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Bioinorganic chemistryw

Ramon VilarDOI: 10.1039/b818285j

This chapter reviews the literature reported during 2008 in the field of

bioinorganic chemistry. The chapter focuses on metalloproteins that

contain d-block metals on their active site and highlights some relevant

bio-mimetic models. The interactions of d-block metals with peptides and

biological chelators are also presented.

1. Highlights

The cobalt trafficking chaperone responsible for the reductive decyanation reaction

of vitamin B12 has been identified.8 An X-ray crystal structure of a copper chaperone

protein has revealed an unusual p-interaction between the metal cation and a

tryptophan residue.30 A new study has shown that changes in pH lead to trans-

formations between blue T1 copper, red T2 copper and the native purple copper CuAwithin the same protein (namely nitrous oxide reductase).44 The X-ray crystal

structures of an unusual mononuclear [Fe]-hydrogenase86 and of human carbonic

anhydrase II with CO2 entrapped139 have been reported.

2. Cobalt

Reviews on cobalamine/cobamide-dependent methyltransferases and riboswitch

effectors (including cobalamin) have appeared.1,2 B12 derivatives are natural

molecular switches depending on the axial coordination of the nucleotide base

(base on and base offsee Fig. 1). Recently, B12 riboswitches were found to

be important components in the regulation of proteins relevant to B12 metabolism.

Therefore, over the past few years there has been continued interest in understanding

the mechanisms of action of these species. In this context, the interactions between

one of these riboswitches (the 202 nucleotide long btuB switch of E. coli) and four

different B12 derivatives (namely coenzyme B12, vitamin B12, adenosyl factor A andadenosyl-cobinamide) have been reported.3 These investigations have shown that the

corrin ring plays an important role in switching the three-dimensional riboswitch

structure. In contrast, the axial ligands on the cobalt centre do not seem to play a

crucial role in inducing the conformational rearrangement of this riboswitch.

The same group has also reported a detailed study on a series of 2 0- 300-nucleotide

conjugates of methylcobalamin and adenosylcobalamin. They have been

prepared with the aim of understanding the role of these systems as ribo-switches.

A guanosyl unit was attached at the 2 0-OH group of the B12 ribose segment yielding

B12-retro-riboswitches (which are defined as riboswitches with an appended

nucleotide that favour the base-off form of B12).4

Department of Chemistry, Imperial College London, London, UK SW7 2AZ.E-mail: [email protected]

{ The HTML version of this article has been enhanced with colour images.

Annu. Rep. Prog. Chem., Sect. A, 2009, 105, 477504 | 477

This journal is c The Royal Society of Chemistry 2009

REVIEW www.rsc.org/annrepa | Annual Reports A

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In addition to their biological relevance, B12 switches have been employed as the

basis for a cyanide sensor allowing for the colorimetric detection of millimolar

concentrations of this toxic anion in water.5 In the base on state of vitamin B12 the

axial positions of the cobalt(III) macrocycle are occupied by cyanide on the b sideand by a benzimidazoleintramolecularly coordinatedon the l side. Upon

addition of cyanide, there is a change in the absorption properties of the system

(detectable by naked eye inspection) due to the displacement of benzimidazole by the

incoming cyanide. The selectivity of the system towards other anions is better than

that for previously reported sensors based on cobalt(III) corrin derivatives.

The biosynthesis of cobalt-containing biomolecules continues to be a topic of

current interest. For example, the mechanism by which cobalt is incorporated into a

low-molecular mass nitrile hydratase from Rhodococcus rhodochrous J1 has been

established. It involves a previously unknown post-translational mechanism in which

a cobalt maturation mediator (NhIAE) is required. An interesting aspect of

this mechanism is that the cobalt-free and non-cysteine-oxidized a-subunit in the

apoenzyme is not retained in the fully functional enzyme. It is eventually substituted

by a different cobalt-containing cysteine-modified a-subunit.6 Another biosynthetic

pathway that has received attention is that of cobalamine. A key step in its

biosynthesis is the attachment of the upper axial ligand to the cobalt centre. This

step requires the reduction of cobalt(II) to cobalt(I) followed by adenosylation

(i.e. transfer of an adenosyl group from ATP). A recent study has identified

an enzyme (cobR) from Brucella melitensis with the ability to reduce the

corrin-coordinated cobalt(II) centre.

7

The protein has been structurally determinedto 1.6 A resolution as a flavoprotein and, together with kinetic and EPR studies, this

has allowed the authors to propose a mechanism for the one-electron reduction of

Co(II)corrin to Co(I)corrin.

A trafficking chaperone (named MMACHC) has been identified as the responsible

protein for the reductive decyanation reaction of vitamin B12. This provides

important insight into the long-lasting question of how cyanide is removed from

the coordination sphere of cobalt in cyanocobalamine. In this process, electrons are

transferred from NADPH via cytosolic flavoprotein oxidoreductases cleaving the

cobaltcarbon bond with reductive elimination of cyanide. These studies have also

shown that the product of the decyanation reaction is bound to MMACHC in thebase-off conformation. The authors of this work propose that this mechanism helps

to explain some observations from patients that are born with deficiencies on

MMACHC namely that fibroblasts from these patients do not respond to vitamin

B12 but show some metabolic correction with the cyanide-free aquocobalamine.8

Fig. 1 Schematic representation of the base on and base off states in B12 derivatives.

478 | Annu. Rep. Prog. Chem., Sect. A, 2009, 105, 477504


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Detailed magnetic circular dichroism and EPR studies have been carried out to

help elucidate the re-activation mechanism of cobalamine-dependent methionine

synthase (MetH). The results reported suggest that the conversion of MetH into the

activation conformation involves cleavage of the CoNH759 bond in MetH-bound

cob(II)alamin. This yields a five-coordinate cobalt complex in which a water

molecule occupies the axial position. A hydrogen bond between the coordinated

water and Y1139 stabilises the cobalt 3dz2-based redox active orbital by elongationof the CoOH2 bond. Addition of S-adenosylmethionine enhances the interaction

between Y1139 and MetH-bound cob(II)alamin causing the partial dissociation of

the axially-coordinated water molecule. The consequence of this is a raising on the

Co2+/Co1+ reduction potential into a range that is accessible to flavodoxin. The

resulting cob(I)alamin intermediate is methylated by S-adenosylmethionine to yield

the His-off form of methylcobalamine which is converted into the six-coordinated

His-on form and, following a conformational change, to the catalytically active

species.9

Kinetic analyses have been carried out to gain insight into the mechanism by

which B12 is delivered to its target enzyme.10

The studies reported provide evidencethat adenosyltransferase (ATR), which is the enzyme that catalyses the ultimate step

in the assimilation of B12 to adenosylcobalamine, is also responsible for delivering

the bound cofactor to its target enzyme (namely methylmalonyl coenzyme A

mutase). This is an interesting study showing the dual role of a protein as a

chaperone and as an enzyme.

3. Copper

3.1 Interaction of copper with peptides and proteins

Interaction of copper(II) with amyloid-b peptide (Ab) can lead to aggregation of Ab

and to the generation of reactive oxygen species (ROS), both of which processes have

been associated to Alzheimers disease pathogenesis. Several new studies have

appeared on the interaction of copper with Ab. A review discussing the molecular

origins of the neurotoxicity of copper(II)Ab aggregates in Alzheimers disease has

appeared.11

Potentiometric, spectroscopic and electrospray mass spectrometric studies have

been carried out on copper complexes of a polyethylene glycol(PEG)modified Ab.

This modification increases the water solubility of the peptide-metal aggregates

which allows their properties to be studied in more detail. These studies have shownthat Ab(1-16)PEG can bind up to four copper(II) ions and some insight has been

obtained regarding coppers coordination environment in each binding site.12 In a

different paper, a low-temperature electron paramagnetic resonance (EPR) study on

both soluble and fibrillar Ab has been carried out to help elucidate the exact

coordination environment of the metal centre. These studies indicate that there

are two distinct copper binding sites on Ab40 (both in its soluble and fibrillar forms).

One of the copper centres binds strongly to Ab and its association to the peptide

seems to be independent of the Ab oligomeric state. The second copper centre is

weakly associated to Ab fibrils and can be removed when fibrils are washed.

