STRUCTURAL AND FUNCTIONAL STUDIES ON TWO MOLYBDOPTERIN AND IRON-SULFUR CONTAINING ENZYMES: TRANSHYDROXYLASE FROM PELOBACTER ACIDIGALLICI AND ACETYLENE HYDRATASE FROM PELOBACTER ACETYLENICUS

ZUR ERLANGUNG DES AKADEMISCHEN GRADES EINES

DOKTORS DER NATURWISSENSCHAFTEN

DEM FACHBEREICH BIOLOGIE, UNIVERSITÄT KONSTANZ VORGELEGTE

DISSERTATION

VON

M. SC. CHEM. GRAŻYNA BERNADETA SEIFFERT

KONSTANZ, MAI 2007

REFERENT: PROF. DR. P.M.H. KRONECK KOREFERENT: PROF. DR. O. EINSLE

Z podziękowaniem dla mojej kochanej mamy oraz Piotrkowi

Table of Contents

I. ZUSAMMENFASSUNG ...... V

I.A. TRANSHYDROXYLASE AUS PELOBACTER ACIDIGALLICI...... VI I.B. ACETYLEN HYDRATASE AUS PELOBACTER ACETYLENICUS ...... VII

II. SUMMARY ...... IX

II.A. TRANSHYDROXYLASE FROM PELOBACTER ACIDIGALLICI...... X

II.B. ACETYLENE HYDRATASE FROM PELOBACTER ACETYLENICUS...... XI

1. INTRODUCTION...... 1

1.1. MOLYBDENUM AND TUNGSTEN IN BIOLOGICAL SYSTEMS ...... 2 1.2. EPR PROPERTIES OF METAL CENTERS IN BIOLOGICAL SYSTEMS...... 8 1.3. ACETYLENE, SUBSTRATE FOR MICROBIAL GROWTH AND METALLOENZYME INHIBITOR ...... 12 1.4. TRANSHYDROXYLASE FROM PELOBACTER ACIDIGALLICI ...... 14 1.5. ACETYLENE HYDRATASE FROM PELOBACTER ACETYLENICUS...... 18 1.6. SCOPE OF THE STUDY...... 18

2. MATERIALS AND METHODS ...... 21

2.1. CHEMICALS...... 22 2.2. ORGANISMS AND CULTIVATION...... 24 2.3. PURIFICATION PROTOCOLS...... 25 2.3.1. Transhydroxylase from Pelobacter acidigallici ...... 25 2.3.2. Acetylene hydratase from Pelobacter acetylenicus...... 26 2.4. ANALYTICAL METHODS ...... 27 2.4.1. Protein...... 27 2.4.2. ICP-MS analysis of metals...... 28 2.5. ENZYMATIC ACTIVITIES ...... 28 2.5.1. Determination of Transhydroxylase activity with HPLC ...... 28

Table of Contents

2.5.2. Photometric determination of Acetylene hydratase activity ...... 29 2.5.3. Experiments under exclusion of dioxygen...... 29 2.6. SPECTROSCOPIC METHODS ...... 30 2.6.1. Electron paramagnetic resonance spectroscopy...... 30 2.6.2. UV-Visible spectroscopy ...... 33 2.6.3. Circular dichroism spectroscopy ...... 33 2.7. PROTEIN CRYSTALLOGRAPHY...... 34 2.7.1. Crystallization conditions...... 34 2.7.2. Data collection and processing...... 35 2.7.3. Graphical representation ...... 36 14 2.8. [ C]-ACETYLENE ASSAY...... 34

3. RESULTS AND DISCUSSION...... 38

3.1. TRANSHYDROXYLASE OF PELOBACTER ACIDIGALLICI ...... 39 3.1.1. Growth of Pelobacter acidigallici ...... 39 3.1.2. Purification of Transhydroxylase...... 40 3.1.3. Spectroscopic characterization of the metal sites ...... 41 3.1.4. HPLC with 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether...... 59 3.2. ACETYLENE HYDRATASE OF PELOBACTER ACETYLENICUS ...... 61 3.2.1. Growth of Pelobacter acetylenicus...... 61 3.2.2. Purification of Acetylene hydratase under exclusion of dioxygen...... 62 3.2.3. Spectroscopic characterization of the metal sites ...... 63 3.2.4. Metal content of Acetylene hydratase...... 70 3.2.5. Reaction with [14C]- acetylene ...... 70 3.2.6. Crystallization of W-AH and three-dimensional structure...... 71 3.2.7. Towards the reaction mechanism of Acetylene hydratase...... 87

4. CONCLUSION...... 92

5. REFERENCES ...... 100

6. ACKNOWLEDGEMENT ...... 109

7. APPENDIX ...... 109

I. Zusammenfassung

Zusammenfassung

In dieser Doktorarbeit wurden zwei neuartige Molybdopterin-abhängige Enzyme aus strikt anaeroben Bakterien untersucht. Sowohl die Pyrogallol-Phloroglucin Transhydroxylase (TH) als auch die Acetylen Hydratase (AH) katalysieren chemisch ungewöhnliche Reaktionen unter dem strikten Ausschluss von Luftsauerstoff. Obwohl es sich bei beiden Reaktionen formal um keine Redoxreaktionen handelt, setzen beide Enzyme komplexe Metallzentren ein für die Katalyse.

I.a. Transhydroxylase aus Pelobacter acidigallici

Das strikt anaerobe Bakterium Pelobacter acidigallici vergärt Gallussäure (3,4,5- Trihydroxybenzoesäure), Pyrogallol (1,2,3-Trihydroxybenzol), Phloroglucin (1,3,5-Tri- hydroxybenzol) oder 2,4,6-Trihydroxybenzoesäure zu drei Molekülen Acetat (plus CO2) [Brune et al., 1990; Schink et al., 1982]. Ein Schlüsselenzym dieses Abbauweges ist die

Pyrogallol-Phloroglucinol Transhydroxylase (TH), welche in Abwesenheit von O2 Pyrogallol zu Phloroglucinol umsetzt – in Anwesenheit von 1,2,3,5-tetrahydroxybenzene als Cosubstrat. Ein möglicher Reaktionsmechanismus wurde von Messerschmidt et al., (2004) auf der Grundlage einer hochaufgelösten Röntgenstruktur vorgeschlagen.

Die funktionelle Einheit der Transhydroxylase von P. acidigallici ist ein Heterodimer bestehend aus einer großen (100.4 kDa) und einer kleinen Untereinheit (31.3 kDa). Dieses Enzym ist nahe verwandt mit Enzymen aus der Familie der DMSO-Reduktasen. Obwohl die von der TH katalysierte Reaktion formal keine echte Redox-Reaktion ist, enthält das Enzym drei [4Fe-4S] -Zentren und ein Mo(MGD)2 als Redox-Kofaktoren.

Die EPR Spektren (Proben eingefroren bei verschiedenen Redoxpotentialen) zeigten einen Satz komplizierter Resonanzen in Abhängigkeit vom eingebauten Molybdän- Isotop, vom pH der Lösung, und von der Anwesenheit von Anionen wie zB. Chlorid. Die EPR Daten der Mo-TH deuteten auf ein Gleichgewicht hin zwischen zwei Mo(V)-

Spezies: Mo(V)-OH2 und Mo(V)-OH. Die Aminosäuren Aspartat und Histidin am aktiven Zentrum scheinen essentiell zu sein für die Katalyse. Inkubationsexperimente mit

VI Zusammenfassung

dem Substrat Pyrogallol und dem möglicher Intermediat 2,4,6,3’,4’,5’- hexahydroxydiphenylether ergaben eine geringe Abnahme des Mo(V) EPR-Signals. Im Falle reduzierter Mo-TH führen diese beiden Verbindungen zu einer partiellen Oxidation der Metallzentren, was durch EPR Spektroskopie gezeigt wurde. Dagegen wurde unter gleichen Bedingungen mit dem Kosubstrat 1,2,3,5-tetrahydroxybenzol eine Abnahme des Mo(V) EPR-Signals und die Reduktion der [4Fe-4S]-Zentren erreicht. Ein möglicher

Elektronen-Transfer-Weg ausgehend von Mo(MGD)2 in der α-Untereinheit via eines Arginin-Restes zum benachbarten [4Fe-4S]-Cluster in der β-Untereinheit wurde vorgeschlagen.

I.b. Acetylen Hydratase aus Pelobacter acetylenicus

Das strikt anaerobe, mesophile δ-Bakterium Pelobacter acetylenicus wandelt den ungesättigten Kohlenwasserstoff Ethin (Trivialname: Acetylen) zu Acetat und Ethanol um, mit Acetaldehyd als Zwischenprodukt [Abt, 2001; Kisker et al., 1998; Schink, 1985]. Der erste Schritt dieses Gärungsweges ist die Anlagerung von Wasser an die Dreifachbindung von Acetylen, die von der Acetylen Hydratase (AH) katalysiert wird [Rosner et al., 1995].

AH aus P. acetylenicus wurde bis zur Homogenität gereinigt. Die funktionelle Einheit ist das Monomer mit einer molekularen Masse der Aminosäurenkette von 81.9 kDa. Sequenz- und Strukturvergleiche ordnen das Enzym der Familie der DMSO-Reduktasen zu. Es enthält ein W(MGD)2-Zentrum und ein [4Fe-4S]-Cluster vom Cubane-Typ. Wolfram kann durch Molybdän ersetzt werden, die entsprechende Mo-AH zeigt eine signifikant geringere Aktivität. ICP/MS, EPR und UV/VIS-Spektroskopie zeigen, dass P. acetylenicus sowohl Wolfram als auch Molybdän in das Aktivzentrum der AH einbauen kann. Die W-AH sticht unter den andere Mitgliedern der DMSO Familie hervor, da sie mit der Hydratisierung von Acetylen zu Acetaldehyd keine Redox- Reaktion katalysiert. Dabei ist zu beachten, dass das Enzym für die Katalyse durch Natriumdithionit oder Ti(III)-Zitrat reduktiv aktiviert werden muss.

VII Zusammenfassung

EPR-spektroskopische Untersuchungen Dithionit-reduzierter W-AH und Mo-AH zeigen die charakteristischen Resonanzen eines [4Fe-4S]-Zentrums, mit gav = 1.966 ± 0.001.

Die mit K3[Fe(CN)6] oxidierte W-AH zeigt das EPR Signal eines W(V)-Zentrums, mit gav = 2.02. Im gegensatz dazu zeigt die Mo-AH („wie isoliert“) Resonanzen eines

Mo(V)-Zentrums, mit gav = 1.99.

W-AH wurde unter striktem Ausschluß von Luftsauerstoff in seiner aktiven, reduzierten Form kristallisiert. Die Kristallstruktur wurde mit einer Auflösung von 1.26 Å erarbeitet.

Das W(MGD)2-Zentrum bindet neben einem Cystein-Schwefel einen Sauerstoff- Liganden – Wasser oder eine Hydroxygruppe – der mit dem benachbarten Aspartat 13 wechselwirkt. Aufbauend auf diesen strukturellen und spektroskopischen Ergebnissen wurde ein erster möglicher Reaktionsmechanismus für die Anlagerung von Wasser an die Acetylen-Dreifachbindung unter Ausschluß von Luftsauerstoff formuliert. Acetylen wird in eine hydrophobe Tasche des Enzyms modelliert, direkt über einem Wasssermolekül, welches an das W(MGD)2-Zentrum gebunden ist. Damit das Substrat Zugang zu diesem neuartigen Aktivzentrum erhalten kann, entwickelt das Protein einen speziellen Substratkanal, weit entfernt von der Stelle, an der dieser normalerweise in anderen Molybdän- und Wolfram-haltigen Enzymen der DMSO Reduktase Familie gefunden wird.

