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Page 1: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

Co

3.13 Vanadium BiochemistryDC Crans and PB Chatterjee, Colorado State University, Fort Collins, CO, USA

ã 2013 Elsevier Ltd. All rights reserved.

3.13.1 Introduction 3243.13.2 Vanadium Chemistry 3243.13.3 Enzymes Containing Vanadium 3253.13.3.1 Nature’s Halogenation Catalysts: VHPOs 3253.13.3.1.1 The peroxidase reaction and general description of the haloperoxidase enzymes 3253.13.3.1.2 Vanadium bromoperoxidase 3253.13.3.1.3 Vanadium chloroperoxidase 3263.13.3.1.4 Comparing the haloperoxidases 3273.13.3.1.5 Substrate activity by haloperoxidases 3273.13.3.1.6 Phosphatase activity by haloperoxidases 3293.13.3.2 Nitrogenases 3303.13.3.2.1 General overview 3303.13.3.2.2 Structural perception and amino acid sequence 3303.13.3.2.3 Enzyme catalysis 3313.13.3.2.4 Dinitrogen reduction 3313.13.3.2.5 Carbon monoxide reduction 3323.13.3.3 Vanabins 3323.13.4 Organisms Accumulating Vanadium 3323.13.4.1 Tunicates 3323.13.4.1.1 General overview 3323.13.4.1.2 Oxidation state of vanadium in tunicates 3333.13.4.1.3 Tunichromes 3343.13.4.1.4 Vanadium accumulation and reduction in tunicates 3343.13.4.2 Amavadine 3353.13.4.2.1 General overview 3353.13.4.2.2 Structure and reactivity 3353.13.4.3 Fan Worm 3373.13.5 Insulin Enhancing Effect of Vanadium Compounds 3373.13.6 Conclusion 338Acknowledgments 338References 338

AbbreviationsABTS 2,20-Azino-bis(3-ethylbenzthiazoline-6-sulfonic

acid)

acac Acetylacetonante

ADP Adenosine 50-diphosphateALG Alginates

ATP Adenosine 50-triphosphateCPO Chloroperoxidase

CSA Chemical shield anisotropy

CWEPR Continuous wave electron paramagnetic

resonance

DFT Density functional theory

DNA Deoxyribonucleic acid

DOPA 3-(3,4-Dihydroxyphenyl)alanine

EFG Electric field gradient

EPA Environmental Protection Agency

ESEEM Electron spin-echo envelope modulation

ESR Electron spin resonance

EXAFS Extended x-ray absorption fine structure

FeVco Iron–vanadium cofactor

mprehensive I

norganic Chemistry II http://dx.doi.org/10.1016/B978-0-08-09777

H3hida N-Hydroxyiminodiacetate

H3hidpa l,l-N-Hydroxyimino-a,a0-dipropionic acidHase Hydrogenase

HOX Hypohalous acid

IR Infrared

MCD Molar circular dichroism

MMA Methyl mercaptoacetate

MW Molecular weight

NADPH Nicotinamide adenine dinucleotide

phosphate

NHE Normal hydrogen electrode

NMR Nuclear magnetic resonance

PS Phenolic substances

QTAIM Quantum theory of atoms in molecules

SEC Size exclusion chromatography

TFA Trifluoroacetic acid

TOPA 3,4,5-Trihydroxyphenylalanine

UV Ultraviolet

VAP Vanadium-associated proteins

VBPO Vanadium bromoperoxidase

4-4.00324-7

323
Page 2: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

324 Vanadium Biochemistry

VCPO Vanadium chloroperoxidase

VHPO Vanadium haloperoxidase

VIPO Vanadium iodoperoxidase

VNase Vanadium nitrogenase

XANES X-ray absorption near edge structure

XAS X-ray absorption spectroscopy

H2O

V

H2O

O

O

-O

Scheme 1vanadium(IV

O 2+

OH2V

H2O

1

3 4 5

2

O-

-

V

O

OH

O--O

V

O

OH

OH-O

OH2

3+

OH2H2OV

H2O

OH2

OH2

OH2

Schematic illustration of major aqueous solution species for) (1), vanadium(III) (2) and vanadium(V) (3, 4, 5).

3.13.1 Introduction

Vanadium is a trace metal that, although present in our vitamin

supplements and healthyMt. Fuji ‘Vanadiumwater’ still remains

an element with less certain functions in biology and biochem-

istry. Although some groups are convinced that vanadium is an

essential element for life,1,2 experiments supporting such role are

difficult to come by and are often accompanied by experimental

limitations. The last 50 years have uncovered the rich bio-

chemistry of vanadium with proteins and organisms requiring

this colorful metal for function in the biosphere.3–9 These pro-

teins include the enzymes haloperoxidases in algae and

seaweed,3,4,7,10–17 vanadium-containing nitrogenases,4,18,19

other enzymes such as periplasmic nitrate reductase,20,21 and

the vanadium-binding proteins, vanabins.4,22–25 Vanadium-

containing tunicates,22–26 also referred to as sea squirts, contain

up to molar concentrations of vanadium where vanadium is

associated with the vanabins.4,22–25 Vanadium-accumulating or-

ganisms including the mushroom, Amanita muscaria,27–33,34,35

together with the tunicates contain the highest concentrations

of vanadium. In addition to its role in biochemistry, vanadium

can take on multiple roles such as vanadium(V)’s analogy with

phosphorus4,36 and vanadium(IV)’s analogy with divalent metal

ions4,37,38 and these are different from the proteins and organ-

isms that require vanadium that are described in this chapter.

Vanadium with element number 23 is a first-row transition

metal between titanium and chromium. It is the 22nd most

abundant element in the earth’s crust surpassing important,

bioactive metals such as Cu and Zn.8 Through its presence in

the soil, it enters the food chain through assimilation into the

vegetation and is a common trace element in the seeds and roots

of plants and crops.39 This propagation of vanadium through

the food chain explains the traces of vanadium that commonly

are found in human and animal foods. For example, in a glass of

beer there is approximately 1 mg of vanadium. It exists in a

number of minerals such as carnotite, vanadinite, and titanifer-

ous magnetite, and is particularly common in rock-containing

crust.40,41 It is also an important component of carbiferous

materials such as oils, shales, tar sands, and coals, and is often

released upon processing of fuels40 which will leach into the

ground water. Vanadium is routinely monitored before certify-

ing the purity of drinking water. Environmental Protection

Agengy (EPA) standards for acceptable vanadium levels in

water are 50 mg l�1, but even in areas with vanadium-containing

rocks, the vanadium content in the ground water is lower.40,41

The concentrations of vanadium in vanadium-accumulating

organisms are significantly higher,4,41 although the vanadium

is complexed and thus not toxic. In contrast, algae and lichen

that contain vanadium haloperoxidases (VHPOs) still only sup-

port an overall low level of vanadium despite the production of

the vanadium-containing enzymes. We herein review vanadium

biochemistry with a focus on the biology and biochemistry that

requires vanadium.

3.13.2 Vanadium Chemistry

As a group 5metal ion, vanadiumhas a range of oxidation states

available to it and several of these are represented in its

biochemistry.42,43 In low oxidation state vanadium com-

pounds are not stable in the presence of oxygen (1 and 2,

Scheme 1) and thus generally not compatible with environ-

ments at the surface of the earth.41,44 Vanadium is abundant in

the earth’s crust generally in the highest oxidation states. The

vanadium nucleus has only two natural isotopes, 50V and 51V,

of which the latter has the natural abundancemore than 99%.45

In the biosphere, the most common forms of vanadium are in

oxidation states (IV) and (V).6,42 The forms of vanadium found

in anaerobic environments can be at oxidation state III and thus

in the lower oxidation state range of vanadium.46 The vana-

dium in nitrogenase and tunicates are in oxidation states (III)

and/or (IV) and testify to the anaerobic environment of these

systems.

The vanadium ion in oxidation state (III) is a d2 metal ion

and in oxidation state IV is a d1 metal ion.46 Both vanadium

(III) and (IV) undergo hydrolysis reactions and therefore form

several species.4,27,47 Studies with these oxidation states in

the absence of ligands and/or in the presence of oxygen will

result in oxidation of vanadium. Vanadium(III) chemistry

is less investigated than vanadium(IV) chemistry.46,48,49

However, several species form from this oxidation state

which can be quite stable even in the presence of oxygen.50

In oxidation state (III) vanadium is always non-oxo. In aque-

ous medium, the hexaaquanonoxovanadium(III) forms exclu-

sively (2, Scheme 1). The vanadium(IV) metal ion generally

exists as an oxo ion, VO2þ (1, Scheme 1), and forms the

classical eight-line profile in EPR spectroscopy.37,51 Bare vana-

dium(IV) ion is rare and has been explored relatively little

because of the extreme inertness of the V¼O terminal bond

Page 3: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

Vanadium Biochemistry 325

and the unusually high V¼O bond energy in vanadyl com-

pounds. Precursors containing this functionality are common

and include VOSO4 and VO(acac)2.

The coordination geometry of a number of oxovanadium

species ranges between square pyramidal and octahedral.

