High‐resolution XANES studies on vanadium‐containing haloperoxidase: pH‐dependence and substrate binding

Abstract

High-resolution X-ray absorption vanadium K-edge spectra were recorded for samples of vanadium-containing bromoperoxidase from the brown alga, Ascophyllum nodosum, at pH 9, 7, 5 and 4, as well as for enzyme samples containing the substrates, hydrogen peroxide and bromide. The well-resolved features of the XANES spectra are discussed. The pH-dependence of the structure of the active site has been studied revealing no significant change of the absorption features. We were able to detect an interaction of HrOr with the vanadium site of the bromoperoxidase using XAS spectroscopy, whereas addition of bromide causes no energy shift of the XANES spectrum. The possible role of vanadium during the enzymatic reaction is discussed on the basis of our results.

Vanadium haloperoxidases: From the discovery 30 years ago to X-ray crystallographic and V K-edge absorption spectroscopic studies

“…Addition of 4 molar equivalents of H 2 O 2 had no effect [42] but a significant shift of the edge to lower energy and a subtle increase in the pre-edge feature were observed with 15 molar equivalents [43]. These changes point respectively to an increase in average V-ligand distance and to a change in symmetry, but not to a change in oxidation state for V. Accordingly, a mechanism was proposed for VHPO-based bromination (Scheme 1)…”

“…While the XANES of A. nodosum VBPOI showed a significant shift to lower energy with a large excess of H 2 O 2 (see above) [43], indicating a longer average Vligand distance, the corresponding spectra for C. inaequalis VCPO with 4 molar equivalents of H 2 O 2 were rather similar. The EXAFS of both enzyme forms was also measured, compared, and analyzed [141] in an approach in which the geometry of the imidazole was constrained [142], i.e.…”

“…For the reduced enzyme, one short (1.63 Å) and 3 medium (1.91 Å) V-O distances were identified, along with two V-N distances of 2.11 Å, which were inferred to belong to imidazole moieties of histidine residues on the basis of advanced EPR (electron spin echo) studies [43], and simulated as such. The results for the native enzyme were similar, but most distances (1.61, 1.72, and 2.11 Å) were shorter, and the more remote atoms were not explicitly assigned to imidazoles but as N or O.…”

“…Since its discovery, AnI was used as a laboratory model enzyme for the understanding of VHPO properties. Numerous further studies were carried out to characterize the V coordination inside the active site of AnI (later called An-VBPOI in this review), based on electron paramagnetic resonance [26,34,35], on 51 V NMR [36][37][38], X-ray crystallography [39,40] and on XAS ( [41][42][43] see below).…”

Halogenation and Vanadium Haloperoxidases

“…In the intermediate H 2 O 2 /enzyme complex, the peroxo bond is oriented versus Phe397 in Ci-VCPO (Messerschmidt, Prade, and , while in An-VBPOI the modelized peroxo bond is hydrogen-linked with the N d1 of His411 (Weyand et al, 1999). The vanadium plays the role of a strong Lewis acid (Arber et al, 1989;, and its oxidation state (V) remains at its highest (Arber et al, 1989;De Boer, Boon, and Wever, 1988;Hormes et al, 1988;Krenn, Tromp, and Wevers, 1989;K€ usthardt et al, 1993;Renirie et al, 2010;Vilter and Rehder, 1987). The reaction with H 2 O 2 does not reduce vanadium(V) to the (IV) state in the native enzyme (De Boer et al, 1986); indeed, when vanadium was reduced to the (IV) oxidation state the enzyme lost its activity.…”