VIII

II. Summary

Summary

In this doctoral thesis, two novel molybdopterin-dependent enzymes from anaerobic bacteria have been investigated. Both pyrogallol–phloroglucinol transhydroxylase (TH) and acetylene hydratase (AH) catalyze rather unusual reactions in the absence of dioxygen. Despite the presence of highly sophisticated metal centers in their active sites, these reactions are formally reactions without a net transfer of electrons.

II.a. Transhydroxylase from Pelobacter acidigallici

The strictly anaerobic bacterium Pelobacter acidigallici ferments gallic acid (3,4,5- trihydroxybenzoic acid), pyrogallol (1,2,3-trihydroxybenzene), phloroglucinol (1,3,5- trihydroxy-benzene), or 2,4,6-trihydroxybenzoic acid to three molecules of acetate (plus

CO2) [Brune et al., 1990; Schink et al., 1982]. A key enzyme in the fermentation pathway is pyrogallol–phloroglucinol transhydroxylase (TH), which converts pyrogallol to phloroglucinol in the absence of O2, in the presence of 1,2,3,5-tetrahydroxybenzene as a cosubstrate. The proposed reaction scheme was described by Messerschmidt et al., (2004) based on a high resolution structure of TH.

Transhydroxylase from P. acidigallici is a heterodimer consisting of a large subunit (100.4 kDa) and a small subunit (31.3 kDa). This enzyme is closely related to enzymes of the DMSO-reductase family. Although the overall reaction of transhydroxylase is no redox reaction it contains three [4Fe-4S] centers and one Mo(MGD)2 as redox-cofactors.

The EPR spectra of the Mo(V) site (samples frozen at different redox potentials) revealed a very complex pattern of resonances dependent on the molybdenum isotope inserted, the pH of the solution, and the presence of anions such as chloride. The EPR study of the

Mo(V) center suggested the presence of two Mo(V) species in equilibrium, Mo(V)-OH2 and Mo(V)-OH, with at 1.996, 1.982, 1.963. The amino acids aspartate and histidine near the active site seem to be essentiell for catalysis. Incubation experiments of Mo-TH (as isolated and reduced by dithionite) with pyrogallol, or the putative intermediate 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether, showed a rather small decrease in intensity of

X Summary

the Mo(V) EPR signal; in the case of reduced Mo-TH, these compounds caused partial oxidation of the metal sites. Surprisingly, the co-substrate 1,2,3,5-tetrahydroxybenzene led to a decrease of the Mo(V) EPR signal, and to a reduction of the Fe-S centers as shown by EPR spectroscopy. A putative electron transfer pathway from the Mo(MGD)2 unit on the α-subunit of TH via an arginine residue to the close by [4Fe-4S] on the β- subunit has been assigned.

II.b. Acetylene hydratase from Pelobacter acetylenicus

The strictly anaerobic, mesophilic, δ-bacterium Pelobacter acetylenicus converts the unsaturated hydrocarbon ethine (trivial name, acetylene) to acetate and ethanol via acetaldehyde as an intermediate [Abt, 2001; Kisker et al., 1998; Schink, 1985]. The first step of the fermentation pathway, the hydration of acetylene to acetaldehyde, is catalyzed by the enzyme acetylene hydratase [Rosner et al., 1995].

Acetylene hydratase (AH) from P. acetylenicus was purified to homogeneity. It is a monomer with a molecular mass of the amino acid chain of 81.9 kDa. Sequence and structure comparisons group the protein into the DMSO-reductase family. It contains a

W(MGD)2 center and a cubane-type [4Fe-4S] cluster. Tungsten can be replaced by molybdenum, the corresponding Mo-AH is quite less active. ICP/MS, EPR, and UV/Vis- spectroscopy revealed that P. acetylenicus is able to insert tungsten as well as molybdenum into the bisMGD cofactor of acetylene hydratase. W-AH stands out from its family class because it catalyzes a non-redox reaction, the hydration of acetylene to acetaldehyde. Note that the enzyme requires reduction by either Na-dithionite or Ti(III) citrate.

EPR spectroscopic investigation of dithionite-reduced W-AH and Mo-AH showed signals of a [4Fe-4S] center, with gav = 1.966 ± 0.001. W-AH oxidized by K3[Fe(CN)6] exhibited resonances of a W(V) center, with gav = 2.02. The “as isolated” Mo-AH showed resonances of a Mo(V) center, with gav = 1.99.

XI Summary

W-AH has been crystallized under the strict exclusion of dioxygen in its active, reduced state. The crystal structure of W-AH was determined at 1.26 Å resolution. The structure showed that the tungsten center binds a water molecule (or OH-group) which can closely interact with a nearby aspartate residue. These structural and spectroscopic results led to the proposal of a reaction mechanism. Hereby, the acetylene molecule is placed into hydrophobic pocket, directly above a water/OH molecule coordinated to tungsten. Two different reactions mmight then take place: electrophilic addition leading to vinyl cation as intermediate or nucleophilic attack yielded vinyl anion with acetylene. Access to this novel W–Asp active site has been made possible by a substrate channel at a position distant from those found in other molybdenum and tungsten enzymes.

XII

1. Introduction

Introduction

1.1. Molybdenum and tungsten in biological systems

1.1.1. Chemistry of molybdenum and tungsten Molybdenum and tungsten belong to the transition metals in Group 6 of the Periodic Table. The atomic and ionic radii of W and Mo, as well as their electron affinity, are virtually identical, and both occur naturally as a mixture of isotopes. Radioactive isotopes are available for both elements (185W and 99Mo), as well as there are stable 183W and 95Mo isotopes with a nuclear spin suitable for the study of hyperfine interactions by magnetic resonance techniques. The high solubility of Mo(VI) and W(VI) oxyanions, 10- 7 and 10-9 M, respectively, in sea water, ensures that both metals are bioavailable [Young, 1997].The difference between both metals can be clearly seen under oxic vs. anoxic 2- conditions. Generally, in the presence of dioxygen, molybdate (MoO4 ) and tungstate 2- (WO4 ) are the principal forms, and these compete with each other for binding sites in proteins and uptake systems. In the absence of dioxygen, in sulfur rich environments, molybdenum can exist in the Mo(V) or Mo(IV) state, usually coordinated to sulfide, such as in MoS2, or to organosulfur ligands, such as the dithiolene moiety of molybdopterin [Sigel et al., 2002]. The organometallic chemistry of Mo and W, respectively, will be described in a separate paragraph when discussing its coordination in metalloenzymes.

1.1.2. Beneficial and toxic effects of molybdenum and tungsten in higher organisms Molybdenum is an essential trace element for all higher organisms where molybdenum enzymes have a number of important roles. Plants for example require Mo in nitrate reductase for proper nitrogen assimilation. In humans Mo is coordinated in the active site of enzymes like xanthine oxidase, sulfite oxidase or aldehyde oxidase, which are involved in diseases including gout, combined oxidase deficiency, and radical damage following cardiac failure. Combined oxidase deficiency is a rare genetic disease responsible for severe neurological disorders observed in infant children. There also exists a link between one of the many proteins required to synthesize the molybdopterin cofactor of these enzymes, and a protein used in neuronal synapses [Burgmayer, ;

2 Introduction

Schwarz et al., 2004]. This illustrates how the biological impact of the molybdenum enzymes may reach beyond the limited context of their unique catalytic function. The impact of tungsten on humans has been studied in the case of workers chronically exposed to hard-metal dust. Tungsten had been shown to accelerate the development of mammary cancer in rats. This was attributed to a decrease in hepatic molybdenum, which severely disturbs the function of the liver, the site of estrogen metabolism [Sigel et al., 2002].

1.1.3. Molybdenum and tungsten in the microbial world According to one possible theory, early life on this planet arose in a hot and reducing environment, with dioxygen being more or less absent [Hille, 2002]. Most likely, transitions metals, such as nickel and tungsten, and sulfur compounds played an important role in building efficient early life catalysts [Kroneck, 2005]. Later, when the concentration of dioxygen increased in the atmosphere, tungsten may have been replaced by molybdenum in enzymes. This hypothesis is supported by the finding that molybdenum enzymes are found in all forms of life in opposite to tungsten enzymes which have been only discovered in anaerobic mesophiles or thermophiles [Hille, 2002]. Depending on the growth conditions, some bacteria can exchange molybdenum by tungsten, which has been described for several enzymes, especially dimethyl sulfoxide reductase [Sigel et al., 2002]. In the case of the tungsten enzyme acetylene hydratase, molybdenum could be inserted, and the corresponding Mo acetylene hydratase was active [Rosner et al., 1995].

The question why some enzymes can function with either metal ion while others seem to be exclusive for one is far from being elucidated. Possible explanations are linked to (i) the redox properties of the enzyme under investigation, and (ii) to the bioavailability of tungsten vs molybdenum. (i) Which metal will be chosen by a microorganism can be determined by the relationship of temperature and redox potential of the metal-sulfur site. The molybdopterin-dependent enzymes show a remarkably small difference of 30

mV at 25 °C for the redox potential E1/2 M(IV)/M(V). The oxidation process M(V)/M(VI) indicated that redox potentials for the tungsten compounds are

3 Introduction

(at 25°C) more positive than the potentials for the molybdenum compound but o the difference is extraordinarily small. At 70 C, is it appears that E1/2(Mo) >

E1/2(W), consequently tungsten is prefer, entially inserted [Schulzke, 2005].

(ii) Concerning bioavailability and insertion of molybdenum or tungsten, several interesting observations have been made. In DMSO reductase from Rhodobacter capsulatus, and TMAO reductase from Escherichia coli, the metal which was of higher concentration in the in the growth medium became incorporated. Similarly, Desulfovibrio alaskensis could insert both molybdenum and tungsten into formate dehydrogenase (Fdh) depending on the concentration in the medium. In the medium where Desulfovibrio alaskensis produced either Mo- or W-FDH, existed other enzymes contained only Mo (aldehyde oxidoreductase) [Andrade et al., 2000]. In contrast, Fdh from both Desulfovibrio gigas and Syntrophobacter fumaroxidans incorporated only tungsten, despite the presence of a suitable concentration of molybdenum in the medium [Moura et al., 2004]. The same relation was obtained for Pyrococcus furiosus containing typical tungsten enzymes (AOR family). The bacterium was grown on medium containing also Mo in an attempt to replace the tungsten, however, in the enzymes only tungsten ion was determined [Sigel et al., 2002].

These experimental findings suggest that bioavailability is not the only parameter which governs the choice of the metal to be incorporated into the active site. Obviously, some enzymes are very selective for either tungsten or molybdenum, whereas other enzymes can function only with a specific ion [Moura et al., 2004].

1.1.4. Molybdenum and tungsten enzymes: classification and general properties Despite the high similarity between the chemical properties of Mo and W, the enzymes containing tungsten represent only a small percentage compared to the molybdenum enzymes [Johnson et al., 1996].