The axial V¼O bond length is in the range 1.57–1.67 A. The

O–V–O bond angles deviate from the ideal value resulting in

the central vanadium being raised slightly above the basal

plane. Molecular orbital description reported by Ballhausen52

describes the bonding properties in oxovanadium species and

the strong terminal V¼O bond which by some is described as a

V�O bond. Other than one axial s-bond there are two axial p-bonds. As in the ion, the p-bonding orbitals are directed axially

resulting in a triple bond between the two atoms, vanadium,

and oxygen. Vanadium(IV) is stable at low pH, but at neutral

pH requires complexation in order to prevent oxidation.53 The

vanadium(IV) species is versatile by forming stable complexes

with a range of ligands with varying donor sites and

denticity.54 Complexes with O-based donors are particularly

stable and N-based donors are also very common.54 Com-

plexes formed with S-based donors are less common,53,54 but

play an important role in vanadium biochemistry.53

In oxidation state (V), vanadium is a d0 metal ion and, as

such, has complex aqueous chemistry.5,54 This oxidation state

has the metal ion existing in both cationic and anionic forms

and forms a wide range of complexes.5 The coordination

chemistry of vanadium(V) is very well understood, because

the diamagnetic nature of this metal ion and its high natural

abundance and desirable quadrupolar moment render it a

very convenient nuclear magnetic resonance (NMR)-active

nucleus.51,55–59 However, the multiple protonation states and

nuclearities of the oxovanadates that form in aqueous solution

make this chemistry very environment dependent and range from

anions at the higher pH values to cationic forms at the lower pH

values.6,60 In nature, vanadium(V) exists in multiple forms of

oxovanadates (3–5, Scheme 1) in a range of minerals.44 This

oxovanadate(V) chemistry is avoided when vanadium is firmly

complexed to a protein such as observed when vanadium is a

cofactor in haloperoxidases.

3.13.3 Enzymes Containing Vanadium

The largest class of enzymes that naturally contain vanadium is

the haloperoxidases.3 VHPOs catalyze a two-electron oxidation

of halide (X�) to hypohalous acid (HOX) in the presence of

hydrogen peroxide via an activated peroxometal intermediate

(eqn [1]). The reaction does not work for fluoride (F�). During

the oxidation process halogenation of a range of organic sub-

strates takes place.

RHþH2O2 þ X� þHþ ! RX þ 2H2O [1]

The second known family of bacterial and archaeal metal-

loenzymes that contain vanadium is vanadium nitrogenases

(VNases). These enzymes are produced under the condition of

molybdenum depletion and they are responsible for enzymatic

dinitrogen fixation. The reaction depicted in eqn [2] shows

conversion of atmospheric dinitrogen (N2) to bioavailable

ammonia (NH3) with concomitant production of hydrogen

(H2) gas:

N � Nþ 8Hþ þ 16MgATPþ 8e�

! 16MgADPþ 16PiþH2 þ 2NH3 [2]

Vanabins are known as vanadium-binding proteins. Using

a specific antibody, these proteins were shown to be located in

the cytoplasm of vanadocytes (vanadium-containing blood

cells) of the ascidian Ascidia sydneiensis samea. These are the

first proteins reported to show specific binding to vanadium

(IV). The role of the vanabins may include transport and

storage processes in tunicates.

3.13.3.1 Nature’s Halogenation Catalysts: VHPOs

3.13.3.1.1 The peroxidase reaction and generaldescription of the haloperoxidase enzymesThere are three known types of haloperoxidases: two contain

either a prosthetic heme group found in mammals or a

vanadate ion as in the case of VHPOs.10 The third type

of haloperoxidases are metal-free haloperoxidases.61,62 The

vanadium-containing haloperoxidases (VHPOs) can be divided

into chloroperoxidases (VCPOs), bromoperoxidases (VBPOs),

and iodoperoxidases (VIPOs),63 with the proteins being classi-

fied according to the most electronegative halide that they

oxidize. VCPOs oxidize Cl�, Br�, and I� in the presence of

H2O2. VBPOs can oxidize Br� and I� while VIPOs catalyze the

oxidation of I�. Haloperoxidases generate oxygen in the absence

of an organic substrate by the disproportionation of hydrogen

peroxide by halide.64 In the presence of bromide, singlet oxygen

is the product.65 The native enzymes require vanadium(V) in

the form of a vanadate covalently linked to a histidine for their

activity. The function of these enzymes is believed to be the

biosynthesis of halogenated natural products in vivo.14,66

VHPOs selectively halogenate the phenyl group of a number of

antifungal, antibacterial, and antitumor compounds.67 Within

the various families of VHPOs only 20% of the primary amino

acid sequences are preserved, whereas the overall structures of

these proteins were found to be different with the exception

of the regions close to the metal active site.68–70

After Vilter’s discovery of VBPO in 1984 from Ascophyllum

nodosum,10,71 the existence of vanadium as a redox cofactor

in other peroxidases was demonstrated.15 Haloperoxidases

are universally present in all types of marine algae and in

many other marine organisms.10,72 Additionally, they are

found in terrestrial fungi and in some lichen.13,73 All the

VHPOs can survive temperatures up to 70 �C. They also remain

functional in many organic solvents and resistant toward oxi-

dative inactivation in the presence of high concentrations of

peroxide.74 Several reviews describe the various aspects of

VHPOs including enzyme structure, function, mechanism,

and functional and structural model chemistry and the reader

is referred there for more detail.4,10

3.13.3.1.2 Vanadium bromoperoxidaseA number of VBPOs have been isolated from different enzyme

sources,75 but the most explored enzyme is the first reported

120.5-kDa isoenzyme of VBPOs from A. nodosum.69 Studies

have focused on substrate specificity and structural, kinetic,

and spectroscopic properties of VBPO. The coordination envi-

ronment around vanadium and the oxidation state of the

metal was investigated using 51V NMR,6,76 XAS,77 continuous

Page 4: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

326 Vanadium Biochemistry

wave electron paramagnetic resonance (CWEPR),78 Electron

spin-echo envelope modulation (ESEEM),79 and x-ray

crystallography.69 The x-ray crystal structure of the VBPO

from the red alga Corallina pilulifera has also been solved in

the presence of phenol red and phloroglucinol.80 In the active

site of VBPOs from A. nodosum, there are two vanadium atoms

per homodimeric structure of the enzyme. X-ray crystallographic

studies at 2.4 A resolution show that the homoenzyme has the

dimension around 90�77�75 A. The two monomeric units

are bridged by three intramolecular and two intermolecular

disulfide bridges.69 The protein fold is mainly a-helical consist-ing of two to four helix bundles and six additional long helices.

The vanadate is bound at the end of a 15 A-deep substrate

funnel. The coordination environment around the vanadium

center is trigonal bipyramidal completed by a NO4 donor set

(Figure 1).69 Carboxylate residues on the protein chains support

the vanadate (VO4) moiety (vanadium(V)) with the His-486

nitrogen atom covalently bound to the vanadate group

(Figure 1). The negative charge on the vanadate is supported

by an extensive hydrogen bond network, which involves differ-

ent protein residues.

3.13.3.1.3 Vanadium chloroperoxidaseThe VCPOs were found in a number of dematiaceous

hyphomycetes,81,82 such as the pathogenic fungus Curvularia

inaequalis.83 The secondary structure of the protein is mainly

all-helical consisting of 2a helices. Of them, two central four-

helix groups are the main structural motifs (mainly a helical).68

The crystal structure of a 67-kDa vanadium-dependent haloper-

oxidase was solved (Figure 2) for the resting state of the native

VCPO at pH 8.0.68,74 The coordination geometry of the vana-

dium is trigonal bipyramidal with three equatorial oxygens and

Asp 278

Asp 490

2.9

2.5

2.7

2.8

2.93.1

3.1

W 773

W 772

W 761His 418

His 411

His 486

Figure 1 Structure of the active site of vanadium bromoperoxidase(VBPO) isolated from the brown alga A. nodosum. Adapted fromWeyand, M.; Hecht, H. J.; Kiess, M.; Liaud, M. F.; Vilter, H.; Schomburg,D. J. Mol. Biol. 1999, 293, 595, with permission. Copyright 1999 Elsevier.

bond distances around 1.65 A. The presumed OH group in one

apical position has a bond length of 1.93 A and the nitrogen

atom from His-496 has a bond length of 1.96 A in the other

apical site (Figure 2). The vanadate-binding site resides at the

bottom of a �10 A solvent-accessible channel. The overall neg-

ative charge on the vanadate is compensated by hydrogen bonds

to several positively charged protein side chains (e.g., Lys-353,

Arg-360, and Arg-490).68 Other important residues including

Lys-353, Arg-360, His-404, and Arg-490 form hydrogen bonds

with the nonprotein oxygens of vanadate (Figure 2). This bond

distance for the apical OH ligand is in the range for vanadium

(V)–hydroxyl bond,84 and its hydrogen bonding with the His-

404 residue suggests the fifth ligand is a hydroxyl group. How-

ever, unambiguous determination of the presence and positions

of hydrogen atoms is difficult. The three equatorial oxygen

atoms also form H-bonds with Ser-402 and Gly-403 of the

enzyme. In the peroxo derivative85 of the enzyme, x-ray crystal-

lography at a resolution 2.24 A reveals that the peroxide is

bound to the vanadium center in an Z2 fashion after the release

of the apical oxygen atom. In this form, the vanadium center is

surrounded by four nonprotein oxygen atoms while the e2nitrogen from His-496 occupying the final coordination site of

the vanadium. The metal cofactor characterized by strongly

distorted trigonal bipyramidal geometry with two peroxo oxy-

gens, an oxo oxygen atom, and the nitrogen atom occupies the

basal position while the other oxo oxygen is in the apical posi-

tion (Figure 3).