Although copper does not seem to regulate fibril structure, it appears to inducefibrilfibril association.13 A range of techniques such as FT-IR, sedimentation assays

and X-ray absorption spectroscopy have been employed to investigate the minimal

copper- and zinc-binding site in Ab peptides. A range of different Ab sequences have

been studied establishing that peptides containing the Ab(1-16) amino acid sequence



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display intrapeptide coordination of copper via three histidine residues

(His6, His13 and His14) and possibly a tyrosine and a water molecule. In the case

of zinc, it seems that a fourth histidine is involved in coordination.14

It has been suggested recently that coordination of copper and zinc to Ab causes

deprotonation; in the case of copper, it has also been suggested that coordination

results in release of water. A new study has been carried out showing with an

osmotic stress method that both copper(II

) and zinc(II

) coordination to Ab

indeedcause dehydration. Interestingly, binding of zinc to Ab causes the release of more

water (two-fold) than coordination of copper; this leads to a more destabilised Ab

which, as a consequence, is more prone to aggregation.15 A series of model peptides

have been used to study in detail (by CD spectroscopy, ThT fluorescence spectro-

scopy, TEM and EPR) the factors that control amyloid formation in the presence of

copper(II) and zinc(II) ions. These studies have shown that, in the model peptides

under study, coordination of metal ions can accelerate (zinc) or slow down (copper)

the amyloid-formation process. In addition, it has been shown that the relative

position of the coordinating residues plays an important role in amyloid-

formation.16

Although the production of ROS by copper(II)Ab aggregates is well documented,

there have been suggestions that Ab acts as an antioxidant. To investigate this

possibility, the generation of ROS by monomeric and fibrillar forms of Ab in the

presence of copper(II) and under aerobic conditions have been investigated. The

system was monitored with and without the biological reductant ascorbate in a

cell-free system. Interestingly, these studies show that Ab does not generate more

ROS than copper(II)/ascorbate controls suggesting that it is not a prooxidant under

these conditions. In contrast, the results reported suggest that Ab displays some

antioxidant-like properties. This may imply that the upregulation of Ab could be a

protective mechanism towards oxidative stress rather than the cause of it in the

early stages of Alzheimers disease. Importantly, the authors of this study also point

out that in the long term, the upregulation of Ab causes the formation of neurotoxic

oligomers which are likely to concentrate copper(II) ions at the membrane surface

allowing for ROS generation, which eventually will lead to neuronal cell death.

These findings could have important implications in the development of drugs for

Alzheimers disease.17

The interaction of copper(II) ions to fragments of prion protein (misfolding of

which leads to transmissible spongiform encephalopathies) has been extensively

studied. However, there are still relatively few investigations of copper bound tofull-length prion protein (PrP). In a recent study, competitive copper-chelators

have been used to determine the interaction of copper with full-length PrP. These

studies have shown that the natively unstructured half of cellular PrP is able to

bind up to six copper(II) ions at physiological pH. This unstructured section of PrP

has been shown to be essential for prion propagation. On the other hand, the results

obtained suggest that the function of PrP could be to scavenge copper(II) ions

released during neuronal depolarisation and in doing so protect the cell against toxic

copper ions.18 In a different study vibrational Raman optical activity and UV

circular dichroism have been used to study the interaction of copper(II) and

manganese(II) ions to full-length murine prion protein (PrP23231). The resultsreported indicate that interactions of both these ions with PrP have a considerable

effect in the proteins global structure. In addition, each ion studied has a

different effect on PrP; while copper(II) tends to destroy the a-helix, manganese(II)

reinforces it.19



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The aggregation ofa-synuclein (a-syn) is associated to Parkinsons disease and it

has been found that metalprotein interaction(s) play an important role in the

aggregation process. Some studies to gain a better understanding of the interaction

between copper(II) and a-syn have appeared. In one, four mutants of the protein

were studied by tryptophan fluorescence measurements, allowing dissociation

constants between copper(II) and the different binding sites within a-syn to be

calculated.

20

In a different study, site-directed and domain-truncated mutants ofa-syn have been used to study this interaction by a combination of NMR, EPR,

UV-vis and circular dichroism spectroscopies as well as by MALDI mass spectrometry.21

3.2 Copper chaperones

The mechanisms of acquisition, distribution and regulation of copper have been

reviewed.22 A number of metallochaperones that transfer copper to other proteins

are known and, over the past year, several studies have appeared aimed at

elucidating their exact mode of action. The role of glutathione (GSH) in the transfer

of copper to and from chaperone Atx1 has been investigated by SDS-PAGEelectrophoresis and spectroscopic methods. These in vitro studies have shown that

GSH is the major source of copper(I) for Atx1 and that the dimer CuI2(GS)2(Atx1)2

is the major conformation of Atx1 in the cytosol.23,24 The copper chaperone

for Cu,Zn-superoxide dismutase is a three-domain protein in which domain 1

(an Atx1-like domain) binds copper(I) via an MXCXXC motif and domain 3

contains a multinuclear cluster formed by two CXC motifs. To gain insight into

the coordination of copper in this chaperon, a series of selenocysteine derivatives

have been prepared and the interaction with copper(I) studied. The results have

allowed some of the key amino acid residues for metal binding in the different

domains of this chaperone to be identified.25 A periplasmic protein (CusF) has been

identified as a metallochaperone for an efflux system in Escherichia coli. Isothermal

calorimetric studies have established that, in the presence of metal, CusF interacts

with a second periplasmic protein (CusB). Using selenomethionine labelling and

X-ray absorption spectroscopy, it has been established that copper(I) is transferred

from CusF to CusB.26

The delivery of copper(I) to Wilson and Menkes disease proteins by Atox1

chaperone and two point mutants has been investigated using a model system

(in which bicinchonic acid acts as a metal acceptor to measure dynamics of copper

transfer). The three proteins under study have shown differences in their ability tobind copper(I) with the mutants losing copper(I) more readily than the wild-type

chaperone. The results obtained in this study suggest that copper chaperones play an

essential role in making copper(I) rapidly accessible; in addition, it has been shown

that the proteincopperchelator seems to be a good mimic of the chaperone

coppertarget complex involved in the in vivo transfer of copper(I).27 A solution

(NMR) structure of copper-binding domains 3 and 4 of Wilson protein and their

interaction with copper(I) and with chaperone HAH1 have been reported.28 It has

been revealed that pigment-cell-specific cuproenzyme tyrosinase acquires the

majority of its copper in specialised organelles called melanosomes (and only

transiently and inefficiently within the trans Golgi network). This study also showsthat ATP7A transporter plays a key role in supplying copper to melanosomes.29

The periplasmic protein CusF is thought to serve as copper chaperone or

regulator. A new study has appeared showing that this protein binds copper(I) via

an unusual and novel interaction, namely p-binding between the metal cation and



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tryptophan. This interaction was detected both by spectroscopic methods in solution

and in the solid state by an X-ray crystal structure (see Fig. 2).30

The transfer of copper(I) from Cox17 to Sco1 (two important proteins in the

metallation of cytochrome c oxidase) has been investigated. It has been shown that

the partially oxidised form of Cox17 with two SS bonds and two reduced

cysteines (HCox172SS) can simultaneously transfer copper(I) and two electronsto human cochaperone Soc1 in the oxidised state. This suggests that copper

trafficking in the mitochondrial intermembrane space is a more complex process

involving different redox states of the proteins.31

3.2 Catalytic roles of copper

3.2.1 Monooxygenases. The oxidation of hydrocarbons by membrane-bound

particulate methane monooxygenase (pMMO) has been reviewed.32 Most evidence

indicates that this protein, which converts methane to methanol in methanotrophicbacteria, is a multicopper enzyme. However, the exact nature of the active centre and

the metal content of this enzyme remain controversial. With the aim of gaining

insight into the activity of this enzyme, pMMO from Methylosinus trichosporium has

been isolated and characterised spectroscopically and crystallographically. These

studies provide strong evidence that this protein contains a dicopper centre,

similar to that found in a previously reported structure for a different bacterium

pMMO. EPR provides evidence that type 2 copper(II) is present as two

distinct species, while EXAFS reveals a coppercopper interaction at 2.52 A .33

The crystal structure to 1.35 A of the methane oxidizing enzyme MopE

from Methylococcus capsulatus has been solved. The structure has revealed thatthe catalytic copper has a distorted tetrahedral geometry coordinated by a

water molecule and two histidine imidazoles. Interestingly, the fourth coordination

position is occupied by kynurenine which is a tryptophan metabolite resulting from

oxidation of Trp-130.34

Fig. 2 X-Ray crystal structure of copper(I)-CusF highlighting the coordination of the coppercentre (sphere) to the sulfur atom of Met47 and Met49, the nitrogen of His36 and the

aromatic ring of Trp44. Figure generated with PyMol (http://pymol.sourceforge.net/) using

crystallographic PDB data.