4 Introduction

Mononuclear centers of molybdenum and tungsten are found in the active sites of a diverse group of enzymes that are involved in important reactions of the biogeochemical cycles of carbon, nitrogen, and sulfur. The metals themselves are catalytically inactive unless they are complexed by a complex cofactor.

Figure 1: The structure of Molybdopterin = Moco cofactor. (A) In prokaryotes, molybdopterin cofactors normally contain a nucleoside bonded via two phosphates. A nucleoside consists of a ribose and one of the four nitrogenous bases shown here. A nucleotide is a nucleoside which is bonded to one or more phosphates; (B) Two molybdopterin guanine dinucleotide (bis-MGD) cofactors coordinated to a molybdenum or tungsten atom, as found in enzymes that belong to DMSO reductase family.

With the exception of nitrogenase, where Mo is a constituent of the so-called FeMo- cofactor, molybdenum or tungsten, is bound to a pterin, thus forming the molybdenum cofactor (Moco, Fig. 1) which is the active compound at the catalytic site of all molybdenum- and tungsten-enzymes [Brondino et al., 2006].

Molybdopterin cofactors (termed either Moco or pyranopterin) are found in several enzymes of both prokaryotes and eukaryotes [Hille, 1996]. Molybdopterin cofactors from 5 Introduction

eukaryotes normally contain only one pterin ring, whereas prokaryotic molybdopterin cofactors may contain either one or two pterin rings and a nucleotide (Fig. 1 A) covalently linked to the pterin moiety. The nucleotides can contain one of four different bases: guanine, adenine, cytosine and uracil. In same bacterial enzymes was found the molybdopterin-guanine-dinucleotide (MGD) as a cofactor, in which each pterin molecule has a guanine nucleotide. One or two MGD molecules can complex the molybdenum or tungsten atom (Fig. 1 B) [Hille, 2002; Moura et al., 2004]. The effect of the nucleotide on the chemical properties of the molybdopterin cofactor is not known. However, it is assumed that molybdopterin cofactors of similar structure have similar chemical properties.

In addition, molybdenum and tungsten proteins may also carry other redox cofactors, such as iron-sulfur centers, hemes, or flavins, which are involved in intra- and intermolecular electron - transfer processes [Mendel et al., 2006]. Based on structural and genomic data, tungsten and molybdenum enzymes have been divided into four families: sulfite oxidase, xanthine oxidase, dimethyl sulfoxide (DMSO) reductase and aldehyde / ferredoxin oxidoreductase [Hille, 2002; Moura et al., 2004] (Fig. 2).

1.1.5. Catalytic center of molybdenum and tungsten enzymes In the dimethyl sulfoxide (DMSO) reductase family, both Mo-containing and W- containing enzymes are found (Fig. 2). In general, the metal is coordinated by the dithiolene sulfur of Moco, and by a variable number of oxygen (oxo, hydroxo, water, serine, aspartic acid), sulfur (cysteine), and selenium ligands (seleno-cysteine). The proteins of the DMSO reductase family exhibit a wide diversity of properties among its members. The crystal structure of DMSO reductase from Rhodobacter capsulatus was the first structure reported for a member of this family [Bailey et al., 1996; McAlpine et al., 1998]. Within the family nitrate reductase (NAR) and formate dehydrogenase (FDH) have been studied most, so far. In this work I will focus on acetylene hydratase (AH) and pyrogallol-phloroglucinol transhydroxylase (TH) which also belong to the DMSO reductase family.

6 Introduction

Molybdenum Tungsten Enzymes Enzymes

Figure 2: Active site structure of the four families of Mo- and W-containing enzymes based on three- dimensional structure. Active site of enzymes belonging to DMSO reductase family are exemplified by (A) molybdenum-containing enzymes, where different types of ligands identified as X: O-Ser by DMSO reductase, transhydroxylase; Se-Cys by formate dehydrogenase; S-Cys by periplasmic nitrate reductase; O- Asp respiratory nitrate reductase; (B) tungsten-containing enzymes including acetylene hydratase (S-Cys ligand) and formate dehydrogenase (Se-Cys ligand) [Hille, 2002; Moura et al., 2004].

1.1.6. Properties of molybdenum and tungsten dithiolene complexes In dithiolene complexes of both tungsten and molybdenum, the interaction between metal and sulfur is very similar. The majority of the oxo-metal-bis(dithiolene) complexes that have been investigated by vibrational spectroscopy, exhibit octahedral geometry in the Mo(VI)/W(VI) state, and square-pyramidal geometry in the Mo(V, IV)/W(V, IV) state. The X-ray structure of FDH from E. coli reveals two MGD coordinated to molybdenum. In the reduced Mo(IV) form of the enzyme the metal carries in the fifth position ligand SH or OH in a distorted square pyramidal geometry, in the oxidized Mo(VI) form the metal binds a seleno-cystein as the sixth ligand, and the geometry changes to distorted

7 Introduction

trigonal bipyramidal [Boyington et al., 1997; Raaijmakers et al., 2006]. Such geometrical differences between the reduced and oxidized states have been also observed in biomimetic complexes for the active site of DMSO reductase which showed a tetragonal pyramidal geometry (coordination number 5) or a trigonal prismatic geometry (coordination number 6) for the Mo(IV) state, compared to a distorted octahedral geometry (coordination number 6) for the Mo(VI) state [Hofmann, in press]. In the case of tungsten enzymes, the active site geometry does not vary significantly, usually a distorted pseudo-trigonal prismatic geometry is found [Stiefel, 2002].

1.2. EPR properties of metal centers in biological systems

1.2.1 Molybdenum The Mo(V) state is paramagnetic, with one unpaired electron. The observed EPR signals are not as sensitive towards changes in temperature compared to the spectra of [4Fe-4S] centers. Nevertheless, the line shapes of Mo(V) EPR signals are complex, usually they are highly anisotropic and show hyperfine structure. Both isotopes 95Mo and 97Mo (natural abundance 25%, nuclear spin I=5/2) give sextets of lines of relatively low amplitude compared to the intense center line resulting from isotopes (92,94,96,98,100Mo), with I = 0. Analysis and assignment of the individual lines in the EPR spectrum will be facilitated by substituting the enzyme with either pure 95Mo,97Mo, or 98Mo. In addition, further lines can be assigned to superhyperfine interactions resulting from coordinated nitrogens and protons at the active site, shown for example in Figure 3. In the latter case, an exchange of the protons by deuterons might help the assignment. Pioneering studies have been performed by R.C. Bray on the enzyme xanthine oxidase [Bray et al., 1966].

8 Introduction

Figure 3: EPR spectra of formate-treated FDH from E. coli obtained at 130 K, (a) The spectrum is a superposition of Mo(V) signal with g-factors of 2.094 and 2.0 (b) The same spectrum as in (a) at higher gain. The gz, gy, and gx components of Mo(V) species with I = 0 are marked by dashed lines. The hyperfine 5 features from Mo(V) isotopes with I = /2 are marked by short sticks. The sample has been frozen after incubation of FDH with 20 mM formate for 10 s [Gladyshev et al., 1996; Khangulov et al., 1998].

1.2.2. [4Fe-4S] centers The first application of electron paramagnetic resonance spectroscopy (EPR) to iron- sulfur proteins was reported by Orme-Johnson et al., (1968). The geometric structure of a generic [4Fe-4S] cluster in a protein environment is a distorted cube, which can be

9 Introduction

depicted as a tetrahedron of four iron atoms interpenetrating a larger tetrahedron of four sulfur atoms (Fig. 4).

Figure 4: The oxidized and reduced state of a [4Fe-4S] cluster presenting the mixed and equal-valence iron atom pairs. The iron and sulfide atoms of the [4Fe-4S] clusters as well as sulfur and Cβ atoms of the cysteines liganting the [4Fe-4S] cluster, are shown with iron atoms as large black spheres, sulfur atoms as light-gray spheres and Cβ as small black spheres [Vassiliev et al., 2001].

The atom-to-atom distances range from 2.67 to 2.81 Å for the iron tetrahedron, and 3.49 to 3.61 Å for the sulfur tetrahedron in different [4Fe-4S] proteins [Vassiliev et al., 2001]. Most commonly, the iron atoms are coordinated to cysteine SH groups which can be detected in the amino acid sequence by the characteristic motif Cys-X2-Cys-X3-Cys [Bruschi et al., 1988]. [4Fe-4S] clusters are diamagnetic in the oxidized state, and paramagnetic in the reduced state. In the oxidized state, the two Fe(III) and two Fe(II) atoms are magnetically coupled, giving rise to an effective total spin of S=0. Any paramagnetism of oxidized iron–sulfur centers at room temperature is due to a population of excited paramagnetic states. In the reduced state, one Fe(III) and three Fe(II) atoms are magnetically coupled, giving rise to an effective total spin of S=1/2. Although in the reduced state a [4Fe-4S]+ cluster may be considered to contain the formal valances of three Fe(II) atoms and one Fe(III) atom, two localized Fe pairs exist: one equal-valence pair (Fe2+- Fe2+), and one mixed-valence pair (Fe2.5+- Fe2.5+) in which the electron is delocalized over the two iron sites. Since EPR signals of [4Fe-4S] clusters have very short relaxation times, they usually can only be detected at temperatures below 30 K. The samples must be frozen and, except in single crystals, the molecules will be randomly oriented. As a result, the

10 Introduction

spectra represent a summation of signals from redox centers oriented in all directions relative to the magnetic field, and are termed ‘polycrystalline powder spectra’. The EPR spectrum of a [4Fe-4S] cluster typically shows three distinct g-values reflecting ‘rhombic’ symmetry. For example the typical rhombic [4Fe–4S]+ signal was obtained by FDH from E. coli (Fig. 5).

A B

Figure 5: EPR spectrum of formate-treated FDH from E. coli obtained at 42 K, (A) The spectrum is a superposition of [4Fe-4S]+ signal with g-factors of 1.840, 1.957, and 2.045, and the Mo(V) signal with g- factors of 2.094 and 2.0; (B) EPR signal of [4Fe-4S] clusters obtained after subtraction of the Mo(V) "2.094" signal from spectrum A; FDH was incubated with 10 mM formate for 1 min [Gladyshev et al., 1996; Khangulov et al., 1998].

Each g-value, gx, gy, and gz, corresponds to one of the value obtained when the magnetic field is parallel to one of the three special directions of the paramagnetic molecule. The principal g-values obtained from powder spectra are therefore approximately equal to the single crystal’s principal g-values. The relationship between the crystal axis and the g- tensor axes is not well understood [Vassiliev et al., 2001].

11 Introduction

1.3. Acetylene, substrate for microbial growth and metalloenzyme inhibitor

Acetylene (C2H2) is a highly flammable gas that forms an explosive mixture with air, and polymerizes exothermically. The carbon-carbon triple bond involves one σ-bond and two orthogonal π-bonds (Fig. 6).