Extended x-ray absorption fine structure (EXAFS) for the

native and peroxo forms of VCPO from C. inaequalis at pH

6.0 combined with the crystallographic and kinetic data

suggests side-on-bound peroxide involving V–O bonds of

1.67 and 1.88 A. The weakly bound oxygen would there-

fore be ’activated’ for transfer.86 Messerschmidt et al. have

His-496

His-404

Arg-490

N

O

O

O

O V

Asp-292

Lys-353

Ser-402

Gly-403

Arg-360

Figure 2 Structure of native VCPO isolated from C. inaequalis.Reprinted from Plass, W. Angew. chem. Int. Ed. Engl. 1999, 38, 909, withpermission. Copyright 1999 Wiley-VCH.

Page 5: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

Lys 353 N

O

O OV

ON

N

N N

N

Arg 360

1.471 1.8551.601

1.9261.887

His 496Arg 490

2.189

Figure 3 Metal cofactor binding site in the peroxo form of VCPOisolated from the fungus C. inaequalis. Relevant bond distances are in A.Adapted from Zampella, G.; Fantucci, P.; Pecoraro, V. L.; De Gioia, L.J. Am. Chem. Soc. 2005, 127, 953. Copyright 2005 American ChemicalSociety, with permission.

His-404His-418

His-411

His-496His-486

Gly-403

Phe-397

Asp-292

Gly-417

Arg-360Arg-349

Lys-353Lys-341

Ser-402Ser-416

Figure 4 Superposition of VCPO from C. inaequalis (CPK color) andVBPO from A. nodosum (pink) to demonstrate the similar geometriesaround the metal active center. Active site residue labels correspond toVCPO (top) and VBPO (bottom). Adapted from Pooransingh-Margolis,N.; Renirie, R.; Hasan, Z.; Wever, R.; Vega, A. J.; Polenova, T. J. Am.Chem. Soc. 2006, 128, 5190. Copyright 2006 American ChemicalSociety, with permission.

Vanadium Biochemistry 327

determined the crystal structure of the apo form of VCPO,

extracted from C. inaequalis followed by its reaction with

p-nitrophenylphosphate, at a resolution of 1.5 A.87 This is a

real reaction intermediate illustrating the inorganic phosphate

release of a dephosphorylation process. The trapped interme-

diate is the metal active site akin to a ‘metaphosphate anion’

covalently bonded to the nitrogen atom of His-496. The apical

water molecule located within the hydrogen-bonding distance

to the phosphorus atom is in a perfect position for a nucleo-

philic attack on the metaphosphate–histidine intermediate

which would generate inorganic phosphate.88

3.13.3.1.4 Comparing the haloperoxidasesOther than the native state of the enzymes (apoenzymes),69,89

the crystal structures of different VHPOs have also been deter-

mined with several small molecules including azidovanadate,68

peroxovanadate,85 tungstate,90 and inorganic phosphate87 in the

active site of the enzyme. Comparison of the crystal structures of

VBPOs and VCPOs reveals an unexpected homology that the

vanadate-binding residues largely overlap (Figure 4) despite the

fact that these enzymes are distinct from each other.15,91 How-

ever, the primary amino acid sequences are conserved to only

�20% across the various families of VHPOs.69 Moreover, there

are a few residues near the active site that differ between VCPOs

and VBPOs such as Arg-395 in VBPOs is a tryptophan in VCPO,

and His-480 in VBPOs is a phenylalanine in VCPOs, while His-

411 residue in VBPO (from A. nodosum) is substituted by Phe-

397 in VCPO (Figure 4). Messerschmidt et al.74 have reported

a tetrahedral vanadate group at the active site in a His-496A

VCPO mutant, representing that the covalent histidine coordi-

nation is a requirement for the modification of the vanadium

coordination with an elongated and weak bond to the axial oxo

group. This hypothesis agrees with the missing haloperoxidase

activity of the H496A VCPOmutant. His-418 from VBPO forms

a hydrogen bond (2.9 A) at the e2 nitrogen to the carboxyl

oxygen of Asp-278,while in VCPO the catalytic histidine residue,

His-404, is hydrogen bonded to the carbonyl oxygen atoms of

Trp-289 and Ala-290. This dissimilar way of acting of the

catalytically active histidine residue may be responsible for the

observed differences in halide substrate specificity92,93 and pH

dependencies83,94 of both haloperoxidases (VBPOs and VCPOs)

due to different pKa values of the catalytic histidine residues.

Despite all these differences, the conserved vanadium-binding

site demonstrates a common genetic origin for both types of

vanadium-dependent haloperoxidases, VBPOs and VCPOs.

From Raman and UV–vis spectroscopic investigation, Molinari

et al. have demonstrated that a peroxo–oxo-bound umbrella

structure is present in VHPO enzymes and metal–organic com-

pounds designed to mimic VHPOs.95

3.13.3.1.5 Substrate activity by haloperoxidasesHaloperoxidases catalyze peroxidative halogenation reactions

and the halide-assisted disproportionation of hydrogen perox-

ide. If there is a convenient nucleophile, the reaction will occur

with HOX to form a diversity of halogenated reaction products.

The catalytic properties of VHPOs have been extensively studied

showing that vanadium remains in the oxidation state of

5 throughout the catalytic cycle.74 Hence, the oxidation of

the bromide ion cannot involve reduction of the metal ion in

the first step. Figure 5(a) shows a mechanism proposed by

Messerschmidt et al.74 for both the VBPO- and VCPO-catalyzed

halide oxidation reactions. The active center binds peroxide

in a side-on fashion and appears to perform as a Lewis acid

in polarizing the bound-activated peroxide for further nucleo-

philic attack by an incoming halide. Lys-353, which is the only

amino acid interacting directly with the peroxo ligated moiety,

polarizes the peroxo bond, making it more susceptible toward

Page 6: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

H

HH HH

H2O2

OH

OH

Cl OH

-OOH

O

OO

O

O

V

V

V

V

O

O

O

O

O

OO

O

O

N(His404)

N(His404)N(His404)

HOCI

N(His404)

N(His496)

N(His496)

N(His496)(a)

N(His496)

Cl-

Cl--2H2O

H+, H2O

Full mechanism

RX + H+

RH

H

H2O2

H2O

H2O2 + 2 OH-

H+

H+

essential

Assisted by H+

Site of substrate attackH(b)

O2 + X−

+ 2 H2OO

O

OO

O

O

OO

OO

OH-

X−

‘X+’ + L-V

L-V

L-V

L-V

Probably protonated

Figure 5 (a) Proposed reaction mechanism for the VHPO-catalyzed oxidation of chlorides. Reproduced from Ligtenbarg, A. G. J.; Hage, R.;Feringa, B. L. Coord. Chem. Rev. 2003, 237, 89, with permission. Copyright 2003 Elsevier. (b) Alternative reaction mechanism for the catalyzed oxidationof halides accounting for the observed Hþ-dependence. (from Zampella, G.; Fantucci, P.; Pecoraro, V. L.; De Gioia, L. J. Am. Chem. Soc. 2005,127, 953).

328 Vanadium Biochemistry

nucleophilic attack.96–98 Indeed, a mutagenesis experiment

supported this possibility for the K353Amutant.99 The hydrogen

bonding of the peroxo vanadium intermediate is important for

the properties of the different enzyme intermediates and further-

more calls for the involvement of charged residues required for

charge neutralization of this system. The Polenova group has

showed using solid-state 51V NMR spectroscopy that the vana-

dium cofactor in the resting state of VCPO contains one hydroxo

group in the axial and equatorial planes. The combination of

density functional theory (DFT) and solid-state NMRparameters

supports the interpretation that the cofactor is likely anionic

in the resting state of the enzyme and is involved in an extensive

H-bonding network.100

Several lines of evidence have been reported pointing to the

charge of the cofactor and the involvement of the proton in

the conversion of H2O2 to H2O using model compounds and

the enzyme. The Pecoraro group examined a number of simple

functional model systems containing the oxo-peroxovanadium

functionality.101 Of these, the complex [VO(O2)(Hheida)]�

(Scheme 2) where H3heida is (2-hydroxyethyl)iminodiacetic

acid, became the gold standard, and has been most exten-

sively studied mechanistically. Studies with this system and

others documented the role of the hydrophobicity of the envi-

ronment.102Theneed forprotonationof the reaction to takeplace

in model systems requires the mechanism in Figure 5(a)101,102

to be modified to that shown in Figure 5(b).96–98

Page 7: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

OO

OO OHO O

N

O

V

Scheme 2 The oxoperoxovanadium(V) complex formed with theH3heida ligand.

Vanadium Biochemistry 329

Quantum mechanical studies first on the resting state of

the haloperoxidase probed the structure of the vanadium

center.96,103 The reaction with H2O2 as well as formation of

various other intermediates were also investigated.96 These

studies show that the formation of the side-on peroxide is

catalyzed by acids and bases.96,97 Computations on the

model system have continued to explore the reaction interme-

diates and transition states comparing the enzyme with the

oxoperoxovanadium(V) compound of H3heida. This leads to

the demonstration that the protonated model peroxide system

is competent to oxidize both bromide and iodide but not

chloride98 in contrast to VCPO that can oxidize chloride, bro-

mide, and iodide. The origin of this discrepancy is due to the

difference of their redox potentials and in part the increasing

basicity of the peroxide resulting from poorer orbital overlap in

the transition state with the halide favoring deprotonation of

the protonated peroxide.98 X-ray absorption near edge struc-

ture (XANES) spectroscopy has been obtained to support this

hypothesis by demonstrating that the peroxo–oxo intermedi-

ate does not contain a protonated oxo-group under catalytic

conditions.98

So far, a distinct halide binding to the vanadiumhas not been

found for any VHPOs. No crystallographic evidence is available

that includes a coordinated halide.104 Further, no kD’s have been

determined for halide binding to these enzymes. The halide

may not interact strongly with the enzyme until the peroxo inter-

mediate is formed suggesting that addition of halide to crystals in

the peroxo form results in enzyme turnover and cracking of the

crystals. When A. nodosum was treated with bromide in the ab-

sence of H2O2, it was proposed that Br is bound to the Ser-416

residue.105,106 However, Feiter et al. employing a combination of

experimental and modeling studies have demonstrated that the

native A. nodosum contains Br in the surface of Tyr-447 and Tyr-

398 residues.107 They proposed that these brominated Tyr resi-

dues are not likely to be reactive intermediates in the VBPOs

catalytic cycle since they are located at the surface of the protein.