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Copper also plays an important role in some neuroregulatory enzymes such as

tyramine b-monoxygenase (TbM). Three mutants of TbM have now been prepared

and their reactivity studied. The three mutants (which differ in the methionine ligand

that coordinates to one of the metal centres) bind copper with similar affinities to the

wild type protein in the oxidized enzyme form, while EPR shows that the coordination

environment of copper(II) is similar in the four proteins. Interestingly, only one of

the mutants (Met471Cys) is able to hydroxylate tyramine. This provides evidencethat a thiol-containing ligand is important for the catalytic activity of the enzyme.35

3.2.2 Oxidases. Two studies have appeared discussing the role of protonated

tyrosine in the cleavage of dioxygen by cytochrome c oxidase. The key step in the

reduction of O2 by this enzyme is OO bond scission which requires four electrons

and one proton. In the first of these two studies, a mutant of the enzyme has been

employed to gain insight into the enzymes mechanism. Using time-resolved optical

and FTIR spectroscopy, it has been shown that the Tyr-280 is the proton donor in

this process. In addition, it is suggested that this residue is also involved in the

donation of the fourth electron for O2 reduction.36,37 In a second publication the

same authors have investigated by IR spectroscopy the entire catalytic cycle of this

enzyme, including the reprotonation of Tyr-280 which is needed to close the cycle.37

A different aspect of the reactivity of cytochrome c oxidase has been investigated,

namely the fact that this enzyme is not inhibited by nitrous oxide produced be

neighbouring NO synthase. A series of functional models for the cytochrome c

oxidase active site have been developed and their reactivities with NO followed by

reactions with O2 and/or O2 have been investigated.38

The reduction of O2 and NO by cbb3-type haemcopper oxidase from Rhodobacter

spaheroides and the corresponding production of transmembrane proton gradients,have been investigated. Using spectroscopic and electrochemical methods, it

was found that the reduction of O2 was coupled to build up a significant electro-

chemical gradient across the membrane, consistent with pumping electrons.

In contrast, reduction of NO did not lead to the build up of such a gradient which

suggests that the protons involved in the reduction of NO (2NO + 2e + 2H+-

N2O + H2O) are not pumped from inside the cell, reversing the expected proton

transfer pathway.39

A new type of bacterial heme copper protein has been characterised. This protein

(SoxAX from Starkeya novella) seems to play an essential role in bacterial thio-

sulfate oxidation. Potentiometry, EPR and enzyme activity assays were carried outshowing that this protein contains two heme groups plus a copper(II) centre with a

distorted tetragonal geometry; the copper is coordinated by 3 to 4 nitrogen ligands,

one of which is histidine. It is proposed that the copper(II) centre enhances the

activity of SoxAX and the electron transfer from a sulfur donor to the protein.40

3.2.3 Miscellaneous. A quantum mechanical/molecular mechanical study of the

catalytic mechanism of tyrosinase (a di-copper protein which converts tyrosine to

dopaquinone with dioxygen) has been reported. This computational study supports

the mechanism previously reported in which the (m-Z2:Z2-peroxo)dicopper(II) speciesplays a central role in the catalysis. It is also shown that a stable phenoxyl radical is

involved in the reaction pathway. By analysing in detail the energy profile of the

process, it is proposed that the rate limiting step is the dissociation of OO. Finally,

in agreement with the previously proposed mechanism, the QM/MM results also



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suggest that the His54 residue plays an important role in the catalysis as a general

base for proton migration.41

The effect of the length of type 1 copper-binding loop in nitrite reductase

(a key enzyme for denitrification) has been investigated. The 15-residues loop in

the original enzyme has been replaced with a 7-residues loop (from cupredoxin

amicyanine) and the effect of this on the enzymes structure and activity studied by

spectroscopic and structural means.42

A series of model compounds have beenreported with the aim of gaining insight into the reaction mechanism of

copper nitrite reductases. The copper(I) complexes are based on tridentate

tris(4-imidazolyl)carbinol ligands with bulky substituents.43

The influence of pH on coppers coordination environment within proteins has

been investigated. In particular, pH-dependent transformations between blue T1

copper, red T2 copper and the native purple copper CuA of nitrous oxide reductase

of Paracoccus denitrificans have been reported. This enzyme was purified as

metal-free apo-protein and its interactions to copper(II) at different pH values

monitored by UV-Vis spectroscopy and EPR. Upon addition of copper at pH 7.5,

blue and red copper centres form initially, which are then transformed into the

purple CuA centre. It was also observed that high pH values favour the formation of

blue and red copper centres and slow down their conversion to purple CuA. These

studies provide some evidence of evolutionary links between the different types of

copper centres in cupredoxins and their pH dependence.44 (Fig. 3)

4. Iron

4.1 Iron uptake, transport and regulation

The coordination chemistry of petrobactin, a hexadentate siderophore synthesisedby Bacillus anthracis and Bacillus cereus during infection to scavenge iron, has been

studied. The affinities of this siderophore and its photoproduct for iron( III) were

determined at physiological pH.45 Another siderophore that makes use of catechol

units to coordinate iron(III) is chrysobactin, which is produced by a plant pathogenic

Fig. 3 Cupredoxin folds with blue, purple and red copper. (A) Blue copper azurin (PDB ID

code 4AZU) with the blue T1 active-site structure (inset). (B) Cupredoxin domain of N2OR

(PDB ID code 1FWX) with purple CuA active-site structure. (C) NC cupredoxin fold monomer(PDB ID code 1IBY) with red-T2-type active site structure (inset). Figure taken from reference

44 with permission. Copyright 2008 National Academy of Sciences, USA.



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enterobacterium. The coordination chemistry of this iron chelator has been studied

by electrospray mass spectrometric, spectrophotometric and potentiometric

methods.46 The interactions between siderophore enterobactin and mammalian

protein siderocalin have been investigated spectroscopically and structurally. These

studies provide interesting insight into the molecular mechanism by which this

protein acts as an antibacterial agent (by sequestering siderophores and their ferric

complexes) and its potential involvement in cellular iron transport.47 (Fig. 4)

The regulation of iron acquisition and storage in mammals has been reviewed with

emphasis on the potential disorders linked to defects in iron homeostasis.48 An

investigation into the interaction between soluble form of hemojuvelin (a recently

identified iron-regulatory protein) and neogenin (a cell surface receptor known to

bind hemojuvelin) has appeared. This study has identified that hemojuvelin binds to

the most membrane-proximal domain of neogenin which might have important

implications in the regulation of the soluble/membrane-bound levels of the protein.49

There have been several reports on the iron storage protein ferritin. UV-Vis and

fluorescence stop-flow spectroscopic studies have been carried out to determine the

kinetics and diffusion pathway of iron(II) in the protein shell. These have shown thatiron(II) transverses the protein with t1/2 of ca. 3 ms via the 3-fold channel. In

addition, binding of iron(II) to the ferroxidase centre and its subsequent oxidation by

O2 have been investigated.50

A range of techniques (such as TEM, SQUID, XANES amongst others) have been

employed to characterise a batch of horse spleen ferritins from which iron had been

gradually removed yielding samples with iron contents varying between 200 and

2200 iron atoms. Interestingly, these investigations show that the iron core in ferritin

is polyphasic (ferrihydrite, hematite and magnetite) and that the proportion of each

phase is dependent on the iron loading. Ferrihydrite dominates when the core

contains 10002000 iron atoms, while magnetite is the dominant phase at lower ironloading (ca. 500 iron atoms).51

The protein nanocage that protects ferritins iron core contains pores which are

gated to control reactions between external reductants and the iron minerals.

Mutation can alter the gating of pores and, therefore, have implications in iron

Fig. 4 Chemical structures of three siderophores.



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metabolism. A study has appeared showing the effect of a pore-unfolding mutation

on the relative amount of59Fe in the mutant protein compared to the wild-type. In

cells expressing the ferritin open-pore mutant increased iron release and increased

chelatable 59Fe was observed.52 A combination of CD, magnetic CD and variable

field magnetic CD have been employed to gain insight into the binding of iron(II) to

the substrate active site of frog M ferritin. These studies have shown that the active

site within each subunit consists of two inequivalent ferrous centres weakly coupledantiferromagnetically. Cooperativity between the different iron centres is observed

which provides some insight into a possible mechanism for the control of iron

loading in ferritin. The proposed diferrous substrate in ferritin features significant

differences with diferrous cofactor sites.53 In another study to understand the

differences between diiron substrate and diiron cofactor, oxidoreductase activity in

ferritin with diiron cofactor residues Gln137 - Glu and Asp140 - His, has been

compared to that of ferritin with natural diiron substrate site variations

(Asp140, Ser140 or Ala140). These investigations have shown that small differences

in the diiron protein catalytic sites have a significant effect on the formation of

diferric peroxo intermediates and whether the iron active site bonds persistthroughout the catalytic cycle or they break to yield iron(III)2O products.

54

Investigations using various types of microscopy have shown that human

transferrin readily forms protein fibrils which allow for the periodic nanomineralisation

of iron along the fibrils length. If this remarkable process can take place in tissues,

these findings could have important implications in the abnormal accumulation

of iron in the brain associated to a range of neurological disorders including

Alzheimers and Parkinsons diseases.55

4.2 Haem proteins

4.2.1 Non-catalytic haem proteins. Several studies have appeared studying the

interaction of O2 to iron in haem proteins. Synchrotron-derived vibrational data has

been used to demonstrate that the oxo ligand in myoglobin compound II,

[Mb(IV)QO], is not protonated.56 QM/MM calculations have been used to probe

the nature of the FeO2 bond in oxy-myoglobin. Two methodologies were

employed, namely in-protein DFT/MM calculations (which take into account the

protein environment) and gas-face calculations using DFT and CASSCF methods.