1st π bond in x, y plane 2st π bond in x, z plane Cylindrically symmetrical set of π electrons Figure 6: Molecular orbitals of acetylene

The hydrogen atoms of alkynes are relatively acidic compared to hydrogen atoms of ethylene or ethane. Acetylene itself has a pKa of about 25. Acetylene has a rather rich chemistry. There exist a number of different reactions, such as reductions as well as electrophilic and nucleophilic additions [Yurkanis-Bruice, 2004]. The role of acetylene as an inhibitor of important microbial metabolic processes is well known [Hyman et al., 1988]. The close association between acetylene and the nitrogen cycle can be traced back to the original description of C2H2 gas as an inhibitor and an alternative substrate of nitrogenase. In the list of prebiotic molecules observed in the interstellar gas, we also find acetylene and higher alkynes [Thaddeus, 2006]. Some interesting information came recently from research on the moon Titan which appears to have environmental conditions similar to those on Earth some 4 billion years ago. This is the time when life most likely originated on Earth. Life may have originated on Titan during its warmer early history and then developed adaptation strategies to cope with the increasingly cold conditions. If organisms originated and persisted, metabolic strategies could exist that would provide sufficient energy for life to persist, even today. Metabolic reactions might include the

12 Introduction

catalytic hydrogenation of photochemically produced acetylene, or involve the recombination of radicals created in the atmosphere by ultraviolet radiation (Fig. 7) [Schulze-Makuch et al., 2005].

Figure 7: Proposed environmental conditions at Titan, schematic. Acetylene and radicals are produced by photochemical reactions in the atmosphere. Due to its high specific gravity acetylene will sink to Titan’s surface and to the bottom of a hydrocarbon reservoir, where it can be used by putative organisms for metabolic reactions (insert). The metabolic end-product methane rises to the atmosphere and is detected to be isotopically lighter than predicted by Titan formation theories [Schulze-Makuch et al., 2005].

The first report on the utilization of acetylene gas (C2H2) by a bacterium was published 75 years ago [Birch-Hirschfeld, 1932]. Later, Norcadia rhodochrous was described to use C2H2 as its sole source of carbon and energy [Kanner et al., 1979]. To date, the major source of atmospheric C2H2 appears to result from automobile exhaust, although, natural sources can not be completely excluded [Whitby et al., 1978]. The solubility of C2H2 gas in water is rather high (1.03 g/l) when compared to other substrate gases, consequently, lowest atmospheric concentrations may allow microbial growth. De Bont and Peck

(1980) reported on the metabolism of C2H2 and methylacetylene by Rhodococcus A1, and mentioned the occurance of the enzyme acetylene hydratase at high levels in cell-free extracts. The enzyme was inhibited by dioxygen but not the product acetaldehyde.

13 Introduction

Yamada and Jakoby (1958, 1959) described the enzymatic utilization of acetylenedi- and monocarboxylic acid, with addition of H2O to the C≡C bond. Later, pure cultures of acetylene-fermenting anaerobes were obtained by enrichment with acetylene from freshwater and marine sources [Schink, 1985].

1.4. Transhydroxylase from Pelobacter acidigallici

Pelobacter acidigallici strain MaGal2 (DSM 2377) is a strictly anaerobic, chemoorganothroph, and gram-negative bacterium that ferments gallic acid, pyrogallol, phloroglucinol, and 2,4,6,-trihydroxybenzoic acid to three molecules of acetate [Hille, 1999; Schink et al., 1982]. It was isolated from black, anaerobic marine mud of Rio Marin, a channel about 2.5 m wide and 70 cm deep, located in the city of Venice, Italy. The cells are rod-shaped with 0.5 – 0.8 x 1.5 – 3.5 µm in size. The DNA base ratio is 51.8% ± 0.4 mol% G + C [Schink et al., 1982]. Pelobacter acidigallici belongs to the group of fermenting bacteria like Eubacterium oxidoreducens, and Pelobacter massiliensis. These bacteria anaerobically degrade trihydroxybenzenes and their carboxylated derivatives, via the intermediate phloroglucinol, which is subsequently reduced and cleaved hydrolytically [Reichenbecher et al., 1999]. A crucial step in the fermentation of pyrogallol is the transhydroxylation of pyrogallol to phloroglucinol [Brune et al., 1992]. This reaction is catalyzed by the Mo/Fe-S dependent enzyme pyrogallol:phloroglucinol hydroxyltransferase (transhydroxylase, TH). Conversion of pyrogallol to phloroglucinol was studied with the molybdenum enzyme transhydroxylase isolated from Pelobacter acidigallici [Baas et al., 1999; Reichenbecher et al., 1994; Reichenbecher et al., 1996]. 18 Transhydroxylation experiments in H2 O revealed that none of the hydroxyl groups of phloroglucinol was derived from water, confirming the concept that this enzyme transfers a hydroxyl group from the cosubstrate 1,2,3,5-tetrahydroxybenzene (tetrahydroxybenzene) to the acceptor pyrogallol, and simultaneously regenerates the cosubstrate [Reichenbecher et al., 1999].

14 Introduction

Pyrogallol * OH

HO OH HO OH OH OH

TRANSHYDROXYLASE OH OH

HO OH HO OH * OH 1,2,3,5-Tetrahydroxybenzene Phloroglucinol

Figure 8: Proposed reaction mechanism of transhydroxylase from P. acidigallici [Brune et al., 1992; Reichenbecher et al., 1999].

This concept requires a reaction which synthesizes the cofactor de novo to maintain a sufficiently high intracellular pool during growth. Some sulfoxides and aromatic N- oxides were found to act as hydroxyl donors to convert pyrogallol to tetrahydroxybenzene. Again, water was not the source of the added hydroxyl groups; the oxides reacted as cosubstrates in a transhydroxylation reaction rather than as true oxidants in a net hydroxylation reaction. No oxidizing agent was found that supported a formation of tetrahydroxybenzene via a net hydroxylation of pyrogallol. However, conversion of pyrogallol to phloroglucinol in the absence of tetrahydroxybenzene was achieved if little pyrogallol and a high amount of enzyme preparation was used which had been pre-exposed to air. Obviously, the enzyme was oxidized by air to form sufficient amounts of tetrahydroxybenzene from pyrogallol to start the reaction. A reaction mechanism has been proposed which combines an oxidative hydroxylation with a reductive dehydroxylation via the molybdenum cofactor, and allows the transfer of a hydroxyl group between tetrahydroxybenzene and pyrogallol without involvement of water. With this, the transhydroxylase differs basically from all other hydroxylating molybdenum enzymes which all use water as hydroxyl source [Reichenbecher et al., 1999]. Transhydroxylase is a heterodimer consisting of a large subunit (100.4 kDa) and a small subunit (31.3 kDa). This enzyme is closely related to enzymes of the DMSO-reductase family. Although the overall reaction of transhydroxylase is no redox reaction it contains three [4Fe-4S] (β-subunit) centers and one Mo-bisMGD as redox-cofactors. It contains

15 Introduction

11.56 ± 1.72 Fe, 0.96 ± 0.21 Mo (atomic absorption spectroscopy), and 13.13 ± 1.68 acid labile sulfur per heterodimer [Baas et al., 1999; Reichenbecher et al., 1994; Reichenbecher et al., 1996]. The isoelectric point is 4.1, the specific activity of transhydroxylase is highest at pH 7.0, and the temperature optimum is between 53 and 58°C [Reichenbecher et al., 1994]. The crystal structure of the reduced enzyme was published by Messerschmidt et al., 2004. It represented the largest structure (1149 amino acid residues per molecule, 12 independent molecules per unit cell), which had been solved so far by the single anomalous diffraction technique (SAD). The crystals were analyzed with synchrotron radiation and the three-dimensional structure of TH could be solved at 2.5 Å resolution. The role of the 3 [4Fe-4S] clusters in the ß-subunit remains unclear at this point. The distance between the closest [4Fe-4S] cluster and the molybdenum with 23.4 Å appears too far for an efficient electron transfer.

Mo-bisMGD

α-subunit

β-subunit

[4Fe-4S]2+/+ ferredoxin cluster

Figure 9: Metal centers in transhydroxylase from P. acidigallici [Messerschmidt et al., 2004].

16 Introduction

In addition, crystal structures of TH in complex with acetate, pyrogallol, and the inhibitor 1,2,4-trihydroxybenzene were successfully solved at a resolution of 2.35 Å, 2.20 Å, and 2.00 Å. In the structure of the complex of TH with its substrate, pyrogallol bound with the O1 oxygen to the Mo centre (Fig. 10). Additionally, pyrogallol was ligated by Asp174 and Arg153. The side chain of Tyr560 can adopt two different conformations and is in the open conformation for cosubstrate, with the substrate pyrogallol bound to the Mo active site. The OH group of Tyr404 and the SH group of Cys557 are in hydrogen bonding distances to pyrogallol and probably play important roles during catalysis. The 3D structure of transhydroxylase supports the participation of a cosubstrate and led to a new possible reaction mechanism [Messerschmidt et al., 2004; Niessen, 2004].

Figure 10: The binding site of pyrogallol in the active site of TH with open conformation of Tyr 560 in channel (pdb VLE).

17 Introduction

1.5. Acetylene hydratase from Pelobacter acetylenicus

Pelobacter acetylenicus is a strictly anaerobic and mesophilic bacterium that is able to grow on acetylene as single energy and carbon source. The first step in the metabolization of acetylene is the transformation of acetylene to acetaldehyde [Schink, 1985]. Growth of P. acetylenicus with acetylene and specific acetylene hydratase activity depended on tungstate or, to a lower degree, on the supply of molybdate in the medium. The specific enzyme activity in the cell extracts was highest after growth in the presence of tungstate [Abt, 2001; ten Brink, 2006]. To date, acetylene is the only hydrocarbon known to be metabolized in the absence and presence of dioxygen in the same manner [Schink, 1985]. The novel tungsten-[4Fe-4S]- enzyme acetylene hydratase (W-AH) is the first enzyme involved in fermentative conversion of C2H2 to acetate and ethanol by the strict anaerobe Pelobacter acetylenicus.

W-AH converts C2H2 to acetaldehyde, a reaction distinct from the reduction of acetylene to ethylene by nitrogenase:

HC≡CH + H2O → [H2C=C(OH)H] → CH3CHO W-AH belongs to the superfamily of molybdopterin-dependent enzymes. The addition of one H2O molecule to the C≡C bond - formally a non-redox reaction - requires a strong reductant and the presence of chemically complex metal centers. When P. acetylenicus grew at elevated levels of molybdate, the less active variant Mo-AH could be isolated [Abt, 2001; ten Brink, 2006]. AH is highly specific for acetylene. AH from P. acetylenicus has been purified to homogeneity [Meckenstock et al., 1999; Rosner et al., 1995]. It is a monomer with a molecular mass of 81.9 kDa (amino acid sequence) compared to 73 kDa by SDS-PAGE. BLASTP searches revealed that the enzyme is highly similar to enzymes of the DMSO-reductase family. W-AH had an isoelectric point at pH 4.2. Per mol of enzyme, 4.8 mol of iron, 3.9 mol of acid-labile sulfur, and 0.4 mol of tungsten but no molybdenum, were detected. The Km for acetylene was 14 µM as assayed in a coupled photometric test with yeast alcohol -1 -1 dehydrogenase and NADH, and the Vmax was 69 µmol ⋅ min ⋅ mg of protein [Rosner et al., 1995]. The optimum temperature for specific activity of the AH(W) was 50°C , and the apparent pH optimum was 6.0 to 6.5 [Meckenstock et al., 1999]. The N-terminus of

AH shows a typical binding motif for a iron-sulfur cluster of the type Cys-X2-Cys-X3- Cys [Rosner et al., 1995]. According to spectroscopic data the midpoint redox potential 18 Introduction

of the [4Fe-4S] cluster was determined at -410 ± 20mV. Redox titrations gave a midpoint redox potential of – 410mV for the [4Fe-4S] cluster, and –340 mV for 50% maximum activity. Setting the potential to ≤ –410 mV brought the iron sulfur center to the [4Fe- 4S]+ state but did not change the activity of the enzyme [Meckenstock et al., 1999]. Model studies demonstrated the likely participation of a W(IV) site in the catalysis of the hydration of acetylene, whereas the corresponding W(VI) remained inactive (Fig. 11), [Yadav et al., 1997].