The observed biochemical difference between VBPO and VCPO

in regard to their CPO activity10 is attributed to the second

histidine residue, His-411 in VBPO, which is in close contact

with the peroxovanadate center. Whether the substrate reacts

predominantly with free HOX or with some form of enzyme-

bound V–OX (X¼halide) intermediate is still uncertain.15,91

Some results dictate that these enzymes can catalyze reactions

with regioselectivity or enantioselectivity, demonstrating the in-

volvement of trapped Xþ equivalents15,108,109; whereas others

show no selectivity or mechanistic flexibility.15

Anderson et al. discovered that the VBPO isolated from the

algaCorallina officinalis catalyzes the oxidation of bicyclic sulfides

to the corresponding sulfoxides with 91% enantiomeric

excess.110 Later, Wever et al. reported generation of chiral sulfox-

ides with high enantiomeric excess employing a range of different

substrates, such as methyl phenyl sulfide, methyl p-tolyl sulfide,

1-methoxy-4 (methylthio)benzene, etc.111–113 The recombinant

VCPO from the fungus C. inaequalis catalyzes the oxidation of

2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), a

typical heme peroxidase substrate.114 Wever et al. also proposed

the mechanistic pathway for the oxidation of sulfides by VBPOs

and VCPOs based on the ternary structure of the peroxo-bound

intermediates (Figure 6). Werncke et al. reported the synthesis of

a novel oxovanadium(V) complex. This complex in the presence

of added H2O2 undergoes a sequence of successive peroxide

formation, and intramolecular thioether oxidation events (sulf-

oxide and sulfone) and form a mixture of five products, which

were all identified unambiguously, partly through an indepen-

dent synthesis and characterization.115 The mechanism for the

formation of thioether and sulfide also involves a critical proton-

ation step of theperoxidemoiety as demonstrated experimentally

and theoretically.98

Wischang et al. reported the synthesis of immobilized en-

zyme of VBPO on magnetic micrometer-sized particles with a

half-life time of about 160 days which acts as a reusable catalyst

for bromide oxidation with H2O2.116 They have also noticed

that VBPOs in the presence of H2O2 and NaBr are effective for

electrophilic aromatic bromination of ester-, cyano-, and

carboxamide-substituted 1H-pyrroles.116 The VHPO extracted

from the brown alga L. saccharina is capable of catalyzing the

formation of a black precipitate, when reacted with L-3-(3,4-

dihydroxyphenyl)alanine (DOPA) in the presence of hydrogen

peroxide and iodide. The L-DOPA oxidation is a multistep

reaction with a crucial role played by the iodide in the

enzyme-catalyzed peroxidative production of dopachrome, a

well-known intermediate in the synthesis of melanin.117 The

interaction between phenolic substances (PS) and alginates

(ALG) was probed via size exclusion chromatography (SEC),

and optical tweezers microscopy has been proposed to be

catalyzed by VBPOs.118

Finally, VCPOs and its mutant P395D/L241V/T343A were

investigated by Renirie et al. for their antibacterial and antiviral

action at pH 8.0 and at a low H2O2 concentration and exhib-

ited a broad spectrum of antimicrobial activity.119

3.13.3.1.6 Phosphatase activity by haloperoxidasesWever and coworkers have demonstrated that the amino acid

residues of the active site of VHPOs are conserved within three

families of acid phosphatases.120 Furthermore, they have shown

that apo-CPO can also function as an acid phosphatase.120

Structural analogy between the two classes of enzymes is readily

illustrated from the crystal structure of the active site of rat

prostatic acid phosphatase (Figure 7) complexed with vanadate

which is bound in the same trigonal bipyramidal geometry as in

VCPO (Figure 2).85,121,122 His-496, which is axially coordinated

to the vanadium in apo-CPO, is a part of an extensive hydrogen-

bonding network in the protein.85 In rat prostatic acid phos-

phatase, the vanadate is also covalently bonded to a His-12

while Arg-11, Arg-15, Arg-79, and His-257 are within

hydrogen-bonding distance.120 VCPO from the fungus C. inae-

qualis exhibits both haloperoxidase and phosphatase activities

and is related to glucose-6 phosphatase.123

Page 8: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

Arg-79

Arg-15

Arg-11

Asp-258

His-12

V O N C

His-257

Figure 7 Active site of rat prostatic acid phosphatase. Adapted fromPlass, W. Angew. chem. Int. Ed. Engl. 1999, 38, 909, with permission.Copyright 1999 Wiley-VCH.

O

OO

O

ON

N

V

His

OO

O

ON

N

V

His

SS .+

SR2

R2

R2S

OR2

R1

R1

R1

S

R2e-

δ+

δ-δ-

δ+

R1

R1

Figure 6 Schematic presentation of the critical sulfoxidation exhibited by VBPO (left) and recombinant VCPO (right). Reprinted from ten Brink, H. B.;Schoemaker, H. E.; Wever, R. Eur. J. Biochem. 2001, 268, 132, with permission. Copyright 2001 Elsevier.

330 Vanadium Biochemistry

Peroxidase activity exerted by the phosphatases has been

investigated by several researchers.120,124–126 Incorporation of

vanadate ion, in the presence of H2O2, into the active site of

phytase, which in vivo mediates the hydrolysis of phosphate

esters, resulted peroxidase activity.125 Whether the dual activity

of these enzymes observed in vitro extends the role of these

enzymes in vivo remains to be explored in future. However,

despite the striking similarity in the active site structures of

the VHPOs and the acid phophatase, the turnover numbers

for the phosphatase from Shigella flexneri and Salmonella

enterica ser. Typhimurium are low.126 Clearly the active sites of

acid phosphatases are not optimized for haloperoxidase

activity.126 Moreover, Lys-76 in glucose-6-phosphatase was

predicted to stabilize substrate by hydrogen bonds as Lys-353

in VCPO, but this residue appears to be in a membrane-bound

section of the protein.4 Given the variation in reaction

mechanism even among the VBPO and VCPO enzymes, diff-

erences between VHPOs and a few phosphatases are not

anticipated.4

3.13.3.2 Nitrogenases

3.13.3.2.1 General overviewThe vanadium(V) nitrogenase (VNase) was isolated and puri-

fied for the first time in 1986 by Hales and coworkers127,128 as

an alternative nitrogenase that is expressed under Mo-depleted

environments.19,129–133 VNases have been found in the free-

living soil bacteria of the genus Azotobacter chroococcum and

A. vinelandii. VNases that have the molybdenum sites are

replaced by vanadium and consequently have unique proper-

ties with distinctive differences in the mechanism of nitroge-

nase catalysis.127,128,134 The VNase is less understood than the

MoNase135; however, as recently described, a new form of

VNase has been isolated, and decreasing the heterogeneity of

these sample is assisting in characterizing the active site geom-

etry and the mechanism of this enzyme.136

3.13.3.2.2 Structural perception and amino acid sequenceIn analogy with the Mo analog (MoNase), the VNase consists

of an iron protein (encoded by vnfH) which is a homodimer of

MW 64 kDa and an iron–vanadium protein (encoded by

nifDGK) (Figure 8) of MW 240 kDa.135,137 The VNases isolated

from A. vinelandii and A. chroococcum have extensive DNA

sequence homology with MoNase. Recently, the isolation of a

homogeneous, His-tagged form of VFe protein from A. vinelan-

dii has a different a2b2g4-heterooctameric composition and is

catalytically distinct from the MoFe protein.136

VNases catalyze the conversion of N2 to NH3. Table 1 sum-

marizes the amino acid sequences for the three vnf-coded VFe

proteins in VNases. As in MoNase, the vnfH-encoded Fe pro-

tein component in VNase (Figure 8) contains two a subunits,

which are bridged by a [Fe4S4] cluster, ferredoxin, and also

includes a MgATP-binding site.137–140 The nifDGK encoded

V-Fe protein (Figure 8) has an a2b2d2 octameric structure that

contains two types of inorganic clusters, a P-cluster (Fe8S7) at

Page 9: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

Vanadium Biochemistry 331

the interface of a and b subunits and a two iron–one vanadium

cofactor (FeVco), located in each a subunits.19 Spectroscopic

and size extrusion studies have revealed a close functional

homology by the P and FeMco clusters (M is either V or

Mo).141 The unique structure of the inorganic cluster

(P-cluster) has led researchers to believe that this cluster serves

as an effective mediator to transfer electrons to the catalytic

component, FeMco cofactor, in all three nitrogenases. The

FeMco clusters may adopt different oxidation states for the

catalytic reduction of a wide variety of substrates.130 Unfortu-

nately, till date there is no structural characterization for VNase

except for solution studies; however, the active centers present

in VFe proteins have been investigated by EPR, Mossbauer,

Fe protein

Fe4S4

MgA

TP

MgA

TP

e-

P-cluster

FeVco

VFe protein

β α

δ δ

Figure 8 Schematic representation of the components of VNase.Adapted from Hu, Y. L.; Ribbe, M. W. Acc. Chem. Res. 2010, 43, 475,with permission. Copyright 2010 American Chemical Society.