Comparison of the results obtained by the two methodologies have established the

importance of the protein environment in the nature of the FeO2 bond.

57

Multifrequency hyperfine sublevel correlation spectroscopy (HYSCORE) has been

successfully used to study the hyperfine interactions between the electron spin and

remote nuclei in the haem site of aquometmyoglobin. These studies revealed the

hyperfine interactions of the proximal histidine ligand, of the mesoprotons and of

the protons of the distal water molecule. In conjunction with previous EPR and

liquid-state NMR data, these investigations allow the spin density in the s and p

orbitals of the system to be calculated.58 Spectroscopic and crystallographic studies

have been carried out to characterise the autooxidation process of Trematomus

newnesi major haemoglobin. The b iron displays a rare pentacoordinated oxidation

state and a series of structural changes take place upon ligand release. The data alsoprovide evidence that the R- T transition is not limited by a major steric barrier

allowing for a facile transition. Put together, these studies provide a detailed

snapshot of haemoglobins allosteric transition.59 A study has appeared on the

differential oxygen affinity and structure of an engineered H-NOX domain in which



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the haem group adopts a flatter haem by mutating proline 115 to an alanine. The

results show that the mutated protein has a higher affinity for oxygen and the haem

group has a decreased reduction potential. The mutation also brings about a

conformational change of the protein at the N-terminus.60

The interactions of CO with wild-type HasASM (a hemophore secreted by some

bacteria to extract haem from host hemoproteins) and its haem pocket mutants

His32Ala, Tyr75Ala and His83Ala have been studied by Raman spectroscopy. Theseinvestigations have allowed the axial coordination in the four haem proteins to be

assessed.61 The NO release mechanism of nitrophorin a protein involved in the

storage and transport of NO in some blood-sucking insects has been studied by

classical molecular dynamics and hybrid QM/MM calculations. The haemNO

structure and FeNO strength have been analysed at different protein conforma-

tions. These calculations indicate that the release of NO from the protein depends on

the differential migration rates of NO rather than on the FeNO strength. This is

achieved thanks to a cage mechanism in this protein which allows the NO to be

trapped at low pH and released at higher pHs.62 The same authors have reported

computational studies to gain insight into the interaction of NO with prokaryotichaem NO oxygen (HNOX) domain.63

4.2.2 Haem enzymes. Electronic structure, hybrid QM/MM and classical

molecular dynamics calculations have been carried out to investigate the egression

pathway of nitrate ion in truncated haemoglobin N. This is relevant to

understanding the detoxification mechanism used by various bacteria in which

NO is oxidised by O2 to the nitrate ion. These calculations have identified specific

amino acid residues that are key in the detoxification process: PheE15 acts as a gate

in the tunnel branch of the protein, TyrB10 and GlnE11 modulate the O2 binding

affinity, the NO positioning and also facilitate the opening of the gate by a

conformational change. Finally, ThrE2 assists the nitrate ion along the egression

pathway.64 Computational studies have also been reported on models of nitric

oxide synthase. The key catalytic steps for the hydroxylation of L-arginine to

No-hydroxo-arginine have been explored by DFT methods. These studies suggest

that the L-arginine acts initially as a proton donor and later as the substrate in the

catalytic cycle. Detailed analysis of the electronic properties of the FeO bond

throughout the cycle are reported.65

New insight into the reactivity of horseradish peroxidase (HRP) has been

provided by two studies. One of these studies analyses the reactivity of HRP towardsamines with the particular aim of understanding why aliphatic amines appear to be

unreactive. This systematic study not only provides insight into the apparent lack of

reactivity but also shows that certain aliphatic amines (containing a tertiary amine

centre prone to undergo one-electron oxidation) are excellent substrates for HRP.65

A combined experimental/theoretical study has been carried out to examine the

activation of H2O2 by HRP. Using oxygen isotope fractionation and modelling

ground- and transition-state structures by DFT, it has been established that OO

cleavage is not the rate limiting step in the reaction of H2O2 with HRP.66

EPR and Mo ssbauer studies have been carried out on MauG oxidized by H2O2 in

the presence and absence of its substrate. MauG is an enzyme that contains twocovalently bound c-type haems, one low-spin and one high-spin, and catalyses the

biosynthesis of cofactor tryptophan to tryptophylquinone. These studies have

revealed the presence of two different iron(IV) centres, one consistent with an

Fe(IV)QO ferryl species and the other one with an Fe(IV) haem species containing



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two coordinated protein residues in the axial positions. The latter is an unusual

species that, according to the authors of this report, had not been previously

observed in vivo.67

Two X-ray crystal structures of cytochrome P450 46A1 (an important enzyme in

the process of cholesterol removal from the brain) have been determined. One of

them (at 1.9 A ) has been solved bound to substrate cholesterol 3-sulfate (see Fig. 5)

while the second one is a 2.4 A

structure of the substrate-free protein. The structuresshow that upon substrate binding important conformation changes take place in the

protein to position the cholesterol for oxygenation.68 Detailed spectral and kinetic

studies have been carried out on an iron(IV)-oxo porphyrin radical cation

(Compound I) formed by photoxidation of a cytochrome P450 119 Compound II

derivative. The results presented provide interesting insights into the active transient

species that are proposed to be generated in P450-catalysed oxidation reactions.69

The main factors that influence the mechanism of alkane hydroxylation by cytochrome

P450 have been investigated by DFT calculations and valence bond modelling, using

eleven different alkanes and model compounds of cytochrome P450.70

Thirteen previously reported X-ray crystal structures of haem catalases (a class ofenzymes that regulate the levels of hydrogen peroxide in living cells) have been

analysed and compared. The aim of this study was to gain insight into the functional

role of NADPH in Clade 3 catalases. The analysis has revealed striking similarities

amongst the Clade 3 NADPH-binding catalases; on the other hand, the structures

show important differences amongst the non-NADPH-binding Clade 1 and Clade 2

catalases. From this analysis and using DFT calculations, a mechanism is proposed

in which electron transfer from the surface-bound NADPH to the haem centre

would be facilitated.71

Fig. 5 X-Ray crystal structure of cytochrome P450 46A1 bound to cholesterol 3-sulfate. The

figure on the left highlights the iron-porphyrin (sticks representation) and the bound substrate

(spheres representation). The figure on the right shows the positioning of the substrate with

respect to some amino acid residues (which hydrogen bond to the sulfate) and the

iron-porphyrin.68 Figure generated with PyMol (http://pymol.sourceforge.net/) using

crystallographic PDB data.



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4.3 Ironsulfur proteins

EPR, Raman and UV-Vis spectroscopic studies have shown that NsrR

(a transcription factor found in various bacteria) contains a [4Fe4S]2+ cluster

which reacts with NO generating dinitrosyl iron complexes. These results are an

important step towards the molecular understanding of the NO sensing mechanism

in NsrR.72 The reduction transcriptional factor FNR is the primary control of the

switch between aerobic and anaerobic metabolism in Escherichia coli. In the absenceof O2, FNR binds a [4Fe4S]

2+ cluster which upon exposure to O2 is converted to a

[2Fe2S]2+ form leading to changes in the structure of the protein. A new spectro-

scopic study has now appeared showing that the cluster conversion in FNR can be

significantly affected by the reaction environment, in particular by the presence of

iron(II)/iron(III) chelators.73 The [FeS] cluster assembly scaffold IscU has been

proposed to play a key role in the preassembly of clusters required for the maturation

of ironsulfur proteins. To investigate this, the intact iron-cluster biosynthesis

machinery from Azotobacter vinelandii has been expressed to levels higher than those

required for the maturation of [FeS] clusters. These investigations indicate that IscU

indeed plays an important role as an in vivo scaffold for the assembly of [FeS] clusters

destined to the maturation of iron-cluster proteins.74 The crystal structure of the

[2Fe2S] oxidative-stress sensor SoxR bound to DNA has been solved. The structural

studies show that the [2Fe2S] cluster is solvent-exposed and surrounded by an

asymmetrically charged environment. The latter is likely to be the cause for the

redox-dependent conformational changes of SocR and the target promoter.75

The mechanism of action of biotin synthase (an enzyme that catalyses the

oxidative addition of sulfur to dethiobiotin and utilizes a reduced [4Fe4S]+ cluster)

has been investigated by a combination of spectroscopic/analytical techniques.