2- 2- NC S O S CN NC S O S CN Na S O W VI 2 2 4 W IV

O NC S S CN NC S S CN

oxidized reduced

VI Figure 11: Reduction of [Et4N]2[W O2(mnt)2], where mnt = 1,2-dicyanoethylenedithiolate [Yadav et al., 1997]

Acetylene hydratase is rather oxygen-sensitive; when purified under air the [4Fe-4S] cluster becomes degraded to a [3Fe-4S] cluster as demonstrated by EPR spectroscopy [Meckenstock et al., 1999].

1.6. Scope of the study

The work described in this thesis has been designed to obtain detailed biochemical, spectroscopic and structural information on the two novel molybdopterin-dependent enzymes acetylene hydratase and transhydroxylase. The data obtained will help to get a deeper insight into the unusual chemistry of both enzymes and their mode of catalytic action. Acetylene hydratase and transhydroxylase catalyze two unusual non-redox reactions, although both enzymes contain sophisticated metal centers. By combining biological, biochemical and biophysical methods we planed to elucidate the 3D-structure of the proteins at high resolution as well as the electronic design and

19 Introduction

function of their metal centers at the atomic level. The knowledge of the architecture and the electronic features of the active sites is essential for understanding the mode of catalysis and the substrate specificity. While the 3D-structure provides the proximity and spatial relationship of amino acid residues and coordinated ligands, the nature of their bonding will be provided by spectroscopic and computational methods. Spectroscopies will yield information on the binding of the redox cofactors in their different oxidation states, and the interaction with the substrate.

20

2. Materials and Methods

Materials and Methods

2.1. Chemicals

If not specified chemicals were obtained in p.a. quality and used without further purification.

(i) Buffers

Fluka: MES (2-(N-morpholino)ethane sulphonic acid), KH2PO4; Merck: K2HPO4,

NaH2PO4; Riedel-de-Haën: Na-citrate dihydrate; Roth: HEPES (N-[2- hydroxyethyl]piperazine-N’-[ethane sulfonic acid]), TRIS (tris-(hydroxymethyl)- aminomethane);

(ii) Chromatographic resins GE Healthcare (Amersham Biosciences): Resource Q15, Resource Q30; Pharmacia Biotech: SuperdexTM 200, HiLoadTM 26/60, Q-Sepharose Fast Flow

(iii) Crystallization factorials Fluka: MPD (2-methyl-2,4-pentanediol, Ultra) Crystal screen solutions were obtained from Hampton Research Corporation (USA), Jena Biosciences GmbH (Germany) and NeXtal Biotechnologies (Qiagen, Germany)

(iv) Dyes Serva: bromphenol blue (sodium salt), Coomassie-brilliant blue G-250

Gas Messer Griesheim: Argon 5.0, Helium 4.6, Acetylene 2.6; Sauerstoffwerk

Friedrichshafen: N2 5.0, N2/CO2 (80:20 v/v), N2/H2 (96:4 v/v). Liquid helium was delivered by the Department of Physics, Universität Konstanz.

22 Materials and Methods

(v) General chemicals

Merck: NaOH, 37.5 % HCl, MgCl2⋅6H2O, MnCl2⋅4H2O; Riedel-de-Haën: Na-acetate,

CuSO4⋅5H2O, Na2S2O4, KH2PO4, Na2CO3, KCl; Roth: NaCl.

(vi) Proteins and enzymes BioRad: low range SDS/PAGE molecular weight standards; Fluka: DNase I (deoxyribonuclease I); Serva: BSA (bovine serum albumin); Sigma: gelfiltration molecular weight marker kit. Boehringer Mannheim: Yeast Alkohol-Dehydrogenase (400 U/mg)

(vii) Reagents

Merck: K3[Fe(CN)6]; Sigma-Aldrich: BCA (bicinchonic acid solution); Boehringer, Mannheim: NADH (nicotinamide adenine dinucleotide); Cayman Chemical: PAPA NONOate

(viii) Non-commercially available compounds 1,2,3,5-Tetrahydroxybenzene was synthesized and kindly provided by Dr. T. Huhn, Universität Konstanz. Purity was checked by NMR. Titanium(III) citrate was synthesized and kindly provided by D. Abt, Universität Konstanz [Zehnder et al., 1976]. 2,4,6,3’,4’,5’-Hexahydroxydiphenyl ether was a generous gift by Prof. J. Retey, Universität Karlsruhe [Paizs et al., 2007].

23 Materials and Methods

2.2. Organisms and cultivation

2.2.1 Pelobacter acidigallici Ma Gal 2 strain DSMZ 2377 was grown in batch cultures (0.1, 1, 50 l) at 30°C in bicarbonate-buffered, sulfide-reduced saltwater mineral medium [Brune et al., 1990]

The medium was sterilized and cooled under a N2/CO2 (80 : 20, v/v) atmosphere. After addition of modified trace element solution SL 10 (contains molybdate) and vitamin solutions, the pH was adjusted to 7.2–7.4 with 1 M HCl [Niessen, ; Widdel, 2- 1980; Widdel et al., 1981]. To replace molybdenum (from natural abundant MoO4 ) by stable molybdenum isotopes 95Mo or 98Mo, the culture was transferred at least four 95 2- 98 2- times in medium containing molybdate MoO4 or MoO4 , respectively (Table 1). Cultures were inoculated with 10% (v/v) of a stock culture. The substrate gallic acid was dissolved in water under exclusion of air, neutralized to pH 7.0 with concentrated NaOH, sterilized by filtration (0.2 µm), and fed at the start (7 mM) and twice (7 mM) during cultivation. Growth was monitored at 578 nm, and the pH of the medium was maintained at pH 7.2 with 2 M Na2CO3. Cells of a 50 l batch culture were harvested at the end of the exponential growth phase after 1-2 days (A578 = 0.8) with a Pellicon ultrafiltration unit (cutoff 100 kDa, Millipore). The concentrate was centrifuged at 10.000 g (30 min, 4°C) and the resulting cell pellet was stored at –70°C prior to use.

2.2.2 Pelobacter acetylenicus WoAcy1 strain DSMZ 3246 was grown in batch cultures (0.1, 1, 20 l) at 30°C in freshwater medium [Schink, 1985]. The medium was sterilized and cooled under a

N2/CO2 (80: 20, v/v) atmosphere, buffered with 30 mM NaHCO3, and reduced with

Na2S. After addition of modified trace element solution SL 10 (contains tungstate) and vitamin solution, the pH was adjusted to 7.0 – 7.4 with 1 M HCl [Niessen, 2004; Widdel, 1980; Widdel et al., 1981]. To replace tungsten by 183W isotope, the culture 183 2- was transferred at least four times in WO4 -containing medium (Table 1). The substrate acetylene was added to the gas phase to provide a concentration of 7-10% in the headspace of the glass bottle. The acetylene consumption during the growth of P. acetylenicus was controlled with a gasometer. Cultures were inoculated with 10% (by vol.) of a stock culture. The growth was monitored at 578 nm, and the pH of the

24 Materials and Methods

medium was maintained at pH 7.0 with 2 M Na2CO3. Cells of a 20 l batch culture were harvested after 2-3 days (A578 = 0.75) Molybdate cultivation of P. acetylenicus was carried out in freshwater medium [Abt, 2001; Schink, 1985] as described above. To replace tungsten by molybdenum, the 2- culture was transferred at least eight times in medium containing 2 µM MoO4 and 2 2- nM WO4 (Table 1) [Abt, 2001]. Cells were harvested after six days (A578 = 0.9) with a Pellicon ultrafiltration unit (cutoff 100kDa, Millipore). The concentrate was centrifuged at 10.000 g (30 min, 4°C) and the resulting cell pellets were frozen in liquid nitrogen and stored at -70°C.

Table 1: Isotope composition of molybdenum and tungsten compounds used in cultivation experiments 95Mo (I=5/2) 97Mo (I=5/2) Mo (I=0) Supplier

2- MoO4 15.92 % 9.55 % 74.53 % Merck

95 MoO3 96.8 % - 3.2 % ORNL

98 MoO3 - - 98.0% Russia/Kroneck 183W (I=1/2) W (I=0)

2- WO4 14.31% 85.69% Fluka

183 WO3 96.5 % 3.5% ORNL

95 98 183 95 2- MoO3, MoO3 and WO3 were diluted in concentrated NaOH giving MoO4 , 98 2- 183 2- MoO4 and WO4 [Greenwood et al., 1990].

2.3. Purification protocols

2.3.1. Transhydroxylase from Pelobacter acidigallici All purification steps were performed in the presence of air at 4°C on a FPLC system (GE Healthcare). Frozen cells were thawed and suspended in 50 mM triethanolamine

25 Materials and Methods

(TEA) pH 7.5 containing a few crystals of desoxyribonuclease I / 10 mM MgCl2·6H2O. Cells were broken by three passages in a French press (137 MPa; Amicon). The crude extract was centrifuged at 100000 g (60 min, 4°C) giving the soluble fraction as supernatant. The solution was applied to a Q-Sepharose column previously equilibrated with 50 mM TEA pH 7.5. After loading, the column was flushed with three bed volumes of buffer containing 175 mM NaCl. A linear gradient of buffer with increasing NaCl concentration from 175 mM to 500 mM eluted transhydroxylase. Fractions showing transhydroxylase on a SDS-PAGE gel were pooled and concentrated to a final volume of 2 ml by ultrafiltration (cut-off 30 kDa, 30-YM membrane, Millipore; Amicon).The concentrate was loaded onto Superdex 200 HiLoad® 26/60 column (GE Healthcare), equilibrated with 200 mM NaCl in 50 mM TEA pH 7.5 and eluted with the same buffer. Active fractions were pooled, concentrated by ultrafiltration (cut-off 30 kDa, 30-YM membrane, Millipore; Amicon), frozen in liquid nitrogen and stored in aliquots of 50-250 µl (10-15 mg/ml) at -70°C.

2.3.2. Acetylene hydratase from Pelobacter acetylenicus All purification steps were performed in the absence of dioxygen. Freshly prepared or frozen cells were brought into the anaerobic chamber, suspended in 50 mM Tris-HCl pH 7.5, to a cell density A578 = 135 containing 1mM PMSF and a few crystals of desoxyribonuclease I / 10 mM MgCl2·6H2O. Cells were broken by three passages in a

French press (137 MPa; Amicon) under Ar or He gas and the lysate collected in N2/H2 containing glass bottles were sealed with a rubber septum. The cell lysate was brought again into the anaerobic chamber and transferred into centrifuge tubes. Cell debris was removed by centrifugation at 10.000 g (30 min, 4°C) giving the crude extract (supernatant).

All chromatographic steps were performed at 18°C in an anaerobic chamber (94% N2,

6% H2; Coy, Pd-Cat) on a FPLC system (GE Healthcare) equipped with a SPD- M10Avp Diode Array Detector.

