V Solid-State NMR Spectroscopy of Vanadium Haloperoxidases and

Chapter 14

51V Solid-State NMR Spectroscopy of Vanadium Haloperoxidases and Bioinorganic Haloperoxidase Mimics Downloaded by UNIV QUEENSLAND on May 13, 2013 | http://pubs.acs.org Publication Date: August 30, 2007 | doi: 10.1021/bk-2007-0974.ch014

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Tatyana Polenova , Neela Pooransingh-Margolis , Dieter Rehder , Rokus Renirie , and Ron Wever 3

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Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716 Institut für Anorganische und Angewandte Chemie, Universität Hamburg, D-20146 Hamburg, Germany Van't Hoff Institute for Molecular Sciences, Faculty of Science, University of Amsterdam, Nieuwe Achtergracht 129, 1018 WS Amsterdam, The Netherlands

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We present V solid-state magic angle spinning N M R spectroscopy as a probe of geometric and electronic environments in vanadium haloperoxidases and in oxovanadium (V) complexes mimicking the active site of these enzymes. In the bioinorganic complexes, V M A S spectra are sensitive reporters of the coordination environment and coordination geometry. In vanadium chloro- and bromoperoxidases, the spectra reveal unique electronic environments of the vanadate cofactor in each species. The experimental N M R observables and DFT calculations of the N M R parameters yield the most likely protonation states of the individual oxygen ligands in vanadium chloroperoxidase. A combination of experimental solid-state N M R and quantum mechanical calculations thus offers a powerful strategy for analysis of diamagnetic spectroscopically silent vanadium (V) states in inorganic and biological systems. 51

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© 2007 American Chemical Society

In Vanadium: The Versatile Metal; Kustin, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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Introduction Vanadium Haloperoxidases Vanadium haloperoxidases (VHPO) catalyze a two-electron oxidation of halides to hypohalous acids in the presence of hydrogen peroxide; the native enzymes require diamagnetic V(V) for their activity (Scheme 1). +

H 0 + X ' + H —• H 0 + HOX 2

2

2

Downloaded by UNIV QUEENSLAND on May 13, 2013 | http://pubs.acs.org Publication Date: August 30, 2007 | doi: 10.1021/bk-2007-0974.ch014

Scheme 1 Haloperoxidases are named after the most electronegative halide they are able to oxidize (i.e., chloroperoxidases oxidize CI", Br", and I"). Vanadium bromoperoxidases (VBPO) are universally present in marine macroalgae; vanadium chloroperoxidases (VCPO) have been found in terrestrial fungi and in lichens (1-4). Vanadium bromoperoxidases are thought to be involved in the biosynthesis of halogenated (brominated and iodinated) natural products; the function of the fungal chloroperoxidase is to damage (through oxidation by HOC1) the protective lignocellulose of plant tissues (1-3, 5, 6). The kinetics and mechanism of haloperoxidases have been the subject of multiple studies (7, 8) due to their potential use as industrial antimicrobial agents and disinfectants (9, 10) as well as halogenation catalysts (//, 12): these enzymes are the most efficient and stable halide oxidants known to date. Despite numerous investigations by multiple laboratories addressing various aspects of chemistry and biology of vanadium haloperoxidases, many questions about their function and mechanism remain open preventing their widespread biotechnological use and evaluation of other areas of applications, in part due to the fact that the enzymes are colorless and diamagnetic in their active form, making the spectroscopic studies difficult. Weak U V bands were detected in the region of 300-330 nm that report on vanadate binding to vanadium chloroperoxidase (VCPO), but these do not permit detailed analysis of the vanadium ligands (13). The factors determining the substrate specificity, i.e. whether a particular enzyme will or will not display chlorinating activity are not understood (14-16). A delicate balance of multiple interactions between the active site residues has been proposed to be responsible for chlorinating activity of the haloperoxidases (15). The architectures of the chloro- and bromoperoxidase active sites are found to be very similar, as illustrated in Figure 1, where the active sites of vanadium chloro- and bromoperoxidases are superimposed using the original X-ray coordinates for the two enzymes (17, 18). Mutation studies suggest that altering the electrostatic potential distribution either reduces or completely abolishes the chlorinating activity in a series of mutant chloroperoxidases, yet the crystal structures for a number of these mutants with the exception of H496A reveal the active site containing vanadate coordinated to His-496, similar to the native protein



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Figure 1. The superimposition of the active sites of VCPO from C. inaequalis (black, ball-and stick) and VBPO from A. nodosum (grey, stick) illustrates their similar geometries. The vanadate cofactor is depicted with larger spheres: vanadium (grey), oxygen (white). Active site residue labels correspond to VCPO (top, black) and VBPO (bottom, grey). The proposed vanadate geometry consists of an axial hydroxo group trans to His-496/His-486 and three equatorial oxo ligands. At pH lower than 8.0 protonation of one of the oxo groups may occur. The figure has been prepared using the original pdb coordinates (pdb codes lidq and lqi9) in DSViewer Pro (Accelrys, Inc.)