These studies have confirmed that the conversion of dethiobiotin to biotin involvesthe reductive cleavage of two equivalents of S-adenosylmethionine. In addition,

a 9-mercaptodethiobiotin intermediate has been identified.76

4.4 Other non-haem proteins

The activation of oxygen by non-haem proteins has been reviewed.77 The X-ray

crystal structures of the effector protein-hydroxylase complex of toluene-4-

monooxygenase in two different redox states (namely in its resting state and the

sodium dithionite-reduced complex) have been reported. Comparison of the three

different structures provides a detailed description of the changes that the proteinundergoes upon binding to the effector. The results show that complex formation

closes access to the active site (and impedes substrate binding) and rearranges the

ligands around the diiron moiety. In addition, it introduces open coordination sites

at the diiron centre facing towards the active site cavity.78 In a different study, CD,

magnetic CD and variable temperature, variable field magnetic CD have been

used to gain insight into the diiron active site of the hydroxylase component of

toluene-4-monooxygenase. In addition, the complex between this enzyme and its

effector has been investigated. These results provide a clearer picture of the diiron

active site of this protein and the changes it undergoes upon binding to its effector

(which modifies the geometry of one of the iron centres and as a consequencechanges its reactivity).79

The X-ray crystal structure at 1.9 A resolution of tryptophan hydroxylase bound

to the amino acid substrate has been reported. The structure has shown that the

tryptophan substrate is bound close to the iron centre in a distinct binding pocket.80



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The thermal unfolding kinetics of apo- and holo-phenylalanine hydroxylase from

Chromobacterium violaceum have been studied by variable temperature CD spectro-

scopy. The kinetic profiles of both the apo- and holo-enzymes showed a single-phase

exponential rise and a first-order dependence on protein concentration. Besides the

iron(II) native enzyme, the studies have also been carried out with zinc(II) and

cobalt(II) as cofactors. To measure the affinity of the protein to each one of these

metals, isothermal titration calorimetry was employed. These studies have shownthat the protein is more stable in its metallated form at physiological temperatures.

In spite of the fact that the native protein contains iron(II) the stability constants for

zinc(II) and cobalt(II) are higher, suggesting that the metal-delivery system in this

bacterium is more efficient for iron(II) than for the other two metal cations.81

The activation of O2 by 1-aminocyclopropane-1-carboxylate oxidase (an enzyme

that catalyses the last step in the biosynthesis of ethylene) has been studied by steady

state kinetics, solvent isotope effects and competitive oxygen kinetic isotope effects.

These studies have provided insight into the nature of the activated oxygen species

produced on the iron catalytic site and its dependence on ascorbic acid. The results

strongly suggest that the formation of Fe(IV)QO (which is the reactive intermediatefor substrate oxidation) is the rate-determining step in this process.82

A review on iron-containing superoxide dismutase with particular emphasis on the

redox tuning of the active site has appeared.83 In addition, two articles have

appeared in which spectroscopic and computational methods have been employed

to understand the effects that second-coordination sphere residues have on the

structural and electronic properties (including redox potentials) of the catalytic iron

site of superoxide dismutase.84,85

The X-ray crystal structure at 1.75 A resolution of an [Fe]-hydrogenase from

Methanocaldococcus jannaschii has been reported. In contrast to other iron-

containing hydrogenases, the active-site in this enzyme is mononuclear with the

iron coordinated to the sulfur of cysteine 176, to two carbon monoxide molecules

and to the sp2-hybridised nitrogen of a 2-pyridinol compound.86 (Fig. 6)

Fig. 6 Active site of the [Fe]-hydrogenase from Methanocaldococcus jannaschii. The iron

centre (central sphere) is coordinated to two CO ligands, to the sulfur of a cysteine and to the

sp2-hybridised nitrogen of a 2-pyridinol compound.



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An [FeFe]-hydrogenase from Clostridium acetobutylicum has been investigated as

a catalyst for the production of H2 in a photoelectrochemical biofuel cell.

Electrochemical studies of the immobilised hydrogenase revealed cathodic proton

reduction and anodic hydrogen oxidation. A catalytic bias towards H2 production is

reported.87

5. Manganese

A special issue of the Philosophical Transactions of the Royal Society (March 27,

2008) compiles articles from a meeting on how Nature uses light to split water. In

this issue, the water oxidation chemistry of photosystem II has been reviewed.88

The functional characteristics of the photosystem II manganese-stabilising protein

(PsbO) have been investigated (using a selection of N-terminal truncation mutants of

the protein with different photosystem II binding abilities). By monitoring O2evolution activity, Ca2+ and Cl retention, and stability of the manganese cluster,

it has been shown that each of the two PsbO subunits bound to photosystem II has a

distinct function. One of them binds to the manganese cluster and enhances theretention of chloride in the O2-evolving complex (OEC), while the second

subunit improves the efficient retention of chloride close to the manganese cluster,

maximizing O2 evolution activity.89

Although it has been previously suggested that bicarbonate is required for the

formation of O2 in the Mn-containing O2-evolving complex of photosystem II,

evidence against this has now been provided. Upon addition of NH2OH to OEC

(which would destroy this complex and release any tightly bound bicarbonate as

CO2), no significant amount of CO2 was detected by mass spectrometry.90 On the

other hand, a time-resolved X-ray absorption study has been carried out to

determine whether elevated O2 partial pressures would lead to product inhibition

in photosystem II. The study focuses on the redox chemistry of the Mn complex in

its catalytic S-state cycle. The results reported indicate that the rate of photo-

synthetic water oxidation is not significantly affected by high partial pressures of O2.91

New structural insights into the mechanism of photosynthetic water splitting have

been reported. A biosynthetically exchanged Ca/Sr photosystem II was prepared and

studied by EXAFS. The structure of the cluster was monitored in the four S

intermediate states of the catalytic cycle (namely from S0 to S3). Significant changes

in MnMn and MnCa(Sr) distances were detected on the transitions from S2 to S3

and from S3 to S0. These investigations indicate that the oxygen atoms involved inthe formation of the OO bond in the water oxidation reaction, come from a

Ca-bound water (or hydroxide) and an oxo-bridging atom with significant radical

character.92

A review on QM/MM computational studies of water binding to OEC of

photosystem II has appeared.93 QM/MM computational studies have been carried

out to gain insight into the water splitting catalytic mechanism in photosystem II.

A series of molecular models of the OEC Mn3CaO4Mn cluster have been

constructed considering the perturbation produced by the surrounding protein.

The results obtained from these calculations have been compared to X-ray

diffraction models of photosystem II and EXAFS data.94In order to gain insight into the role that Glu162 plays at the tetrameric interface

of human Mn superoxide dismutase (MnSOD), two site-specific mutants of human

MnSOD in which Glu162 has been replaced by Asp (E162D) and Ala (E162A)

have been prepared and structurally characterised. In addition, the thermal stability



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and catalytic properties of the MnSOD and the two site-specific mutants have been

investigated. The results obtained emphasise the important role of the dimeric

interface in MnSOD. It is shown that the side chain of Glu162 indeed influences

several properties (structure, stability, catalysis and inhibition) of MnSOD.95 A

comparison of the kinetic mechanism of MnSOD from Deinococcus radiodurans

(a bacterium with a remarkable ability to tolerate high levels of ionising radiation),

humans and Escherichia coli has been reported. It has been found that under equalenzyme to superoxide ratios, the dismutation efficiency is highest for Deinococcus

radiodurans. It is proposed that the higher efficiency of MnSOD in this bacterium is

not due to this enzyme reacting at a faster rate with O2, but due to differences in the

mechanism of the reductase step.96 The potential antioxidant mechanism of

manganous ions that might take place in vivo, has been re-investigated using two

different methods to generate superoxide in solution, namely pulse radiolysis and

gamma irradiation using a 60Co source. It was found that at physiological

relevant concentrations only manganous phosphate (not chloride, sulfate and

pyrophosphate) remove superoxide from solution in a catalytic fashion.

A disproportionation mechanism has been proposed for this transformation whichis completely different to that used by MnSOD. This suggests that manganous ions

might indeed play a distinct antioxidant role in vivo.97

The catalytic cycle of extradiol oxygenase has been investigated by X-ray

crystallography98 and EPR.99 Using two nearly identical enzymes with different

metals (manganese and iron) their catalytic activity for the ring opening of

homoprotocatechuate by O2 has been monitored. The X-ray crystal structure of

Mn-HPCD (where HPCD = homoprotocatechuate-2,3-dioxygenase) at 1.7 A has

been resolved and compared to the Fe-HPCD (the latter being the native protein).

The two structures have been found to be indistinguishable. Comparing the activity

of both the iron and manganese enzymes, it was proposed that in the catalytic cycle

an electron is transferred from the substrate to O2 via a manganese(II) centre

(implying that a change in oxidation of the metal centre is not needed for the

reaction to take place). In contrast, the same group has reported a further EPR study

which provides evidence for the rapid formation of a manganese(III)-radical species,

which indicates that a redox transformation of the metal centre does take place

in the cycle.99 It is proposed that this species is then converted into a manganese(II)-

alkylperoxo intermediate, implying that the OO bond cleavage from this

intermediate is the overall slow step in the cycle.