The crude extract was subjected to precipitation by (NH4)2SO4. In the first precipitation step, 4 M (NH4)2SO4 in water was added slowly to a final concentration of 2.3 M. The solution was stirred on ice for 30 min. After centrifugation (30 min, 10000 g) acetylene

26 Materials and Methods

hydratase was precipitated in a second step by adding 4 M (NH4)2SO4 (final concentration of 3.2 M) and stirring on ice for 30 min. After centrifugation (next day) the pellet was resuspended in 50 mM Tris/HCl pH 7.5 and desalted by ultrafiltration (cut-off 30 kDa, 30-YM membrane, Millipore; Amicon) with the same buffer. The desalted protein was centrifuged at 10000 g (5 min, 4°C) and the supernantant was loaded onto a Resource 30Q column (HR 10/16, GE Healthcare) equilibrated with 50 mM Tris/HCl pH 7.5. Fractions containing AH were pooled, again desalted by ultrafiltration (cut-off 30 kDa, 30-YM membrane, Millipore; Amicon) with the same buffer and loaded onto a high resolution Resource 15Q column (HR 10/16, GE Healthcare) equilibrated with 50 mM Tris/HCl pH 7.5. A linear gradient (0.1-0.3 M NaCl) led to the elution of AH at 0.2 M NaCl. Active fractions were pooled and concentrated by ultrafiltration (cut-off 30 kDa, 30-YM membrane, Millipore; Amicon) to 1.5 ml. The concentrate was loaded on a HiLoad® 26/60 Superdex 200 column (GE Healthcare), equilibrated with 200 mM NaCl in 50 mM Tris/HCl pH 7.5 and eluted with the same buffer. The pure protein was concentrated with a Vivaspin ultrafiltration spin column (30 kDa PES, Vivascience), frozen in liquid nitrogen and stored in aliquots of 50-250 µl (10 mg/ml) at -70°C.

2.4. Analytical methods

2.4.1. Protein Protein was determined by the bicinchoninic acid (BCA) method [Smith et al., 1985]. 100 µl of unknown or standard proteins (5 − 20 µg) Bovine serum albumin (BSA) were mixed with 1 ml of a solution containing 50 : 1 (v/v) BCA and CuSO4 · 5H2O (4%, w/v). The reaction mixture was incubated for 20 min at 60°C. The calibration curve was recorded on a Cary 50 spectrometer (Varian, Darmstadt) and the absorbance was monitored at 562 nm.

27 Materials and Methods

2.4.2. ICP-MS analysis of metals Metals were determinated by Inductively Coupled Plasma Mass Spectrometry (ICP- MS) at the Spurenanalytisches Laboratorium Dr. Baumann (Maxhütte-Haidhof). Iron, molybdenum and tungsten were determined in samples of acetylene hydratase purified from different cultivations (200 µl; approx. 2 mg/ml).

2.5. Enzymatic activities

2.5.1. Determination of Transhydroxylase activity with HPLC Transhydroxylase activity was measured by a discontinuous assay [Brune et al., 1990; Reichenbecher et al., 1999]. All steps were performed under exclusion of dioxygen in an anaerobic chamber. In a typical assay 775 µl of 100 mM potassium phosphate buffer (KPi), pH 7.0 were mixed with 100 µl pyrogallol (100 µM in KPi), 100 µl 1,2,3,5- tetrahydroxybenzene (100 µM in KPi), and incubated for one minute. The reaction was started by addition of 25 µl transhydroxylase solution. Aliquots (100 µl) were taken after 3 and 5 min incubation and immediately added to 100 mM H3PO4 (400 µl). Samples were transported from the anaerobic chamber in a gas-tight syringe and analyzed quantitatively for pyrogallol and phloroglucinol on a HPLC system (Sykam) equipped with a Gromsil C-18 Reverse Phase column (Grom Analytik + HPLC GmbH); temperature 40°C, solvent methanol/phosphate (12.5% methanol, by vol.; 100 mM KPi pH 2.6). Samples (20 µl) were injected and eluted at a flow rate of 1.5 ml min-1. Aromatic compounds were detected at 205 nm. Peak identification was performed by comparison of retention times and UV spectra with those of standard samples and quantified by comparison with standards of known concentration. The same procedure was applied to analyze the potential intermediate 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether (syntheses by Prof. J. Retey).

28 Materials and Methods

2.5.2. Photometric determination of Acetylene hydratase activity Acetylene hydratase activity was measured photometrically in a coupled assay with alcohol dehydrogenase [Meckenstock et al., 1999; Rosner et al., 1995]. The reaction was performed in quartz cuvettes sealed with rubber stoppers under N2/H2. In a typical assay, 10 µl acetylene hydratase solution was added to 960 µl of 1.5 mM titanium (III) citrate in 50 mM Tris/HCl pH 7.5. After addition of 20 µl of 10 mM NADH and 10 µl of yeast alcohol dehydrogenase (2000 U ml-1) the mixture was incubated for 30 min at 30°C. The reaction was started by addition of 2 ml acetylene to the gas phase. Depletion of NADH was followed at 365 nm. Activity was calculated -1 -1 using Beers Law and ε365 (NADH) = 3.4 mM cm [Ziegenhorn et al., 1976]. One unit is defined as the amount of enzyme required for conversion of 1 µmol acetylene to acetaldehyde / min under the assay conditions.

2.5.3. Experiments under exclusion of dioxygen Experiments under exclusion of dioxygen were carried out in an anaerobic chamber

(94% N2, 6% H2, Coy) equipped with a Palladium catalysator type K-0242 (0.5 %

Pd/Al2O3, ChemPur) to remove traces of dioxygen. The content of dioxygen in the anaerobic chamber was below 1 ppm, which was experimentally determined [Beinert et al., 1978]. Glass and plastic items were stored in the anaerobic chamber for at least 24 h prior to use. Dioxygen from buffers and solutions was removed by 8-10 cycles of degassing / flushing with Argon at a vacuum line [Beinert et al., 1978]. Traces of dioxygen were removed from argon via passage through a glass/copper system filled with BTS Catalyst R3-11 (BASF). Buffers were stored for at least 24 h in the anaerobic chamber prior to use, in order to equilibrate with the N2/H2 atmosphere.

29 Materials and Methods

2.6. Spectroscopic methods

2.6.1. Electron paramagnetic resonance spectroscopy A detailed discussion of electron paramagnetic resonance spectroscopy (EPR) was beyond the scope of this work and can be found in relevant textbooks [Weil et al., 1994; Abragam et al., 1986; Pilbrow, 1990] and reviews [Beinert et al., 1962; Hagen, 2006].

Theoretical aspects The EPR effect was discovered by E.K. Zavoisky in 1944 at Kazan State University. Two important consequences of the early history have a significant influence on the application of EPR in biology. The g-values are calculated from the resonance condition:

h ⋅ v g = (eq. 1) β ⋅ B

with h = 6.6262⋅10-34 J s (Planck constant) β = 9.274096⋅10-24 J T-1 (Bohr magneton) [ν] = s-1 (Microwave frequency) [B] = T (Magnetic field) g-Value. The electronic g-value contains information about the electronic structure (energy levels and symmetry) of the molecule carrying one ore more unpaired electrons. The g-value can therefore give valuable information as a means of identifying a paramagnetic species as well as determining its electronic environment. Transition metals can be used as intrinsic probes to study proteins and enzymes. Detailed information on the nature of ligands, coordination geometry of the metal site, its oxidation and spin state can be obtained [Hagen, 2006].

Hyperfine interaction. The electron-nuclear hyperfine interaction reveals the local nuclear environment of the unpaired electron, and thus helps to unravel the molecular structure. As the number of hyperfine interactions from different nuclei increases, the

30 Materials and Methods

EPR spectrum becomes increasingly more complex. In this case multifrequency spectroscopy (2 – 95 GHz) and advanced EPR techniques have to be applied [Hagen, 2006].

Computer simulation. The simulation of the resonances associated with the major species with an orthorhombic spin Hamiltonian (eq. 2) and the g matrix (for the species

Mo-OH, Mo-OH2 and Mo-X, where X may represent an anion) yields the molybdenum (98Mo and 95,97Mo) spectra of TH enzymes.

H = βBgS + ∑ (SA i I i − g n β n BI i ) (eq. 2) i =1,2 where S and I are the electron and nuclear spin operators, respectively, g and A are the electron Zeeman and hyperfine coupling matrices, respectively, β the Bohr magneton and B the applied magnetic field [McDevitt et al., 2002].

Practical aspects EPR spectra were recorded on a Bruker Elexsys 500 with an ER 049 X microwave bridge. The system was equipped with an Oxford Instruments ESR 900 helium cryostat controlled by the ITC 503 temperature device. The measurements were performed with the 4122 SHQE cavity at ≈ 9.38 GHz, modulation frequency 100 kHz, modulation amplitude typically 1.0 mT. The samples (180-250 µl) were transferred to Suprasil quartz tubes 706-PQ-9.50 (∅out 4 mm, Wilmad) in the anaerobe tent.

EPR monitored redox titrations of TH were performed under the exclusion of dioxygen (Argon, or anaerobe chamber) at 25°C, by stepwise addition of sodium dithionite (or potassium ferricyanide) to 52 µM TH in 5 mM Hepes pH 7.5 buffer containing 50 µM of the following redox mediators: NNN´N´-tetramethylphenylenediamine (E’0 = +260 mV), 2,6-dichlorophenolindophenol (E’0 = +217 mV), phenanzinmethosulfate (E’0 = +80 mV), indigo trisulfonate (E’0 = -70 mV), indigo disulfonate (E’0 = -125 mV), 2- hydroxy-1,4-naphtochinone (E’0 = -152 mV), antrachinone-2-sulfonate (E’0 = -225 mV), phenosafranine (E’0 = -252 mV), methylviologen (E’0 = -440 mV). All solutions were made anoxic through several cycles of vacuum/argon. The potential was measured

31 Materials and Methods

with a combined Pt, Ag/AgCl electrode (EMC 50 NSK, Meinsberg, Germany), connected to an E620 Metrohm potentiometer. The electrode was calibrated with a saturated quinhydrone solution at pH 4.0 (E0 = +255 ± 5 mV) and pH 7.0 (E0 = +75 ± 5 4 – 3– 0 mV) as well as with the redox standard solution [Fe(CN)6] / [Fe(CN)6] (E = +220 ± 5 mV, Mettler-Toledo, Germany). The potential was read on the instrument when the value changed by less then ±2 mV within 5 min. The EPR spectra were corrected for contributions of the redox mediators; potentials are quoted vs the standard hydrogen electrode (SHE).

The simulations of the spectra from [4Fe-4S] clusters were performed with the program WEPR [Neese, 1995]. Computer simulations of the EPR spectra were performed using the version 1.0.4 of XSophe [Hanson et al., 2004]. At this point, the simulation of the Mo(V) spectra did not attempt to reproduce the full set of molybdenum hyperfine splittings. The computational program SOPHE employs a number of methods, including matrix diagonalization, SOPHE interpolation, and homotopy for the analysis of randomly oriented EPR spectra. In this research, the matrix diagonalization was employed in conjunction with mosaic misorientation to simulate the randomly oriented EPR spectra of the Mo(V) sites. Comparisons of simulated and experimental spectra and data manipulation were performed with Xepr.

EPR spectra were quantitated by the following procedure.

Measurement of a copper standard: 50 µM and 200 µM CuSO4 in NaClO4/HCl.

