2.24 Â resolution) reveals a distorted tetragonal pyramid in which vanadium(V) is coordinated by peroxide in an η fashion (1.87 Â V-O bond lengths; 1.47 À O-O bond length), His 496 (2.19 À V - N bond length) and an oxygen atom (1.93 Â) in the basal plane and by an oxo ligand (1.60 Â) in the axial position (Figure 3B (15)). His 404 is no longer hydrogen bonded to the vanadium center, however, one of the peroxide oxygen atoms is hydrogen bonded to Lys 353. The shortening of the apical V-O bond length from 1.93 Â to 1.60 Â upon 2

In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


204

Ν Η α-synderol L. snyderae [3λ]

R = Η, indigo R = Br, e.e'-dibromoindigotin

Br NCS Thiocyanate and isothiocyanate sesquiterpenes [32]

Η L. brongniartii [33]

Figure 1. Selected Marine Natural Products (Reproduced with permission from reference 8. Copyright 1997 Springer-Verlag.)

Figure 2. The V-C1PO Active Site Channel

In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

205 coordination of peroxide is cited to support the shift from hydroxide coordination (VOH) to oxide coordination (V=0) (15). The Reactivity of Vanadium Bromoperoxidase V-BrPO catalyzes peroxidative halogenation reactions (e.g., equation 1) (2) and the


halide-assisted disproportionation of hydrogen peroxide, producing dioxygen (19-23): Χ" + 2 H 0 -> 0 + 2 H 0 + X 2

2

2

Eqn2

2

Direct disproportionation is not observed with V-BrPO.

The V-BrPO catalytic

mechanism involves first coordination of hydrogen peroxide to the vanadium(V) center (24). Halide oxidation follows (Scheme 1) and because halide saturation kinetics are observed (for both V-C1PO (26) and V-BrPO (A. nodosum) (20,25)) halide binding must occur. One possible halide binding site is the vanadium center (15), although other sites are possible (14). The nature of the oxidized halogen intermediate, such as hypobromous acid (HOBr), bromine (Br ), tribromide (Brç"), or an enzyme-trapped bromonium ion 2

equivalent (e.g., Enz-Br, Venz-OBr, Enz-HOBr, etc), in the case of bromide, has been the subject of much speculation (25,27-29). Under the optimum catalytic conditions for V-BrPO (A. nodosum) (i.e., pH 6.5, 2 m M H 0 , 0.1 M K B r , 50 uM MCD), an 2

2

intermediate cannot be observed, because the halogenation of the substrate or the oxidation of a second equivalent of hydrogen peroxide by the oxidized halogen intermediate is very fast (19-21). At pH 5.0, Br

Vanadium Compounds - American Chemical Society

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