Table 1 Amino acid sequences flanking His-442 of the a subunitin VFe proteins

Source Sequence Ref.

A. vinelandii Tyr-Val-Asn-Gly-His-Gly-Tyr-His 138A. chroococcum Tyr-Val-Asn-Gly-His-Gly-Tyr-His 139An. variabilis Tyr-Val-Asn-Gly-His-Gly-Tyr-His 140

Ac2v(MgATP)2 + Aclv

Ac2v

Ac2v(MgADP)2

2MgADP

2MgATP

k+1

KDMgADP

KiMgATP

k–1

Ac2v(MgATP)2A

Figure 9 MgATP-induced electron transfer from Fe protein (Ac2V) to VFe pThorneley, R. N. F.; Bergstrom, N. H. J.; Eady, R. R.; Lowe, D. J. Biochem. J.

MCD, XANES, and EXAFS studies. The resonance at g¼5.5

(from EPR) suggests the spin state in the protein is 3/2 and

the available studies suggest that the oxidation state of vana-

dium lies between (II) and (IV).4,19,142,143 However, with the

report of a homogeneous, His-tagged form of VFe protein from

A. vinelandii differences is observed compared to the MoFe

protein suggesting that this form of the protein is slightly

different from the older forms described above.135,136

3.13.3.2.3 Enzyme catalysisMgATP-induced electron-transfer kinetics of VNase has long

been a benchmark for dinitrogen chemists and this process

was studied very systematically by Thorneley et al.144 for

VNase isolated from A. chroococcum by using stopped-flow

spectrophotometry at 23 �C and at pH 7.2 (schematically

represented in Figure 9). The first two steps of the catalytic

cycle of Nase turnover, that is, the binding of MgATP to the Fe

protein and the electron transfer from the Fe-protein to the VFe

protein, result in the formation of a complex similar to those

formed by MoNase isolated from Klebsiella pneumoniae.145 At

high MgATP concentration, first-order kinetics is observed with

a rate constant of kobs¼46 s�1. MgATP binds at the interface

between VFe and Fe proteins with the apparent binding con-

stant kD¼230�10 mM. The dependence on MgATP concentra-

tion and its competitive inhibition by MgADP were very

similar to those of MoNase isolated from K. pneumoniae.145

Because of these and other similarities between the VNase with

MoNase, it is not surprising that VNase will catalytically resem-

ble MoNase and will be a proficient catalyst for a range of

substrate turnovers.

3.13.3.2.4 Dinitrogen reductionThe biological function of VNase is the production of ammo-

nia from dinitrogen, which sequesters dinitrogen gas in a

biologically accessible form. Like MoNases, the alternative ni-

trogenase, VNase, uses MgATP.19 Both the electrons and the

ATP in this system are provided through photosynthesis. The

absence of dioxygen and a low-potential reductant, usually

dithionite (S2O42�), are the other two requirements for this

enzyme complex for enzyme activity. Although in general,

Nase activity toward N2 is depicted by eqn [2], for VNase the

distribution of e�, proton, and MgATP and the overall compo-

sition of the products of the reduction reaction is somewhat

modified and is represented by eqn [3].19 More H2 is generated

for each NH3 produced and, therefore, more ATP is required

for N2 reduction by the VNase as compared to the MoNase

k+2clv Ac2v (MgADP + Pi)2 Aclvred.ox.

rotein (Aclv) and inhibition of this reaction by MgADP. Adapted from1989, 257, 789, with permission. Copyright 1989 Portland Press Limited.

Page 10: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

332 Vanadium Biochemistry

(eqn [2]).146 The rate-determining step for this reduction reac-

tion is the dissociation of the MgADP–Fe protein bond in the

transition state.19 Equations [2] and [3] demonstrate that

Nases also hold an inherent hydrogenase (Hase) activity that

results in a mandatory production of hydrogen gas.

N � Nþ 12Hþ þ 40MgATPþ 12e�

! 40MgADPþ 40Piþ 3H2 þ 2NH3 [3]

VNase, unlike MoNase, has the propensity to form low but

significant amount of hydrazine (N2H4) following eqn [4] (up

to 0.5% of the reduction equivalent resulting in the dinitrogen

reduction) during its catalytic dinitrogen fixation.147 Figure 10

summarizes the catalytic conversion of N2 to NH3 along with

the minor generation of N2H4 as a side product. When the

VFe protein was combined with purified Fe protein of either

VNase or MoNase, N2H4 was detected while combination of

MoFe protein with two Fe proteins does not form N2H4.148

N � Nþ 4Hþ þ 4e� ! N2H4 [4]

3.13.3.2.5 Carbon monoxide reductionVNase reduces the substrate N2 but can also reduce carbon

monoxide (CO) to hydrocarbons with the concomitant reduc-

tion of the proton to H2 gas149 as shown in eqn [5]. The

formation of hydrocarbons by VNase was inhibited by the

addition of increasing amounts of H2, which implies that H2

is an unlikely hydrogen source for this reaction. Considering

the isoelectronic properties of N2 and CO and the similarities

between the reactions shown in eqns [4] and [5]

some similarities for both N2 and CO reduction reactions

will take place with both substrates. More distinct differences

have recently been observed with varying enzyme composi-

tion, and it is possible that some of the early studies may

need to be revisited with this new purer form of the

VNase.135,136 The intramolecular hydrogenation mechanism

earlier proposed for N2 reduction (including H-atom tunnel-

ing) can also effect reduction of CO. The possible steps for the

overall reaction are: (1) CO binding, (2) CO to HCO conver-

sion, early hydrogenation, (3) persisting hydrogenations of

CO at both C and O to form HCOH and H2COH, (4) O

eliminations of H2O, and (5) the formation of a C–C bond.

Intermediate organic fragments can migrate around the FeVCO

N2

NH3

M

M-NH2

N2H4

NH3

M=N-NH2M-NH-NH2

M=NH

M-N N M-N NH

(A)(B)

M=N-NH3-

Figure 10 Cycle for the stepwise protonation of coordinated N2 onVNase to give NH3 or N2H4. Reproduced from Dilworth, M. J.; Eady, R. R.Biochem. J. 1991, 277, 465, with permission. Copyright 1991 PortlandPress Limited.

active site, and hydrogen bonding between COH functions and

S or SH components of FeVCO protein can occur

and contribute to the stabilization and orientation of the

intermediates.150 The ability of VNase to catalyze both CO

and N2 reductions shows that similar reactions can occur in

the carbon cycle as it takes place for nitrogen cycles. Indeed, in

the reactions using CO as a substrate the difference between

the MoFe and VFe enzymes is underlined,136,151 particularly

with regard to the higher product alkanes that have been

reported recently.151

COþHþ þ ATP ! C2H6 þ C2H4 þ C3H8 þ C3H6

þ ADPþH2 [5]

3.13.3.3 Vanabins

Several vanadium-containing proteins from the cytoplasm

of the vanadium-containing blood cells referred to as vana-

docytes from A. sydneiensis samea have been identified by

Michibata et al.22–26 When first discovered, these proteins

were known as vanadium-associated proteins (VAPs).25 Of

these proteins, vanabin1 and vanabin2 with apparent molec-

ular weights 12.5 and 15 kDa, respectively, are rich in lysine

and cysteine residues. Michibata et al. have determined the

affinities of vanabin1 and vanabin2 for vanadium(IV). They

have shown that the two vanabins bind multiples of 10 and 20

vanadium atoms per protein with dissociation constants of

2.1�10�5 and 2.3�10�5 M, respectively, indicating differ-

ences in amino acid sequences in these proteins.25,152

Vanabin1 and vanabin2, which are respectively composed

of 87 and 91 amino acids, each contain 18 cysteines and 12

and 14 lysines, respectively.23 Only Cu(II) reduces vanadium

binding while other metals, for example, Mg(II) or Mo, do not.

Two other vanabin-like-proteins, known as vanabin3 and

vanabin4, have primary structures which closely resemble

those of vanabin1 and vanabin2.152 Recently, Michibata et al.

have isolated and analyzed the fifth type of vanadium-binding

protein known as vanabinP (vanabin in plasma) available in

blood plasma of the ascidian A. sydneiensis samea.152 Based on

EPR spectroscopic titrations and ESEEM studies (Figure 11), it

has been shown that vanabin2 can bind up to 23.9 vanadium

ions per protein where the vanadium ions are in mononuclear

state and coordinated to amine nitrogen.153 Regardless of the

high number of cysteines in these proteins, no evidence was

found for thiolate coordination to the vanadium. Michibata

suggested that the roles of these vanabin proteins are to trans-

port the vanadium to the vacuole.153

3.13.4 Organisms Accumulating Vanadium

3.13.4.1 Tunicates

3.13.4.1.1 General overviewTunicates, also referred to as sea squirts or ascidians, so named

because they release jets of water through their siphons when

disturbed are a class of common marine invertebrate organ-

isms that selectively accumulate metal ions such as V, Fe, Mo,

and Nb from seawater.27,154 Tunicates get their name from

their tough, often translucent, tunic-like body. It was Martin

Henze154 who first discovered that the Mediterranean ascidian

Page 11: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

260 300 340

B (mT)

380

1:20

3.9

00

3τ (μs)2.8 7.1

13.7= ν(1H)

[Vanabin] : [V]

420

0

0Four

ier

amp

litud

e (A

.U.)