The concentration of EPR detectable copper (cSA) was then determined by:

(ST ) J SA g AV TSA MAST PST f ST GST SCST cSA = cST ⋅ ⋅ (SA) ⋅ ⋅ ⋅ ⋅ ⋅ ⋅ (eq. 3) J ST g AV TST MASA PSA f SA GSA SCSA

where SA = sample; ST = standard; J = double integral of simulated spectrum; gAV = Åasa Vänngård value (eq 4) [Åasa et al., 1975]; T = temperature; MA = modulation amplitude; P = microwave power; f = sample tube diameter; G = receiver gain; SC = number of scans.

32 Materials and Methods

2 1 1 g = ⋅ ⋅()g 2 + g 2 + g 2 + ⋅()g + g + g (eq. 4) AV 3 3 x y z 9 x y z

Hereby the filling value of the standard and the sample tube should be identical, i.e. the sample volume and tube diameter should be the same. Care must be taken not to saturate the spectrum of either the standard or the unknown sample. At 10 K the spectrum of the copper standard will be started to saturate at about 2.007 mW. Ideally, spectra taken at several different microwave powers and temperatures should be employed for the quantification.

2.6.2. UV-Visible spectroscopy UV/Vis spectra were obtained on a Cary 50 spectrometer (Varian, Darmstadt). The spectrometer was equipped with a thermostated cell holder and a temperature control unit, allowing temperatures of up to 80°C.

2.6.3. Circular dichroism spectroscopy CD spectra were recorded on a Jasco J-810 spectropolarimeter at room temperature, using a cell with path-length of 1 mm, before measurment the cell compartment was flushed with nitrogen. The spectra were averaged and the spectrum was corrected for the applied buffer. The content of α-helix was analyzed using the software CDNN [Böhm, 1997].

33 Materials and Methods

2.7. Protein crystallography

Protein crystals were grown by the vapour diffusion method, where the protein solution was mixed with a precipitant solution and equilibrated against the precipitant reservoir in a closed environment. Under regular conditions, using non-volatile precipitants such as polyethylene glycol, MPD, or salts, equilibrium was reached by diffusion of water from the protein drop to the reservoir, thus slowly increasing the concentration of all components in the drop [McPherson, 1982]. Sitting drop experiments were carried out in Cryschem plates (Charles Supper Company), hanging drop setups in Costar Model 3424 plates with siliconized cover slides (Hampton Research). The filling volume per well was 300-500 µl of reservoir solution. Buffer exchange was carried out on NAP gravity flow columns (GE Healthcare) with 5 mM HEPES pH 7.5 as buffer of low ionic strength. Protein solutions were concentrated using Centricon YM 30 tubes (Amicon). For the buffer exchange and concentration step a 5 % loss of the amount of protein was estimated.

2.7.1. Crystallization conditions

Transhydroxylase from Pelobacter acidigallici

Crystallization was done in the anaerobic chamber in N2/H2 (94% / 6%) atmosphere at 18°C using the sitting drop vapour diffusion method. 3 µl protein solution (12 mg/ml, pH 7.5), 12 equivalents Na-dithionite, and 10% (v/v) additive (0.1 M sodium cacodylate, pH 6.5, 1.4 M sodium acetate) were mixed with 3.5 µl reservoir solution (0.05 M potassium phosphate pH 7.5, 20% PEG 8000). Crystals formed over a period of 1 to 6 weeks and were frozen directly from the crystallization solution after adding 2- methyl-2,4-pentanediol (MPD) to a final concentration of 25% [Messerschmidt et al., 2004].

34 Materials and Methods

Acetylene hydratase from Pelobacter acetylenicus

Crystallization was done in N2/H2 (94% / 6%) atmosphere at 20°C using the sitting drop vapour diffusion method. Crystals formed over a period of 1 to 3 weeks from 10 mg/ml protein solution in 5 mM HEPES/NaOH, pH 7.5 reduced by sodium dithionite to a final concentration of 5 mM. 2 µl protein solution was mixed with 2.2 µl of reservoir solution (0.1 M sodium cacodylate, pH 6.5; 0.3 M magnesium acetate; 21% polyethylene glycol 8000; 0.04 M sodium azide). 15% -methyl-2,4-pentanediol (MPD) was added as a cryoprotectant and the crystals were frozen in liquid nitrogen.

2.7.2. Data collection and processing Diffraction data of acetylene hydratase crystals were collected at beam lines BW6 (MPG-ASMB) and X11 (EMBL) at DESY, Hamburg, Germany. One data set was collected at a wavelength of 1.738 Å on the high-energy side of the K-edge of iron, and a high resolution data set was subsequently collected from a different crystal at a wavelength of 1.05 Å. Heavy metal sites were located with the program SHELXD [Schneider et al., 2002], which produced five clear solutions, corresponding to the four iron atoms of the [4Fe- 4S] cluster and the tungsten atom. At the energy of the iron K-edge, the anomalous signal of tungsten corresponds to approximately 6.8 e-, such that this atom appeared as the strongest peak in an anomalous difference Patterson map. All data sets were indexed, integrated, and scaled using the programs DENZO and SCALEPACK [Otwinowski, 1996]. For all further steps of structure solution data sets were converted with CCP4, including conversion of intensities to structure factor amplitudes and to suitable file formats.

Model building and refinement - Phase calculations were carried out with SHARP [De la Fortelle et al., 1997]. SOLOMON [Kroneck, 2005] was used for electron density modification. An initial model comprising 448 of the 730 amino acid residues of AH was built automatically using RESOLVE [Terwilliger, 2004]. All subsequent manual

35 Materials and Methods

rebuilding steps were carried out in COOT [Emsley et al., 2004]. The model was refined using REFMAC5 [Murshudov et al., 1997].

2.7.3. Graphical representation Illustrations of structures were prepared using the programs DSVIEWER (Accelrys Ltd, UK), COOT [Emsley et al., 2004] and “The PyMOL Molecular Graphics System” by Warren L. DeLano (DeLano Scientific LLC, San Carlos, CA, USA) with its implemented ray-tracer. Electrostatic surface was calculated in the program APBS and displayed in PYMOL (DeLano Scientific LLC, San Carlos, CA, USA).

2.8. [14C]-acetylene assay

14 14 Pure acetylene hydratase was treated with [ C]-acetylene synthesized from Ba CO3

[Hyman et al., 1988]. The Ba/BaC2, fusion mixtures were hydrolyzed in vials which had been sealed with butyl rubber stoppers / aluminum crimp seals and evacuated using a vacuum manifold. Hydrolysis was initiated by adding water to the samples. In all cases the reaction was allowed to continue until completion of gas evolution indicating complete hydrolysis of the Ba/BaC2 mixture.

For a solution of acetylene in DMSO, Ba/BaC2 mixtures (either labeled or unlabeled) were hydrolyzed in a sealed serum vial (14 ml) which had been modified by cementing an open 1-ml vial inside of the reaction vessel. The Ba/BaC2 mixture was placed in the inner chamber and the reaction was started by adding 200 µl of water. After gas evolution had ceased and the hydrolysis reaction had reached completion (typically after 2 h), 500 µl DMSO was injected into the outer chamber of the reaction vial and the vial was then placed on an orbital shaker (75 rpm) for 15 min. The DMSO was then removed by a gas-tight syringe and was replaced with fresh DMSO, and the vial was returned to the shaker. This procedure was repeated four times.

36 Materials and Methods

14 The carbide-generating reaction mixture consisted of 1 mCi Ba CO2, (3.46 mg) with a specific activity of 57.5 mCi/mmol. The resulting metal/carbide mixture was hydrolyzed with 200 µl water in the inner chamber of a double-chambered, stopped 14- ml vial. After completion of the DMSO-trapping procedure, 1 µl of the 2 ml of the recovered DMSO / [14C]-acetylene solution contained 6.65 X 105 dpm, as determined by liquid scintillation counting. Protein samples (50 µg) were separated on a 10% polyacrylamide gel at a constant current of 15 mA. Gels which had been stained with Coomassie blue were destained using methanol-acid water. Afterwards they were dehydrated by placing them in DMSO for 30 min. The dehydrated gel was then soaked in a DMSO/PPO (2,5-diphenyloxazole in DMSO) for 2h, and the PPO (2,5-diphenyloxazole) was recrystallized by immersion of the gel in running cold water. The radioactive polypeptides were visualized by fluorography as described by Bonner & Leasky (1974). Fluorographs were produced using Hyperfilm ECL (Amersham Biosciences GmbH, Freiburg) film and a 14-day exposure time.

37

3. Results and Discussion

Results and Discussion

3.1. Transhydroxylase of Pelobacter acidigallici

3.1.1. Growth of Pelobacter acidigallici P. acidigallici was grown in 50 l batch cultures. The bacterium grew in saltwater medium containing molybdate with different molybdenum isotopes. The substrate gallic acid, as energy and carbon source, was fed 5 mM or 7 mM initially, and added two or three times during cultivation (Fig. 3.1).

0.9

0.8

0.7

0.6

0.5 578 nm 578 0.4 OD 0.3

0.2

0.1 Gallic acid 0.0 5mM 7mM 7mM 5mM 5mM

0 5 10 15 20 25 30 35 40 45 time [h] Figure 3.1: Growth curves of P. acidigallici. The medium contained molybdate ■ 95Mo, ▶ 98Mo, □ 95,97Mo; the solid arrows indicate the addition of gallic acid to cultures with 95Mo, 95,97Mo; the dashed arrow indicate the addition of gallic acid to culture with 98Mo.

The cells of P. acidigallici enriched with 98Mo grew for a longer time in comparison to the two other cultures, finally reaching an optical density of 0.85 at 578 nm (Fig. 3.1).

39 Results and Discussion

3.1.2. Purification of Transhydroxylase TH was purified in the presence of air to homogeneity according to SDS-PAGE in a three-step procedure [Niessen, 2004]. A typical protocol is documented in table 3.1.

Table 3.1: Purification of TH from molybdate (98Mo) grown cells from P. acidigallici. 25 g cells (wet weight) were used. 1U = 1µmol phloroglucinol · min-1. SDS-PAGE (12.5%): Lane 1, Crude extract; Lanes 2 and 4, molecular weight markers; Lane 3, elution from Q-Sepharose; Lane 5, elution from Superdex 200; bands on the weight of the α and β unit of the TH (86 and 38 kDa).

Cultivation Protein Yield Specific activity 1 2 3 4 5 kDa 98Mo [mg] [%] [U· mg-1]

Crude extract (CE) 750 100 0.87 (0.80) -86

-38 Q-sepharose (QS) 304 40 2.02 (1.92)

Superdex (S ) 54 7.2 4.15 (4.60) 200 * Specific activities in brackets are taken from Abt ( 2001). SDS-PAGE of purified protein gave bands at 86 and 38 kDa. They were assigned to the α- and β-subunit of TH, which is a heterodimer with an apparent molecular mass of 133 kDa [Reichenbecher et al., 1994]. Depending on the culture and the molybdenum isotope used, the yield of pure TH amounted to 2.6 - 3.9 mg out of 1 g wet cell mass (Table 3.2). The enzyme obtained by the purification procedure of Niessen (2004), was highly active (4.15 U· mg-1), similar to the previously reported TH activity of 4.60 U· mg-1 [Abt, 2001]

Table 3.2: Purification statistic of TH depended on the molybdenum isotopes

Cultivation Cells Pure Protein Yield (Protein) [g] [mg] [%] 95,97 Mo 20 78 9.1 95Mo 20 51 6.7 98Mo 25 54 8.5

40 Results and Discussion

3.1.3. Spectroscopic characterization of the metal sites

3.1.3.1. Circular Dichroism (CD) CD over 300-700 nm provided valuable information on the coordination of the Mo sites and the types of Fe-S centers. The room-temperature CD spectrum of as isolated TH consisted of five broad negative bands at 400, 525, and 630 nm and three explicit shoulders at 325, 370, 430, 495, 600 nm (Fig. 3.2). The sample used in the CD experiments was identical to the samples studied by EPR (EPR spectrum-Mo(V) signal, Fig. 3.5).

