Vanadium K-edge x-ray absorption spectroscopy of bromoperoxidase from Ascophyllum nodosum

Abstract

Bromoperoxidase from Ascophyllum nodusum was the first vanadium-containing enzyme to be isolated. X-ray absorption spectra have now been collected in order to investigate the coordination of vanadium in the native, native plus bromide, native plus hydrogen peroxide, and dithionite-reduced forms of the enzyme. The edge and X-ray absorption near-edge structures show that, in the four samples studied, it is only on reduction of the native enzyme that the metal site is substantially altered. In addition, these data are consistent with the presence of vanadium(IV) in the reduced enzyme and vanadium(V) in the other samples. Extended X-ray absorption fine structure data confirm that there are structural changes at the metal site on reduction of the native enzyme, notably a lengthening of the average inner-shell distance, and the presence of terminal oxygen together with histidine and oxygen-donating residues.

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

“…The prominent pre-edge peak at 5469 eV ( Figure 5A) is ascribed to a 1s-3d transition which is dipole-forbidden and absent in octahedral VO [129] but very strong in tetrahedral vanadate ( Figure 5A). For the native bromoperoxidase of A. nodosum, the feature is somewhat weaker than for vanadate ( Figure 5A, [42]) which is consistent with the trigonal bipyramidal coordination symmetry established in the crystallographic study [39]. A similar change was observed in the XANES of vanadate upon binding to the chloroplast H + -ATPase CF1 together with Mg 2+ and ADP; this creates an enzyme-bound transition-state analogue for ATP-hydrolysis in which the V has trigonalbipyramidal coordination geometry [130].…”

“…The haloperoxidase XANES is not affected by addition of the substrate Br -(90 molar equivalents), but upon reduction the edge shifts to lower energy and the pre-edge feature is A c c e p t e d M a n u s c r i p t 18 reduced in intensity [42]. 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].…”

“…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)…”

“…nodosum VBPOI in a k range of 3-10 Å -1 [42] at a time when no crystallographic information was yet available. 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.…”

“…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).…”