Some of the factors that modulate the catalytic activity of DNA gyrase(an enzyme involved in managing bacterial DNA topological changes during

transcription and replication) have been investigated by circular dichroism, protein

melting experiments and enzyme activity assays. It has been found that the nature of

the divalent metal ions has an effect on the activity of the enzyme: while Ca2+ and

Mg2+ do not have a significant effect on the structure of the protein, Mn2+ was

found to trigger considerable modifications. In addition to the role of metal ions on

the enzymes structure and activity, this study also shows that DNA plays an active

role in the formation of the catalytic complex (rather than being simply a passive

substrate).100

EXAFS and DFT investigations have been carried out on the manganese(IV)/iron(III) cofactor of ribonucleotide reductase (from Chlamydia trachomatis). EXAFS

indicates that in this co-factor the metal-metal distance is of 2.92 A while a short

MnO bond (of 1.72 A ) has been found. The computational (DFT) studies are

consistent with a m-oxo/m-hydroxo/m-carboxylato core.101



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An intriguing question in bio-inorganic chemistry is how metallo-proteins acquire

selectively metal ions directly from pools of cations (especially when no chaperon

protein is linked to the metallation process). Some insight to this question has been

provided by a study investigating the binding of Mn2+ and Cu2+ to proteins in the

periplasm of cyanobacterium Synechocystis PCC 6803. The first part of the study

focuses on identifying which soluble proteins bind most Mn2+ and Cu2+ in the

bacterium periplasm. These were found to be cupins (CucA and MncA) which werethen expressed in Escherichia coli and exposed to Mn2+ and Cu2+ to confirm that

they indeed bind the cations. The coordination environment of the metal centres in

these metallo-proteins was then studied showing that in both cases identical ligands

are used within a cupin fold to bind the respective metal. Cation exchange studies

with the metallo-proteins were undertaken showing that once the metal is bound to

the folded protein, it is not exchanged (e.g. Mn2+ is not displaced by Cu2+ in spite

of the later expected to bind more strongly to proteins according to the

Irving-Wallace series). The results obtained in this study show that the compart-

ment in which a protein folds defines the metal ion that will be bound, which

controls the content of a specific metal cation in a given protein.102

It hasbeen previously proposed that prion protein binds manganese. A new investigation

has provided some insight into the binding site of manganese in PrP.

Using isothermal titration calorimetry it has been shown that wild-type PrP has

two binding sites for manganese which are associated to His-95 (which is normally

associated to copper binding). In addition, this investigation has shown that

manganese can displace copper from PrP which brings about a change in protein

conformation.103

6. MolybdenumThe biosynthesis of the iron-molybdenum cofactor of nitrogenase has been

reviewed.104,105 The role of MoaB protein in the biosynthesis of molybdenum and

tungsten cofactors has been studied. It has been found that this protein catalyses the

adenylation of metal-binding pterin (MPT) in the presence of Mg2+ and ATP. The

adenyl transfer activity displayed by MoaB is shared by other homologous proteins

such as MogA (bacterial) and Cnx1G (eukaryotic) involved in the insertion of Mo or

W in the maturation process of the corresponding cofactor.106

A new study has provided further insight into the role played by nitrogen fixation

(nif) genes and the corresponding proteins (Nif) for the biosynthesis of nitrogenaseiron-molybdenum cofactor (FeMo-co). More specifically, this study provides direct

in vitro evidence that NifQ protein carries a molybdenum-iron-sulfur cluster,

[Mo3Fe4S], which serves as a molybdenum donor for the synthesis of

FeMo-co.107 On the other hand, the role of a chaperone protein (TorD) for the

biosynthesis of the molybdoprotein trimethylamine-oxide reductase (TorA) has been

studied. This chaperone protein allows the molybdenum cofactor insertion by

directly interacting with the core of the apo-TorA protein.108

A computational DFT study that provides insight into the reaction mechanism of

formate dehydrogenase a molybdoenzyme that catalyses the oxidation of formate

to CO2 has appeared.109 The two proposed mechanisms (which differ in the directcoordination or not of a SeCys) have been investigated in vacuum and in protein

environment. The energy profiles for both mechanisms suggest that the most

favourable (lower energy barriers) pathway is that in which SeCys is not directly

coordinated to the metal centre.



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The X-ray crystal structure of polysulfide reductase (Psr) from Thermus

thermophilus (at 2.4 A resolution) has been determined.110 In addition to the native

protein, the structures of complexes of Psr with three different quinone analogues are

reported providing important information on how the enzyme recognises and

reduces its substrate. The crystal packing has revealed a dimer configuration

(molecular weight ofca. 260 kDa) which is likely to be physiological. The monomers

of this dimer consist of three subunits two of which (PsrA and PsrB) are associatedto the membrane while the third one (PsrC) in an integral membrane subunit. The

catalytic PsrA subunit contains two molybdopterin guanine dinucleotide cofactors

(bis-MGD) and a cubane-type [4Fe-4S] cluster. The PsrB subunit belongs to a

superfamily of [4Fe4S]-containing electron-transfer subunits while PsrC consists of

eight transmembrane helices anchoring the protein to the membrane.

The molybdenum(V) centre of the pathogenic R160Q mutant of human sulfite

oxidase (hSO) has been studied by EPR (using 17O and 33S labelling).111 This

investigation provides confirmation for the presence of three different molybdenum-

containing species, their abundance being dependent on the pH. One of these species

(which is significant at physiological pH and predomininant at pH o =6) is asulfate-bound complex that represents a catalytic dead end. This species is proposed

to contribute importantly to the lethality of the R160Q mutation.

Structural characterisation of xanthine oxidoreductase showing the orientation of

its substrate has been carried out. The structure shows the molybdenum active site

interacting with the substrate.112

7. Nickel

Hydrogenases are generally air-sensitive being inactivated by O2. However, recent

studies have shown that the production of H2 in the presence of atmospheric levels of

O2 is possible. The ability of membrane-bound [NiFe] hydrogenases from Ralstonia

species to produce H2 under aerobic conditions has been reported. This O2-tolerant

enzyme is not usually good at producing H2 since it undergoes product inhibition.

However, in this study it has been shown that by adsorbing this enzyme onto a

rotating disk graphite electrode it produces H2 efficiently if the H2 product is

constantly removed to avoid product inhibition. An important observation from

these investigations is that O2 is a worse inhibitor that H2, therefore it is possible to

perform the reaction under aerobic conditions.113,114 Protein film voltammetry

studies have shown that [NiFeSe] hydrogenase from Desulfomicrobium baculatum,in the presence of 100% H2, has the highest ratio of H2 production to H2 oxidation

activity if compared to conventional [NiFe] hydrogenases. This enzyme can continue

producing H2 even if traces (up to 1%) of O2 are present.115

The maturation of hydrogenase in Escherichia coli has been studied, in particular

the role of the accessory protein HypB. This protein has two different metal binding

pockets that are used to bind nickel(II) and their physiological relevance has been

investigated. The results obtained show that the two sites are indeed important for

nickel delivery and signalling.116 A series of novel NiFe hydrogenase model

compounds have been prepared, structurally characterised and their reactivity

studied.117 Detailed electronelectron double resonance-detected NMR (EDNMR)studies with 61Ni have been carried out on a hydrogenase from Desulfovibrio (D.)

vulgaris. This work has shown the potential of EDNMR in measuring metal

hyperfine interactions in metalloproteins, which in turn provides electronic and

geometrical information of the active site of the metalloenzyme under study.118



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Methyl-coenzyme M reductase (MCR) is a nickel enzyme that catalyses the key

step in the production of methane in archaea. Electronnuclear double resonance

(ENDOR) and hyperfine sublevel correlation spectroscopy (HYSCORE) have been

used to investigate the coordination environment around the paramagnetic nickel

centres at different states of the proteins (in particular MCRred2 and MCRred2a).

These studies suggest that a Ni(III)-hydride is present in the MCRred2a state which

forms by oxidative addition to nickel(I

). The catalytic implications of the hydridecomplex are discussed.119

Acetyl coenzyme A synthase/CO dehydrogenase is an enzyme found in anaerobic

archae and bacteria with an active site containing a [Fe4S4] cluster linked to a

di-nickel subcomponent. A combined Mo ssbauer and EPR investigation has been

carried out to provide some insight into the oxidation state of the metals in the

enzyme. Upon reduction of the system by Ti(III) citrate, the {[Fe4S4]+1 Nip

+1} state

forms together with what appears to be a semireduced {[Fe4S4]+1 Nip

+2} state.