5 10 15Frequency (MHz)

Figure 11 EPR (left) and ESEEM (right) and its time domain data (right, inset) spectra of VO2þ and vanabin2. Reproduced from Fukui, K. I.; Ueki, T.;Ohya, H.; Michibata, H. J. Am. Chem. Soc. 2003, 125, 6352, with permission. Copyright 2003 American Chemical Society.

OHOH

OH

OHOH

OH

O

NHHO

NH

HO

HO

HO

H2N

Figure 12 Structure of tunichrome. Redrawn from Bruening, R. C.;Oltz, E. M.; Furukawa, J.; Nakanishi, K.; Kustin, K. J. Am. Chem. Soc.1985, 107, 5298, with permission. Copyright 1985 American ChemicalSociety.

Vanadium Biochemistry 333

Phallusia mammillata, phylogenically belonging to the

Chordata, accumulated high levels of vanadium in its blood

cells. The black tunicate, Ascidia nigra, sequesters vanadium

in its various cells as pentavalent monomeric vanadate and

concentrates it by �106-fold after reduction of the vanadium.27

It was early on suggested that low-molecular- weight pigments

known as tunichromes (Figure 12) serve as complexing agents

for the accumulation and storage of the intracellular

vanadium.27,155,156 The tunichromes constitute a series of com-

pounds isolated from the blood cells of ascidians that form the

major class of the subphylum Urochordata (Tunicata).6 The

distribution of vanadium and tunichrome among different cell

types has been studied by electron microscopy,157–159 cell

sorting,160,161 and by 2,2-bipyridine staining for vanadium

(III).159 In general, the signet ring and compartment cells contain

the bulk of the vanadiumwhile themorula cells are the source of

the tunichromes.160 Therefore, the possibility that the tuni-

chrome is bound to the vanadium and stored as chelates in the

tunicates is no longer supported by experimental results.

Ascidians provide the oldest known example of vanadium

sequestration in nature, and despite one century of research

since Henze’s discovery, a function for the metal remains

elusive. Considerable efforts have been made162 to understand

in what form, and why, vanadium is stored in these sessile

marine organisms. Researchers know that the sequestered

vanadium(V) is actually stored in its reduced forms either

as vanadium(IV) or as vanadium(III) at physiological pH

(�7.2), possibly in the hexaaqua form or as some other

complex species in specialized blood cells referred to as

vanadocytes.22,27,163–166 The vanadium concentration in the

vanadocytes is 0.15 M; however, in some vanadium-containing

vacuoles in the vanadocytes, the vanadophores, it can reach up

to 1 M.6 These organisms are the only organisms in which the

formation and existence of V(III) in biological systems have

been reported. Since it is not trivial to form vanadium(III)

under physiological conditions, even under reducing condi-

tions, the presence of a strong reductant that also serves as a

complexing agent would maintain vanadium in such a low

oxidation state.27 The generally accepted mechanism for this

sequestration process involves a reductive chelation reaction.27

For this, vanadocytes use a yellow green and fluorescent organic

chromogen named tunichrome (Figure 12), a powerful physi-

ological reducing agent which reduces V(V) to V(IV) or V(III) in

vitro and presumany facilitate the reduction before the vana-

dium reaches its final location in the vanadocytes.155,156

3.13.4.1.2 Oxidation state of vanadium in tunicatesVanadium is a versatile element that can exist in oxidation

states ranging from þ5 to �3 but þ5, þ4, and þ3 are the

oxidation states that exist at physiological pH.36,167,168 The

first suggestion by Henze154 that the vanadium existed in þ5

oxidation state in these organisms was rejected. Studies using

noninvasive physical methods, including spectroscopic (e.g.,

ESR, XAS, and NMR)169–173 and magnetic (SQUID)166,174 of

isolated extracts166,174 and whole cells,169–173 indicated that

the vanadium ions in ascidian blood cells were predominantly

in the þ3 and þ4 oxidation states.

V(III) is usually unstable toward air and moisture based on

its standard reduction potential value of 0.337 V (vs. NHE) in

strongly acidic solutions and are easily hydrolyzed to V(OH)2þ

above pH 2.5.27 V(III) species are generally not stable in neu-

tral and alkaline solutions and are maintained in aqueous

solution only in an acidic pH range under reducing conditions

or in the presence of strong complexing agents. Tunichromes,

the bright yellow blood pigments from A. nigra and other

various tunicates, can complex vanadium(III) and readily

maintain the vanadium in the low oxidation state at neutral

pH.175,176 However, considering that the tunachrome and va-

nadium are found in different cells,157–159 complexation may

facilitate reduction of the vanadium(III) but does not serve to

store the vanadium. Alternatively, vanadium ions are stable

under acidic conditions within the blood cells of ascidians.

The possibility whether vanadium existed in two oxidation

Page 12: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

334 Vanadium Biochemistry

states (þ3 and þ4) in one type of blood cell, or is each state

formed in a different cell type was investigated by Michibata

et al.177,178 when they conducted noninvasive EPR measure-

ments of blood cells of A. gemmata. Their study showed that

the oxidation state of vanadium in these vanadocytes is pre-

dominantly þ3, with some vanadium being in the þ4 oxida-

tion state. The ratio of V(III) to V(IV) was 97.6:2.4.177

3.13.4.1.3 TunichromesTunichromes were discovered and characterized in the quest

for compounds that are involved in vanadium accumulation

and reduction in ascidian blood cells.156,160 Evidence would

also be formed via blood cytolysis.156,160 In the crude state,

tunichromes are extremely labile and consist of a complex

mixture of closely related compounds, which is why the com-

pounds eluded isolation attempts.22–27 Tunichromes contain

TOPA (3,4,5-trihydroxyphenylalanine) residues, and reducing

amino acids with strong metal chelating prospective through

their catechol and pyrogallol moieties, respectively.27,155,156

The first reported tunichrome, An-1, was isolated from the

phlebobranch ascidian A. nigra.155 It contains a modified tripe-

ptide of three TOPA units (Figure 12) while the structures of

An-2 and An-3 were determined, differing only in the degree of

hydroxylation.156 Once isolated in the pure form, the tuni-

chromes become more stable. Three more tunichromes desig-

nated as Pm-1, Pm-2, and Pm-3 were identified from the blood

cells of P. mammillata.27 The presence of the pyrogallol moiety

in tunichromes provides the reduction potential in these com-

pounds making it kinetically competent to reduce V(V) to

V(IV) or V(III). For some time it was believed to resolve the

long-standing controversy regarding the mechanism of V(V) to

V(II1) reduction in ascidians. From the structures of the tuni-

chromes it is clear that their biosynthesis occurs through cou-

pling of three amino acids, enzymatic dehydrogenation, and

sequential hydroxylation. The major observations supporting

the existence of tunichromes in intact blood cells is the yellow

color of tunichromes, and this is also the case for the morula

35 nM V(V)in seawater

Metal transporter

V

V

V

NAD

V-ATPase

Branchial sac

Vanabin

Vanadocyte

Figure 13 Schematic presentation of vanadium accumulation and reductionUeki, T. Coord. Chem. Rev. 2003, 237, 41, with permission. Copyright 2003 E

cells, which can be observed by light and fluorescence

microscopy.28 Recently, the location of the tunichromes and

vanadium has been found to deviate, which suggests that

although tunichromes may be involved in bioprocessing of

the vanadium, tunichromes do not serve to store the vanadium

by complexation.22

3.13.4.1.4 Vanadium accumulation and reductionin tunicatesAscidians accumulate vanadium to levels more than 10million

times higher than that in seawater and have attracted the

curiosity of researchers for decades. Two suggestions have re-

ceived most consideration: vanadium(III) is stabilized in a

strongly acidic intracellular milieu,163,169,179–181 or vanadium

(III) is stabilized by chelation to an endogenous ligand.182,183

These suggestions have been evaluated considering cellular

energy requirements, suitable ligands, and the local pH.

The million-fold concentration of vanadium from seawater

constitutes a sizable metabolic energy allocation. The uptake

likely follows one of the two routes: (1) distribution of vana-

dium(V) between the plasma and blood cells governed by

direct uptake from the plasma into the blood cells, or alterna-

tively, (2) via vanadium influx into a tissue and a slow uptake

into the blood cells.29 The second pathway results in either loss

of vanadium from remote sites into the plasma along with

incorporation into the blood cells or alternatively production

and development of blood cells. Neutron activation analysis

by Michibata et al. showed that the vanadium accumulation

begins abruptly 2 weeks after fertilization, and continues so

that after 2 months, the amount of vanadium in larvae is

600 000 times higher than that in the unfertilized egg.184

The accumulated vanadium is mainly stored in vacuoles.

The vanadium-binding proteins, vanabins, facilitate the trans-

port and accumulation of the vanadium into these vacuoles,

as shown in Figure 13. 51V NMR shows that vanadium(V)

persists in the blood plasma before the accumulation takes

place. Because vanabins bind vanadium(IV) stronger than

350 mM V(III)

Metal transporter

III

PH

H+

IV

IV

H+

Cytoplasm

Vacuole

500 mM SO42-

SO42-

SO42-

pH 1.9

in ascidians. Adapted from Michibata, H.; Yamaguchi, N.; Uyama, T.;lsevier.