181 (13, 15, 19). Therefore, these mutagenesis results provide indirect evidence that the electronic structure of the vanadate cofactor is modulated by the protein, thus affecting its chemical reactivity. The X-ray crystal structure determined at pH 8.0 revealed that in the resting state of VCPO, the vanadate cofactor is covalently bound to the N E 2 atom of a histidine residue (His-496 in the V C P O from C. inaequalis) (17). The negative charge of the vanadate group is compensated by hydrogen bonds to several positively charged protein sidechains (Lys-353, Arg-360, and Arg-490) (17). Ser402 and Gly-403 form hydrogen bonds with the equatorial oxygens of the vanadate cofactor; an additional hydrogen bond may exist between the axial hydroxo group and His-404. However, the presence and positions of hydrogen atoms could not be unambiguously determined from the crystal structure due to the inherently limited resolution; therefore, the nature of the vanadium first coordination sphere ligands (i.e., whether a particular group is oxo- or hydroxo-) still remains the subject of debate. Recently, the electronic structure and geometry of the vanadium center in the resting state (20, 21) and peroxo forms (21, 22) of a series of V H P O active site models were addressed quantum mechanically via Density Functional Theory. Subsequently, hybrid Q M / M M calculations were conducted independently by two groups of investigators addressing extended active site models of V C P O treated quantum mechanically and significant portions of the protein treated with classical mechanics (23, 24). In parallel, DFT calculations were performed on large models of V C P O active site (25). These exciting DFT and Q M / M M studies have led to several very interesting conclusions. A l l of the above calculations suggest that in the resting state, at least one equatorial oxygen needs to be protonated to stabilize the metal cofactor (20, 23-25). According to the DFT results, the equatorial hydroxo group is likely to be coordinated to Ser-402 (25). As anticipated, Q M / M M calculations indicate that the protein environment is crucial for creating the long-range electrostatic field necessary for the stabilization of the resting state (23). Interestingly, the Q M / M M calculations conducted by Carlson, Pecoraro and Kravitz suggest that the protonated equatorial oxygen is accepting two hydrogen bonds from Arg-360 and Arg-390 residues (23). A hybrid resting state consisting of the two lowest-energy minima is likely based on the energetic considerations (23). The roles of the individual amino acid residues and of the oxo atoms of the cofactor have been re-examined, and a revised mechanism for V C P O catalyzed halide oxidation reaction has been proposed (23). In another Q M / M M study, Raugei and Carloni examined the early intermediates and the transition state of the halide oxidation in VCPO, by including a somewhat smaller number of atoms in the Q M region of the calculation, and treating the rest of the protein at the M M level (24). They concluded that one of the equatorial oxygens is protonated, in agreement with the above studies, but the equatorial hydroxy group is hydrogen bonded to either Ser-402 or to Lys-353 residue (24). Thus, the coordination environment of the vanadate cofactor including the protonation states of the individual oxygen atoms still remains an open question.


182 Based on the above studies, it is clear that there is a need to address experimentally the coordination environment of the vanadium cofactor in vanadium chloro- and bromoperoxidases. Understanding the protonation states of the oxygen atoms is important for elucidating the chlorinating activity in haloperoxidases. The lack of direct site-specific spectroscopic probes has so far prevented gaining further insight on the electronic environment of the vanadium center intimately related to the enzymatic mechanism of vanadium haloperoxidases. In this work, we introduce V solid-state magic angle spinning N M R spectroscopy as a direct spectroscopic probe of the diamagnetic vanadium (V) sites of vanadium haloperoxidases. V is a half-integer quadrupolar nucleus (I = 7/2) whose high natural abundance (99.8%), relatively high gyromagnetic ratio (Larmor frequency of 157.6 M H z at 14.1 T), and a relatively small quadrupole moment (-0.052* 10* V/m )(26) make it favorable for direct detection in the N M R experiments. The V solid-state spectra are typically dominated by the anisotropic quadrupolar and chemical shielding interactions. These interactions in turn report on the geometric and electronic structure of the vanadium site (2729). As demonstrated for the model bioionorganic oxovanadium complexes mimicking the active site of haloperoxidases, the N M R parameters extracted from numerical simulations of the V M A S spectra are sensitive to the coordination environment of the vanadium atom beyond the first coordination sphere. The N M R fine structure constants calculated for the crystallographically characterized complexes using Density Functional Theory, are in good agreement with the experimental results, illustrating that a combination of solidstate N M R experiments and quantum mechanical calculations presents a powerful approach for deriving coordination geometry in these systems. 5 ,