Although these two states had been previously proposed (the latter as a catalytic

intermediate) this study is the first experimental evidence of their existence.120

An X-ray structure of a distinct class of Ni-dependent carbon monoxidedehydrogenase (Ni-CODH) from methanogenic archae has been reported. The

structure has revealed the presence of an FeS domain not present in other

Ni-CODH. The structure also provides details of a C cluster intermediate that

contains both a bound CO and water. These structural details provide support for a

mechanism in which the formation of CO results from coupling between the CO

bound to nickel and a H2O/OH bound to iron (see Fig. 7)121

Structures of the metal-binding domain of the nickel regulatory protein (NikR) in

the apo form and loaded with Cu(II) and Zn(II) have been determined. These

structures have been compared with that of the previously reported protein with

Ni(II) and insight into the high selectivity of the protein for the latter metal cation

has been obtained. The findings indicate a correlation between the ability of the

metal to order the alpha3 helix upon coordination and the corresponding metals

ability to induce DNA binding.122

8. Vanadium

Vanadate-dependent haloperoxidases and their models have been reviewed.123

A vanadyl ion has been incorporated into the biotin-binding pocket of streptavidin

yielding an artificial metalloenzyme with the ability to catalyse the enantioselective

oxidation of prochiral sulfides. The conversion of a range of substrates using this

Fig. 7 Proposed coupling of CO and H2O based on the structural information obtained in this

study.



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artificial enzyme takes place with good enantioselectivities.124 The crystal structure

of the apo form of a vanadium chloroperoxidase from Curvularia inaequalis reacted

with para-nitrophenylphosphate has been determined at a resolution of 1.5 A . The

structure has revealed the trapped intermediate PO3 anion of the phosphohydrolase

reaction. This species is covalently bound to the nitrogen of His496 of the protein.125

9. Zinc9.1 Structural roles of zinc

Zinc fingers continue to attract great interest. Several reviews have appeared

covering topics such as the design of new zinc fingers,126 structural aspects of

DNA-zinc finger recognition127 and zinc finger nucleases as gene therapy

agents.128,129 A novel CCCH-zinc finger protein family has been reported to regulate

the activation of macrophage (which plays an important role in many inflammatory

diseases).130 In a different study, zinc finger ZBTB20 has been identified as a key

repressor of alpha-fetoprotein (AFP) gene transcription in liver. Although this

protein is the major serum protein in the mammalian fetus, shortly after birthAFP gene is greatly repressed with a 10,000-fold decrease in transcription. However,

AFP is reactivated in certain conditions such as hepatocellular carcinogenesis. The

identification of ZBTB20 as a major repressor of AFP gene will, therefore, help to

understand the reactivation of this gene with the potential impact that this could

have on better diagnosis of hepatic cancer.131

The structure of an unusual knotted zinc finger motif has been reported. More

specifically, the solution structure of Rds3p (an essential protein for pre-mRNA

splicing) has been determined revealing the presence of 13 cysteine residues

(10 of which are present within five CXXC motifs) in its primary structure. Mass

spectrometry has shown that Rds3p binds three zinc(II) ions. The most interesting

structural feature of this protein is its unprecedented knotted fold.132

A computational study has investigated the role of zinc in folding and stabilising

zinc finger proteins, in particular the second finger of the human transcription factor

Sp1, a Cys2His2-type zinc-finger. An important feature of these molecular dynamic

calculations is that they have been carried out accounting explicitly for charge

transfers between zinc(II) and surrounding ligands, and also metal induced

protonation/deprotonation effects. The calculations provide important insight into

the folding mechanisms of the protein under study.133

The role of zinc in the formation of bio-films by Staphylococcus epidermidis andStaphylococcus aureus (two of the bacteria often associated to hospital infections)

has been investigated by analytical ultracentrifugation and circular dichroism

spectroscopy. Two cell-surface proteins (Aap and SasG), which contain sequence

repeats known as G5, are needed for these bacteria to grow bio-films. This domain

dimerises in the presence of zinc(II) incorporating 23 ions in the dimer interface.

This suggests that G5 domain-based intercellular adhesion in bacterial bio-films is

mediated by zinc which, as suggested by the authors of this work, gives rise to a

zinc zipper mechanism.134

As indicated in a previous section, the interaction of copper and zinc with

amyloid-b peptide can lead to aggregation of Ab and to the generation of ROS,both of which processes have been associated to Alzheimers disease. The Ab

molecules isolated from Alzheimers disease patients contain several post-

translational modifications, one of them the most abundant being the

isomerisation of Asp7 residue. In order to study the relevance of this modification



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wire, which in the Y7F mutant is more directional and less branched than in the

wild-type enzyme. This study has shown that in the Y7F mutant (in which Tyr7 has

been substituted by phenylalanine) the absence of Tyr7 alters the stability of His64

and the active site waters yielding a more efficient enzyme.141

The reaction mechanism of the di-zinc enzyme dihydrooratase has been

investigated by hybrid density functional theory. The calculations were carried out

on a model of the active site of the enzyme based on the X-ray crystal structure of the

protein. A hybrid functional B3LYP was employed to calculate potential-energy

surface identifying the different transition states of the reaction. These calculations

have established that the substrate (dihydrooratate) binds to the active site via

hydrogen bonding interactions with Arg20, Asn44 and His254, and by coordination

to one of the zinc centres. This is followed by nucleophilic attack of the bridging

hydroxide on the substrate. The rate-limiting step of the reaction is the protonationof the amide nitrogen coupled with CN bond cleavage.142

The catalytic activity of matrix metalloproteinase (MMP)-12 has been studied

using catalytic assays and NMR footprinting methods. It has been shown that,

similarly to other MMPs, this enzyme hydrolyses the triple helical peptide derived

from the collagen V that it cleaves.143 The structural characterisation of MMP-12

both in the solid state (X-ray crystallography to 3 A resolution) and in solution

(NMR and SAXS) has been carried out. These studies show that full-length

MMP-12 display relative mobility of its catalytic and hemopexin domains.144

The activity of a di-zinc aminopeptidase from Vibrio proteolyticus (AAP) has been

investigated. A detailed structural and kinetic analysis has been carried out to studythe modulation of the catalytic activity in this enzyme as a function of the

coordination environment around the metal centres. More specifically the

correlation between charge/dipole polarity and ligand binding affinity has

been investigated on the M180A, S228A and D118N variants of AAP. A series of

Fig. 8 X-Ray crystal structure of carbonic anhydrase with CO2.139 The structure shows the

CO2 oriented so that the carbon is primed for nucleophilic attack from the zinc-bound

hydroxide. Figure generated with PyMol (http://pymol.sourceforge.net/) using crystallographic

PDB data.



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high-resolution crystal structures together with measurements of binding affinities

towards leucine phosphonic acid (a transition state analogue) and leucine (product)

are presented. A structure-function relationship has been drawn up supporting the

idea that the coordination number and polarity tune the electrophilicity of zinc.145

Insulin-degrading enzyme (IDE) a zinc-containing protein that hydrolyses a

range of different peptides such as insulin and b-amyloid peptide is inhibited by

compounds that react covalently with cysteine residues. In this study insight into therole of thiol-alkylating reagents in inhibiting IDEs activity has been carried out.

A comprehensive mutational analysis of the 13 cysteine residues in IDE is presented.

Three residues (C178, C812 and C819) have been identified as those that confer

thiol-sensitivity to this enzyme. Interestingly, two of these residues are quite far from

the catalytic zinc centre, suggesting that the active site of IDE comprises two

different domains which need to be positioned nearby for the enzyme to be active.146

The selectivity of aminopeptidase N from Escherichia coli has been structurally

investigated. This enzyme has the ability to cleave selectively peptide bonds that are

next to the large and non-polar amino acids Phe and Tyr. In addition, it is able to

cleave next to the polar residues Lys and Arg. To gain some insight into this unusualselectivity the X-ray crystal structures of aminopeptidase N in the presence of these

four amino acid residues have been solved.147 Dipeptidyl-peptidases III (DPP III)

are a family of zinc-dependent enzymes with the ability to cleave selectively the first

two amino acids of a range of different peptides. In this study the 1.95 A crystal

structure of yeast DPP III is reported. It shows that this enzyme contains a novel

fold with two domains forming a wide cleft which hosts the catalytic zinc centre.148

A comparison between two different Salmonella Cu,Zn-superoxide dismutases

SodCI and SodCII has been carried out providing interesting insight into the role

these two enzymes play in Salmonellas virulence. The study demonstrates that the

two superoxide dismutases display different protease resistance, metal affinity and

peroxidase activity. Dimeric SodCI has superior stability and activity while mono-

meric SodCII has been shown to be unable to retain its catalytic copper in the

presence of zinc. These observations are consistent with SodCI playing a key role in