Page 13: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

Vanadium Biochemistry 335

vanadium(V), it is likely that the reduction of the vanadium

happens before the vanabins bind the vanadium. Hence the

process of accumulation of the vanadium into the vacuoles

facilitates the reduction of V(V) to V(IV) and V(III). It has

also been suggested that this reduction involves NADPH.4,22

3.13.4.2 Amavadine

3.13.4.2.1 General overviewAmavadine is the only known vanadium-containing natural

product and consist of a 1:2 complex of a vanadium(IV) with

two ligands l,l-N-hydroxyimino-a,a0-dipropionic acid

(H3hidpa) (1 and 2, Scheme 3).185–191 It is isolated from a

mushroom of the genus A. muscaria and it does not resemble

the vanadium in the tunicates (also referred to as hemovana-

din) with respect to valence state or ligand.192 Identification of

the ligand (H3hidpa) associated with amavadine was done

using a combination of spectroscopy and synthesis. In 1986,

Bayer et al. reported a non stereoselective synthesis of the

ligand (H3hidpa), including a nonroutine separation proce-

dure of the ligand diastereomers, followed by insertion of the

vanadium to generate amavadine.193 The presence of the

hydroxylamido and carboxylate groups makes this microbial

ligand, H3hidpa, a very efficient chelating agent for a range of

metal ions of which vanadium ion has the highest affinity

(log K2¼23).189 Other metal complexes with H3hidpa (e.g.,

Mo, U, Nb, Ta, Ti, Zr, etc.) show chemical and structural

properties similar to amavadine.194–197 This natural product

has a unique structural feature, chemical environment, and

properties, and has generated interest to further examine this

class of compounds, and their chemistry.

3.13.4.2.2 Structure and reactivityThe fly agaric toadstool, A. muscaria, is remarkably rich in

vanadium with up to 400 mg kg�1 (dry weight) of

OO

OHOH

OH

N

N

O

OH

O

H

O

O

OO

N

N

V

O

V

OO

O

N

NO

O

O

HO

1 2

4

Scheme 3 The structures for the ligand (1), the vanadium(IV) complex (2),t(IV) model compound (5) and the ligand diacetyl hydroxylamine (6).

vanadium,198 mainly in the bulb of the mushroom. In 1972,

Bayer and Kneifel185 first isolated a blue, and very stable

vanadium-containing compound from A. muscaria and

named it amavadine (structure 2, Scheme 3).185 Since then,

several reports describe the structural investigations of the

natural product amavadine,35,187,193,199 its role in

biology,35,187,193,199–202 investigations into the properties of

the ligand H3hidpa,195–197 and identification of new species

and its complexes with other metal ions.195–197,203–205

The initially proposed molecular composition of amava-

dine was [VO(H2hidpa)2] (3, Scheme 3) where an oxovana-

dium(IV) center contains two molecules of N-hydroxy-2,20-iminodipropionic acid (H3hidpa) as a bidentate (O,N) donor

ligand, in monoprotonated form with a square pyramidal

coordination geometry surrounding the metal active center.185

In 1986, Bayer et al. reported the total synthesis of amavadine

leading to the structural revision and characterization of the

eight-coordinate, non-oxo complex currently known as ama-

vadine (2, Scheme 3).35,193 Comparing formation constants

between iminodipropionic acid (4, Scheme 3) and H3hidpa

with vanadium(IV) demonstrate the necessity of expanding the

coordination sphere and involving the N–OH group in coor-

dination to the vanadium.4,200 An analog of amavadine (5,

Scheme 3) based on the ligand N-hydroxyiminodiacetate

(H3hida) (6, Scheme 3) did not contain any IR band at

980 cm�1. This clue led to the hypothesis that no V¼O moiety

was present in the natural product amavadine.4 The bond

lengths for the V¼O bond lie typically between 1.57–

1.67 A.54,84 The lack of a short V¼O bond in the EXAFS spec-

trum of amavadine indicated the absence of an oxo group at

the vanadium center.193

The non-oxo V4þ containing natural product has a novel

octacoordinated geometry with the highly distorted cubic

geometry containing two hidpa3� ligands, each bonded via

a Z2-N,O group, and two unidentate carboxylate donors

N

N

N

V

O

OH

OH

HO

HO

O

O

OH HO

O

O

O

O

OO

O

O

O

O

2-

O

O

OO

O

O

H2-

3

5 6

he original proposed structure (3), diisopropanyl amine (4), the vanadium

Page 14: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

336 Vanadium Biochemistry

(1, Scheme 3).187 Prior to this report, Carrondo et al. decribed

the crystal structure for the amavadine analog [V(IV)(hida)2] in

which the alanine amino acid is replaced by a glycine.199

Garner et al.206 reported the crystal structure of amavadine

(Figure 14) isolated from A. muscaria on samples of natural

amavadine crystallized as phosphoric acid derivative or as its

Ca2þ salt. We now recognize that amavadine contains five

chiral centers both in the neutral and in the anionic forms:

four chiral carbons all having ‘S’ configuration associated with

the amino acid ligand and the fifth chiral center at the vana-

dium generated by the chelation of the multidentate ligands

about the vanadium. The racemic form contained equal

amounts of the Δ and the L helical forms of amavadine.187

Upon oxidation, the coordination geometry of the vanadium

center in amavadin and its analog does not change (Figure 15).

The oxidized amavadin complex and its analog thus offer a

unique opportunity to study the eight-coordinated vanadium

geometry as well as fundamental reactivity of the com-

pound.207–209 The x-ray structure of meso-amavadin revealed

O(8

O(

O(5)

C(6

C(5)

O(4)

O(14)

O(15) O(9)-

O(11)

O(13)bO(13)aO(12)

Ca(1)

C

Figure 14 Crystal structure of [Ca(H2O)5][Δ-V((S,S)-hidpa)2]2H2O. ReprodErtok, S. N.; Helliwell, M.; Garner, C. D. Angew. Chem. Int. Ed. 1999, 38, 795

Figure 15 Molecular structures of [PPh4][V(V)(hidpa)2]�H2O (left) and [PPh4atoms, white; carbon atoms, gray; nitrogen atoms, blue; oxygen atoms, red;Baruah, B.; Choudhary, M. A.; Crans, D. C.; Polenova, T. Dalton Trans. 2009,

that the negatively charged anionic species is joined into infi-

nite hydrogen-bonded chains, counterbalanced by cationic

hydronium species.210

The biological function of amavadine is still not known.

Amavadine is a weaker inhibitor for the sarcoplasmic reticu-

lum, Ca-ATPase,211 and a weaker effector of glucose uptake212

than compared to vanadium compounds that readily lose their

ligand.213 In addition, it was not found to show any anti-

diabetic properties and failed to lower elevated blood–glucose

levels in STZ-induced diabetic rats. Studies have shown that

amavadine can act in peroxidative halogenation, hydroxyl-

ation, and oxygenation of alkyl and aromatic substrates in

the presence of H2O2.4,214–216 Amavadine catalyzes the electro-

catalytic oxidation of thiols217 such as cysteine, glutathione,

mercaptoethanol, and other thiols in acidic solution.202 Sur-

prisingly, little work has been done including a stereochemical

analysis of amavadine during its catalytic action. Amavadine’s

hydrolytic stability is accompanied by its reversible one-

electron transfer reactions that has been linked to functions

C(9)

C(8)

N(2)

C(10)

C(12)

C(2)

C(3)

C(11)

O(9)

O(10)

O(1)

)

O(7)

6)

V(1)

O(3))C(1)

O(2)C(4)

N(1)

(7)

uced from Berry, R. E.; Armstrong, E. M.; Beddoes, R. L.; Collison, D.;, with permission. Copyright 1999 Wiley-VCH.

][V(V)(hida)2]�CH2Cl2 (right). Hydrogen atoms are color coded: hydrogenand vanadium atoms, green. Adapted from Ooms, K. J.; Bolte, S. E.;3262. Copyright 2009 Royal Society of Chemistry, with permission.

Page 15: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

Vanadium Biochemistry 337

such as iron–sulfur proteins, cytochromes, and blue copper pro-

teins known to retain their structure during a one-electron redox

change.35,186,187 Both amavadine and its analog, [V(IV)(hida)2],

undergo a reversible one-electron redox process that is very sen-

sitive to solvent variation.201,209 The solvent sensitivity is attrib-

uted to the solvent interactions with the carboxylate groups of the

complex. The electron transfer reaction of amavadine and its self-

exchange rate constants have been determined.208,209

Information about the electronic properties of the vana-

dium center is important for understanding amavadine’s

chemical properties and its function. Oxidation using different

metrical parameters show smaller change indicating outer-

sphere electron transfer reaction in the V(IV)–V(V) redox cou-

ple of amavadine.217 DFT computations and quantum theory

of atoms inmolecules (QTAIM) analysis of the electron density

differentiate the stereoelectronic features of the V(IV) (inactive)

and V(V) (active) states of amavadine. DFT calculations di-

vulge that protonation of V(V) amavadine at a carboxylate

oxygen is not involved in the vanadium coordination, instead

favoring methyl mercaptoacetate (MMA) binding into the first-

coordination sphere of vanadium, by substitution of the ama-

vadine carboxylate oxygen. The kinetic and thermodynamic

feasibility of the V(V)-MMA intermediates formation is consis-

tent with the biological role as a redox mediator, which is

supported by electrochemical data.217

Reports have also appeared on the efficacy of amavadine in

converting methane into acetic acid in the presence of a perox-

odisulfate salt and trifluoroacetic acid (TFA).215,218 The catalyst

can remain active upon multiple recycling of its solution; how-

ever, the yields for this reaction are very low. Carboxylation

proceeds through free radical mechanisms. The one-pot carbox-

ylation of ethane to propionic and acetic acids with the former as

the main product has recently been reported from the laborato-

ries of Pombeiro and coworkers.216 Two mechanistic pathways

have been proposed for the formation of acetic acid, the leading

one via oxidation of ethane with conservation of the C–C bond,

and the other through breaking of this bond and carbonylation

of the methyl group by CO. The C–C bond is maintained in the

formation of propionic acid upon carbonylation of ethane. The

reactions proceed via both C- and O-centered radical formation.