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5 1

5 ,

5 ,

We present V M A S N M R spectra of vanadium chloroperoxidase from C. inaequalis and of vanadium bromoperoxidase from A. nodosum. The spectra reveal different electronic environments of the vanadate cofactor in each species. In both enzymes, the spectra are dominated by a large quadrupolar interaction, providing the first direct experimental evidence of the asymmetric electronic charge distribution at the vanadium site. The isotropic chemical shifts in V C P O and V B P O are profoundly different, suggesting that the vanadate oxygens are likely not to have the same protonation states. In VCPO, the N M R observables were extracted from the spectra, and the DFT calculations of these observables in the extensive series of the active site models whose electronic structure and energetics were previously addressed by De Gioia, Carlson, Pecoraro and co workers (20) indicate that one equatorial and one axial oxygen are protonated resulting in an overall anionic vanadate. Our experimental results are in remarkable agreement with the quantum mechanical calculations discussed above (20, 23-25). We anticipate that our approach combining V solid-state N M R spectroscopy and quantum mechanical calculations will yield a thorough understanding of the salient features of the vanadium haloperoxidases' active sites. 5 ,


183 5 1

V Solid-State N M R Spectroscopy 5 ,

V is a half-integer quadrupolar nucleus (1=7/2). Two tensorial interactions dominate the V solid-state N M R spectra: the quadrupolar and chemical shielding anisotropics. Due to their different magnitudes and symmetries, both tensors can be extracted from a single N M R spectrum (27, 28, 30, 31). It is worth noting that in solution, both interactions are averaged due to molecular tumbling, and only the isotropic component of the chemical shift tensor is observed. Therefore, valuable geometric and electronic information is lost in solution. In the solid state, the total Hamiltonian can be expressed as: Downloaded by UNIV QUEENSLAND on May 13, 2013 | http://pubs.acs.org Publication Date: August 30, 2007 | doi: 10.1021/bk-2007-0974.ch014

5 ,

"

=

" Zeeman + H RF +

H

DIP +

H

Q +H

0)

C S A

The first three terms are the Zeeman, the radiofrequency field, and the dipolar interactions. The dipolar interaction is typically much smaller than the quadrupolar and chemical shielding anisotropy and will be omitted in the subsequent discussions. The last two terms are the quadrupolar and C S A interactions, which determine the spectral shape. They are commonly expressed in a spherical tensor notation in terms of the spatial (R ) and spin (T ) variables (32): mn

(V

,

H

C

R

S

and

T

T

g y 2m 2-m[ 2m- 2-m]

"CSA -Mo Tw H^Q

R

mn

(3)

( 4 )

+^2o)=te + » T h

are the first- and second-order quadrupolar interactions. The

quadrupolar and CSA tensor elements are defined in a spherical harmonics basis set according to the standard notation (33, 34):

„ yy' xx . 7h

HN^ Ph

SJZ0060 (V)

SJZ0069 (IV)

SJZ0068 (VI)

A HS001 (VIII)

SJZ00108(VII) P h

^

s

" ST I OH,

«

M

M

0

HS003 (IX)

SalSOEt (X)

VQ [acpy-inh] (XI) 2

Figure 2. Chemical structures of the oxovanadiumfV) compounds mimicking the VHPO active sites.



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siV Frequency (kHz) 51

Figure 3. Top: Experimental V solid-state MAS NMR spectrum of XI (MAS frequency of 11 kHz). The overall width of the spectral envelope is determined by CQ, the shape- by n^; the width and the shape of the central transition- by 8

V Solid-State NMR Spectroscopy of Vanadium Haloperoxidases and

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