Salmonellas virulence.149

List of abbreviations

a-Syn a-synuclein

Ab Amyloid-b peptideATR AdenosyltransferaseCASSCF Complete active space self-consistent fieldCD Circular dichroismDFT Density functional theoryENDOR Electron-nuclear double resonanceEPR Electron paramagnetic resonanceEXAFS Extended X-ray absorption fine structureGSH GlutathioneHYSCORE Hyperfine sublevel correlation spectroscopyMALDI Matrix-assisted laser desorption/ionisationMM Molecular mechanicsMMO Methane monooxygenaseOEC O2-evolving complexPEG Polyethylene glycolPrP Prion protein



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PSII Photosystem IIQM Quantum mechanicsROS Reactive oxygen speciesSDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresisSOD Superoxide dismutaseSQUID Superconducting quantum interference deviceTEM Transmission electron microscopy

ThT Thioflavin TXANES X-ray absorption near edge structure

References

1 J. C. Cochrane and S. A. Strobel, RNA, 2008, 14, 993.2 R. G. Matthews, M. Koutmos and S. Datta, Curr. Opin. Struct. Biol., 2008, 18, 658.3 S. Gallo, M. Oberhuber, R. K. O. Sigel and B. Krautler, ChemBioChem, 2008, 9, 1408.4 S. Gschosser and B. Krautler, Chem.Eur. J., 2008, 14, 3605.5 F. H. Zelder, Inorg. Chem., 2008, 47, 1264.6 Z. Zhou, Y. Hashimoto, K. Shiraki and M. Kobayashi, Proc. Natl. Acad. Sci. USA, 2008,

105, 14849.

7 A. D. Lawrence, E. Deery, K. J. McLean, A. W. Munro, R. W. Pickersgill, S. E. J. Rigbyand M. J. Warren, J. Biol. Chem., 2008, 283, 10813.

8 J. Kim, C. Gherasim and R. Banerjee, Proc. Natl. Acad. Sci. USA, 2008, 105, 14551.9 M. D. Liptak, S. Datta, R. G. Matthews and T. C. Brunold, J. Am. Chem. Soc., 2008,

130, 16374.10 D. Padovani, T. Labunska, B. A. Palfey, D. P. Ballou and R. Banerjee, Nat. Chem. Biol.,

2008, 4, 194.11 A. Rauk, Dalton Trans., 2008, 1273.12 C. A. Damante, K. Osz, Z. Nagy, G. Pappalardo, G. Grasso, G. Impellizzeri,

E. Rizzarelli and I. Sovago, Inorg. Chem., 2008, 47, 9669.13 J. W. Karr and V. A. Szalai, Biochemistry, 2008, 47, 5006.14 V. Minicozzi, F. Stellato, M. Comai, M. Dalla Serra, C. Potrich, W. Meyer-Klaucke and

S. Morante, J. Biol. Chem., 2008, 283, 10784.15 H. Yu, J. Ren and X. Qu, ChemBioChem, 2008, 9, 879.16 K. Pagel, T. Seri, H. von Berlepsch, J. Griebel, R. Kirmse, C. Boettcher and B. Koksch,

ChemBioChem, 2008, 9, 531.17 R. C. Nadal, S. E. J. Rigby and J. H. Viles, Biochemistry, 2008, 47, 11653.18 M. Klewpatinond, P. Davies, S. Bowen, D. R. Brown and J. H. Viles, J. Biol. Chem.,

2008, 283, 1870.19 F. Zhu, P. Davies, A. R. Thompsett, S. M. Kelly, G. E. Tranter, L. Hecht, N. W. Isaacs,

D. R. Brown and L. D. Barron, Biochemistry, 2008, 47, 2510.20 J. C. Lee, H. B. Gray and J. R. Winkler, J. Am. Chem. Soc., 2008, 130, 6898.21 A. Binolfi, G. R. Lamberto, R. Duran, L. Quintanar, C. W. Bertoncini, J. M. Souza,

C. Cervenansky, M. Zweckstetter, C. Griesinger and C. O. Fernandez, J. Am. Chem.Soc., 2008, 130, 11801.

22 B.-E. Kim, T. Nevitt and D. J. Thiele, Nat. Chem. Biol., 2008, 4, 176.23 D. Roger, C. Fillaux, R. Miras, S. Crouzy, P. Delangle, E. Mintz, C. Den Auwer and

M. Ferrand, J. Biol. Inorg. Chem., 2008, 13, 1239.24 R. Miras, I. Morin, O. Jacquin, M. Cuillel, F. Guillain and E. Mintz, J. Biol. Inorg.

Chem., 2008, 13, 195.25 A. N. Barry, K. M. Clark, A. Otoikhian, W. A. van der Donk and N. J. Blackburn,

Biochemistry, 2008, 47, 13074.26 I. Bagai, C. Rensing, N. J. Blackburn and M. M. McEvoy, Biochemistry, 2008, 47, 11408.27 F. Hussain, J. S. Olson and P. Wittung-Stafshede, Proc. Natl. Acad. Sci. USA, 2008, 105,

11158.28 L. Banci, I. Bertini, F. Cantini, A. C. Rosenzweig and L. A. Yatsunyk, Biochemistry,

2008, 47, 7423.

29 S. R. G. Setty, D. Tenza, E. V. Sviderskaya, D. C. Bennett, G. Raposo and M. S. Marks,Nature, 2008, 454, 1142.

30 Y. Xue, A. V. Davis, G. Balakrishnan, J. P. Stasser, B. M. Staehlin, P. Focia, T. G. Spiro,J. E. Penner-Hahn and T. V. OHalloran, Nat. Chem. Biol., 2008, 4, 107.

31 L. Banci, I. Bertini, S. Ciofi-Baffoni, T. Hadjiloi, M. Martinelli and P. Palumaa, Proc.Natl. Acad. Sci. USA, 2008, 105, 6803.



Downloadedb


2011


039/B818285J

View Online


25/28


26/28

69 X. Sheng, J. H. Horner and M. Newcomb, J. Am. Chem. Soc., 2008, 130, 13310.70 S. Shaik, D. Kumar and S. P. de Visser, J. Am. Chem. Soc., 2008, 130, 10128.71 W. Sicking, H.-G. Korth, H. de Groot and R. Sustmann, J. Am. Chem. Soc., 2008, 130,

7345.72 E. T. Yukl, M. A. Elbaz, M. M. Nakano and P. Moenne-Loccoz, Biochemistry, 2008, 47,

13084.73 J. C. Crack, A. A. Gaskell, J. Green, M. R. Cheesman, N. E. Le Brun and A. J. Thomson,

J. Am. Chem. Soc., 2008, 130, 1749.

74 E. C. Raulfs, I. P. OCarroll, S. P. C. Dos, M.-C. Unciuleac and D. R. Dean, Proc. Natl.Acad. Sci. USA, 2008, 105, 8591.

75 S. Watanabe, A. Kita, K. Kobayashi and K. Miki, Proc. Natl. Acad. Sci. USA, 2008, 105,4121.

76 A. M. Taylor, C. E. Farrar and J. T. Jarrett, Biochemistry, 2008, 47, 9309.77 E. G. Kovaleva and J. D. Lipscomb, Nat. Chem. Biol., 2008, 4, 186.78 L. J. Bailey, J. G. McCoy, G. N. Phillips, Jr. and B. G. Fox, Proc. Natl. Acad. Sci. USA,

2008, 105, 19194.79 J. K. Schwartz, P.-p. Wei, K. H. Mitchell, B. G. Fox and E. I. Solomon, J. Am. Chem.

Soc., 2008, 130, 7098.80 M. S. Windahl, C. R. Petersen, H. E. M. Christensen and P. Harris, Biochemistry, 2008,

47, 12087.81 A. Loaiza, K. M. Armstrong, B. M. Baker and M. M. Abu-Omar, Inorg. Chem., 2008, 47,

4877.82 L. M. Mirica and J. P. Klinman, Proc. Natl. Acad. Sci. USA, 2008, 105, 1814.83 A.-F. Miller, Acc. Chem. Res., 2008, 41, 501.84 L. E. Grove, J. Xie, E. Yikilmaz, A. Karapetyan, A.-F. Miller and T. C. Brunold, Inorg.

Chem., 2008, 47, 3993.85 L. E. Grove, J. Xie, E. Yikilmaz, A.-F. Miller and T. C. Brunold, Inorg. Chem., 2008, 47,

3978.86 S. Shima, O. Pilak, S. Vogt, M. Schick, M. S. Stagni, W. Meyer-Klaucke, E. Warkentin,

R. K. Thauer and U. Ermler, Science, 2008, 321, 572.87 M. Hambourger, M. Gervaldo, D. Svedruzic, P. W. King, D. Gust, M. Ghirardi,

A. L. Moore and T. A. Moore, J. Am. Chem. Soc., 2008, 130, 2015.88 G. W. Brudvig, Philos. Trans. R. Soc. London, Sect. B: Biol. Sci., 2008, 363, 1211.

89 H. Popelkova, A.

2009, review,vilar

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