The oxidized amavadin and its analog offer a unique

opportunity to study the electronic properties of an eight-

coordinate vanadium geometry by comparing the effect such

coordination has on the electric field gradient (EFG) and chem-

ical shift anisotropy (CSA) on the solid-state 51V NMR spectra.198

Crans and Polenova have reported large downfield chemical shift

of the 51V resonance and they attribute this effect to the unusual

coordination geometry. Buhl et al. have reported DFT calculation

results for the oxidized form of amavadine to account for its

observed 51V NMR chemical shift.219 DFT methods have also

been employed to calculate the electronic g tensors and hyperfine

coupling tensors for amavadine.220 We attribute the continued

interest in amavadine to the unique structural and electronic

properties of this complex.

3.13.4.3 Fan Worm

Pseudopotamilla occelata, fan worm of the polychaeta class

reported in 1993,221 was found to contain high levels of vana-

dium. The vanadium concentration is the highest in the

bipinnate radiole.222 Most of the vanadium is believed to be

in oxidation state (III) and a high level of sulfate is found in the

vanadium-containing cells.223,224 The role of vanadium in

these animals is to regulate the oxidation–reduction reactions

at the surface of the radiole, in the absorption of O2, and in

detoxification mechanisms. The vanadium-storage environ-

ment and form appear to be the same in this fan worm and

in ascidians.223,224

3.13.5 Insulin Enhancing Effect of VanadiumCompounds

In the sections prior to this, we have described in detail each

enzyme for which vanadium is a cofactor. These sections con-

cerned studies of systems in which the role of the vanadium

was determined by how it interacts with a protein. However,

vanadium is known to exert effects other than those that can be

traced back to an enzyme. This is undoubtedly due to vana-

dium’s ability to act as a phosphate analog when in oxidation

stateþ5, and as a divalent cation when in oxidiation stateþ4.4

Indeed, vanadium compounds have been reported to have

anti-diabetic, anti-cancer, and anti-cardiovasular effects.4 Al-

though description of these effects are less straightforward,

because there are several targets and the action of the com-

pounds is less defined, undoubtedly the mode of action of

these compounds will at some point involve interactions

with proteins and metabolites that can be understood and

used for further development. In this section, we describe one

such example, namely when vanadium compounds or salt

exhibit insulin-enhancing effects.4,47,225

The early discoveries by Shechter in cell studies226–229 fol-

lowed by the groups lead by Sakurai230–234 and McNeill235–243

demonstrated that vanadium found in simple salts had a ben-

eficial effect in animal systems. These studies launched an

effort to not only investigate how vanadium exerted its

insulin-enhancing action, but also the potential development

of this system into a drug. The latter studies were fueled by

the efforts of the Posner group244–253 which demonstrated

that vanadium as a peroxovanadium derivative was tenfold

more active than the simple salt, and as such had potential

as a drug. These studies formed the basis on which many

subsequent studies were based in our group and many

others,4,47,212,225,254–259 and have resulted in several studies

with both salts and a coordination complex in human

beings.242,243,260–264 Most well-known is the development of

a vanadium-containing coordination compound, bismaltolato

oxovanadium(IV), BMOV, subjected to phase I and II clinical

trials.237,242,243,265–268 Unfortunately, this endeavor has re-

cently been abandoned mainly because this compound will

no longer be protected by patent in 2015. As a result, this area

now needs new compounds with improved efficacy.242,243

During the early studies vanadium was believed to be able

to replace insulin; however, more recent reports have demon-

strated that the vanadium acts by enhancing the effect of

insulin, and thus less effective in systems in which no insulin

is present. For several decades, the focus was on investigation of

vanadium(IV) coordination complexes.230,231,233,235–243,269–275

However, more recent studies have demonstrated that several

vanadium complexes in oxidation states (V) and (III) also can be

Page 16: Comprehensive Inorganic Chemistry II || Vanadium Biochemistry

338 Vanadium Biochemistry

effective as insulin-enhancing drugs.240,258,276 Regardless of the

oxidation state, the active vanadium compounds are potent in-

hibitors for protein tyrosine phosphatases, and since most phos-

phatases are inhibited by vanadium compounds, the selectivity

exhibited are based on thedegree of inhibition. That is, inhibition

of the protein tyrosine phosphatase, 1B (PTP1B), is very potent,

whereas the inhibition of other phosphatases such as serine

and threonine phosphatases is much less.246,259,277–281 Studies

have demonstrated that these studies are nontrivial because

often the vanadium compounds are not compatible with the

assay conditions used for such studies and complicating

the importance of these studies, and the results obtained will

not show a pattern.278,279

Recent studies in this area include exploring the distribu-

tion of vanadium compounds. Although vanadium first binds

to transferrin, some binding to other proteins in the blood

stream and formation of ternary complexes have been ob-

served under a range of conditions.282–289 These studies com-

bined with biochemical investigations into the cellular

responses290–292 are suggestive of a mechanism involving

some of the proteins associated with the signaling in the

cell293 which may involve action at the membranes.47,294–295

Regardless of the mode of action of these compounds, it ap-

pears that the ligand dissociated from the metal ion, and as a

result small differences between complexes efficacy has been

reported. Undoubtedly, if the mechanism for the beneficial

effects of these vanadium compounds can be elucidated,

there is a potential of using such information to develop new

and effective drugs against diabetes.

3.13.6 Conclusion

Vanadium is abundant in the biosphere. Nature has chosen vana-

diumas cofactor, for a range of enzymes that contain vanadium in

oxidation states (III) through (V) in their active sites, highlighting

the different roles for vanadium in biology. As a result a number

of reviews exist in the literature on different aspects of functional

and/or structural description of vanadium-containing biomole-

cules. This chapter focuses on the protein-based aspects of bioi-

norganic vanadium chemistry describing the coordination

chemistry of different classes of proteins that contain vanadium.

Markeddiversity inbiochemical activityof vanadium is associated

with its oxidation state. In order to illustrate how the chemistry of

vanadium contributes to life process in the biosphere, vanadium

interactions in the biological systems are detailed.

Beginning from the discovery of the first vanadium-

containing enzymes, VHPOs, in 1984 to the current under-

standing of the details in the five-coordinate vanadium com-

plex with the side-on peroxo group has uncovered the the

importance of protonation for the catalytic process. The com-

bination of structural characterization withmechanistic studies

together provides a more complete description of the systems

allowing a comparison between the VBPO and VCPO, and

furthermore allows a comparison of relationship between per-

oxidases and phosphatases. Two classes of enzymes that once

were so distinct have now been demonstrated to have signifi-

cant sequence, structural, and functional analogy.

Microorganisms use vanadium as an essential trace element

for their enzymatic processes such as two-electron oxidation of

halide to HOX and nitrogen fixation via biological conversion

of atmospheric dinitrogen to bioavailable ammonia. The

vanadium-containing nitrogenase, VNase, is an anaerobic en-

zyme that forms under condition when molybdenum is defi-

cient. The vanadium will then replace the position of the Mo in

the cofactor and as such the protein is competent to do many

of the reactions the MoFe enzyme carries out. There have been

some model studies in this area generally involving the low

oxidation states of vanadium.

Metal accumulation by living organisms is an important

aspect of bioinorganic chemistry, but the mechanisms for

these processes remain underexplored in vanadium biochem-

istry. Despite the fascination with tunicates and their function

of accumulation of vanadium from seawater, the mechanism

for concentration is poorly understood, perhaps in part be-

cause information on oxidation state(s), location of the vana-

dium, and the molecular biology was the initial focus of these

studies. In the coming years the role and action of the vanabins

will be further elucidated and the transport of vanadium from

the seawater to the vacuoles will be further elucidated. Further-

more, information on how the concentration of the metal ion

is directed so that it can carry out one or more of its biochem-

ical functions will be characterized.

The presence of vanadium in several accumulators such as

the mushroom A. muscaria, marine invertebrate tunicates, and

the fan worn P. occelata has also drawn significant interest in

vanadium biochemistry. Much less is known about these sys-

tems, and the majority of the work carried out thus far focuses

on identification of what form the vanadium is in. In the case of

the mushroom, the vanadium is placed in a dodecahedral coor-

dination environment with two tetra-coordinated ligands and is

a non-oxo complex. This compound has reversible redox chem-

istry in aqueous solution, and as such serves as an ideal tool to

investigate fundamental aspects of redox chemistry. The natural

product is believed to be involved as redox mediator.

The insulin-enhancing mechanism of vanadium com-

pounds is a complex topic, which continues to evolve. The

compounds that have been front-runners for the past decade

have not only shown promise in animal studies but some of

them have also been investigated in human studies. Indeed,

the ability of vanadium compounds to lower elevated blood–

glucose levels but not go significantly below normal glucose

levels is a very desirable effect of an anti-diabetic drug. Increas-

ing information on the mode of action of these compounds

continues to be investigated yielding clues to the unique prop-

erties of vanadium compounds and provides inspiration for

drug development in diabetes.

Acknowledgments

We thank National Science Foundation under Grant 0628260

(CRC) for funding this work. We thank Dr. P. S. Pharazyn for

editing this chapter.

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