4

2

0 ) -1

*cm -2 -1 (M

Δε -4

-6

-8 300 350 400 450 500 550 600 650 700

Wavelength (nm) Figure 3.2: Room temperature CD spectrum of “as isolated” transhydroxylase from P. acidigallici grown on (98Mo). Enzyme 2 mg·ml-1 in 10 mM Tris pH 7.6, recorded under exclusion of dioxygen.

A comparison of the CD spectra of TH as isolated form and of the glycerol inhibited Mo(V) form of DMSO reductase from Rhodobacter sphaeroides revealed similarities of the bands in the range 500-700 nm which are assigned to π-dithiolene→Mo(V) charge- transfer transitions (CT). The CD spectrum of the glycerol inhibited Mo(V) form of DMSO reductase had three broad negative bands at 700, 540, and 395 nm, which are corresponding to negative MCD bands and therefore expected to exhibit CD spectrum under the parent C2v symmetry of Mo(V)-dithiolene fragment [Finnegan et al., 1993]. The MCD spectra of DMSO reductase from Rhodobacter capsulatus showed CT transitions, which originate from dithiolene sulfur-orbitals forming σ-bonds to Mo(V).

41 Results and Discussion

Thus the bands between 500 and 700 nm in DMSO reductase undoubtedly arise from the presence of sulfur ligation [Benson et al., 1992]. Upon reduction of sulfite oxidase to Mo(V) by sulfite, or dithionite, significant changes of the CD spectrum between 430 and 600 nm were observed similar to those found for TH [Garner et al., 1982].

Transitions around 350-450 nm commouly originate from iron-sulfur centers. The comparison of the shoulders in the CD spectrum of TH (325, 370, 430 nm) with those obtained for an enzyme with two [4Fe-4S] clusters - Ferredoxin (Fd) from Chlorobium tepidum - revealed similar features. The shoulders and bands of Fd at 330, 370, 420, 570 nm, originated from oxidized enzyme they are similar to the transitions observed for TH from P. acidigallici, were attributed to oxidized [4Fe-4S]2+ clusters in Fd the bands around 370 and 425 nm decreased in intensity after reduction of the enzyme. The mentioned pair of CD bands is believed to arise from an excitation coupling of the strongest transition [Yoon et al., 2001]. These bands have been observed in the UV/Vis spectra of TH around 384 nm and were assigned to S-Cys-Fe charge transfer transitions [Abt, 2001].

3.1.3.2. Electron Paramagnetic Resonance (EPR)

Mo center of Transhydroxylase “as isolated”

The EPR spectrum of the as isolated enzyme exhibited a high intensive signal at gav = 1.98, with no significant broadening in the temperature range 10-80K, which was assigned to molybdenum in the S=1/2 Mo(V) state (Fig. 3.3). Contrary to TH, most molybdenum enzymes do not show such a Mo(V) signal in the as isolated form of the protein. The signal usually becomes visible after reduction or oxidation depending on the conditions of purification.

Depending on the molybdenum isotope used for the preparation, the signal at gav = 1.98 differs with respect to intensity, anisotropy, and hyperfine splittings. Spin quantization revealed that 48, 46 and 16 % of the Mo is present as Mo(V) in transhydroxylase from cells grown with 98Mo, 95,97Mo, and 95Mo, respectively (Fig. 3.3).

42 Results and Discussion

g-Value A 2.12 2.08 2.05 2.02 1.99 1.96 1.93 1.90 1.87 1.85 1.82 B

Mo98

Mo95, 97

95

Mo Mo(V) Signal

x20 relative intensity line relative line intensity

3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 3650 Magnetic Field [G] Magnetic Field [G]

Figure 3.3: EPR spectra of as isolated Transhydroxylase from P. acidigallici, enriched with different molybdenum isotopes. (A) TH (7 mg/ml), in 50 mM TEA pH 7.5 buffer, 200 mM NaCl; (B) Temperature dependence of EPR spectra of 95,97Mo-TH at 10K (gray) and at 80K (black). Instrument settings: 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude.

98 The spectrum of Mo-TH gave a strong line centered around g⊥= 1.981 and showed no hyperfine splittings as expected (98Mo, I = 0) (Fig. 3.3 A). In contrast, the spectra of 95Mo-TH and 95,97Mo-TH showed multiline resonances indicative for interaction of the unpaired electron of Mo(V) with (1°) its nuclear spin I =5/2, and (2°) with a neighboring ligand carrying a I =1/2 nucleus . The use of different molybdenum isotopes in EPR studies of molybdenum enzymes, mainly xanthine oxidase, was introduced by Bray [Bray et al., 1966; George et al., 1988]. The EPR parameters of TH in the as isolated state from P. acidigallici have been compiled in Table 3.3, along with parameters of related enzymes from the corresponding family of molybdenum enzymes. Apparently, the parameters of TH were most similar to those of the DMSO reductase from Rhodobacter capsulatus (high g split signal), to the parameters of three forms of the DMS dehydrogenase from Rhodovulum sulfidophilum (Mo-X, Mo-OH, Mo-X), and parameters reported for the low pH nitrate reductase NarGH.

43 Results and Discussion

Table 3.3: Mo(V) EPR parameters of Transhydroxylase from P. acidigallici and of enzymes of the DMSO reductase family

Enzyme gz gy gx gav. gzz – gxx gzz – gyy /gzz – gxx anisotropy rhombicity

TH 95,97Mo 1.997 1.984 1.961 1.9806 0.036 0.36

TH 95Mo 1.985 1.981 1.938 1.9773 0.043 0.30

TH 98Mo 1.989 1.981 1.969 1.9796 0.020 0.40

DMSOR 1.9922 1.9818 1.9673 1.9804 0.0249 0.42 High g split 1) DMS DH 2.0006 1.9846 1.965 1.9834 0.0356 0.45 Mo(V)-aqua 2a) DMS DH 1.9989 1.9805 1.9600 1.9798 0.0389 0.47 Mo(V)-X 2b) DMS DH 1.9914 1.98 1.9627 1.9785 0.0287 0.40 Mo(V)-OH 2c) NarGHI 1.9997 1.9851 1.9642 1.9830 0.0355 0.41 Low pH 3) NarGHI 4) Low pH 1.9989 1.9855 1.9628 1.982 0.036 0.41 High pH 1.9870 1.9805 1.9612 1.976 0.026 0.25 All enzymes contained molybdenum in natural abundance; 1) DMSOR: dimethyl sulfoxide reductase from Rhodobacter capsulatus. Mo(V) high g split, [Bennett et al., 1994]; 2) DMS DH: Dimethyl Sulfide Dehydrogenase from Rhodovulum sulfidophilum: a. DMS DH: dimethyl sulfide dehydrogenase. Mo(V)- aqua: two equivalent protons; b. X may represent an anion, such as chloride; c. Mo(V)-OH, [McDevitt et al., 2002]; 3) NarGHI: nitrate reductase from E. coli. Mo(V) low pH nitrite, [George et al., 1989]; 4) NarGHI: nitrate reductase from E. coli. Mo(V) low pH and high pH, [Vincent et al., 1978]

The enzymes listed in Table 3.3 all carry Mo(MGD)2 sites. The differences in g-values observed for the various enzymes are most likely linked to the additional ligands coordinated to the molybdenum center. For DMSO reductase similar enzymes, depending on the redox state of molybdenum Mo(IV), Mo(V) and Mo(VI), the fifth ligand originates from a Ser oxygen, and the sixth ligand is most likely water or hydroxide anion [Raitsimring et al., 2003; Temple et al., 2000].

44 Results and Discussion

According to different reaction conditions (reaction time, pH, presence/nature of reducing or oxidizing agent), different types of molybdenum EPR signals could be distinguished and will be described in the following chapter.

Effect of Cl - ions Anion binding to EPR active Mo(V) centers has been reported earlier for nitrate reductase (Nar GHI) [Gutteridge et al., 1983] and sulfite oxidase [Bray et al., 1983]. In the case of TH there was a significant effect of chloride (200 mM) on the EPR spectra ions is ־Fig. 3.4). The splitting observed in the spectrum of TH in the presence of Cl) similar to that observed for nitrate reductase (NarGHI) from E.coli [George et al., 1985]. Contrary to NarGH, the anion effect was not so strong, and was analyzed only in pH 7.5 of 95Mo-TH and 95,97Mo-TH.

A B 95,97Mo 95Mo

+ NaCl + NaCl

relative line intensity line relative relative line intensity line relative

3200 3300 3400 3500 3600 3200 3300 3400 3500 3600 Magnetic Field [G] Magnetic Field [G]

Figure 3.4: Effect of chloride on the Mo(V) EPR spectrum of Transhydroxylase from P. acidigallici. (A) 95,97 Mo(V) signal; (B) 95Mo(V) signal: upper spectra 200 mM NaCl in 50 mM TEA/HCl, pH 7.5 and lower spectra 5 mM Hepes/NaOH, pH 7.5. Instrument settings: 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature.

Effect of pH To examine the effect of the pH on the EPR spectrum of TH, samples with both isotope enrichments (98Mo and 95,97Mo) were prepared in buffers covering a range from 6.5 to

45 Results and Discussion

8.5. In the 98Mo(V) signal examination of I = 0 resonances as a function of pH (Fig. 3.5 A) revealed shoulders on the three resonances only at low pH, which might have arise from multiple species and/or proton hyperfine coupling. Increasing the pH from 6.5 to 8.5 enhanced the broad of the vertical bar shoulders and the formarly observed splitting disappeared. Contrary to that, in the spectrum of 95,97Mo-TH containing natural occurring molybdenum (data not show) no changes in spectral line shape depending of the pH were observed.

A g-Value B 2.15 2.10 2.05 2.01 1.97 1.93 1.89

98 Mo Asp 174 – + pH 6.5 H donor

sim

His 144 – +

pH 7.5 H acceptor

sim relative line intensity

pH 8.5

sim

3100 3200 3300 3400 3500 3600 Magnetic Field [G] Figure 3.5: Effect of pH on the 98Mo(V) EPR signal of Transhydroxylase from P. acidigallici. (A) EPR spctra of 98Mo-TH in different buffer a) Na-Cacodylate/NaOH pH 6.5; (b) Tris/HCl pH 7.5; (c) Tris/HCl pH 8.5; (a’, b’, c’) simulation spectra of a, b, c respectively; the simulation parameters are compiled in Table 3.4. Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature; (B) View of active site of Mo-TH (pdb VLE) with amino acid as potential proton donor Asp174 or acceptor His144 [Messerschmidt et al., 2004].

The pH dependence of the Mo(V) EPR spectra was reported previously for nitrate reductase from E. coli, as low-pH form and high-pH form. Parameters for Mo(V) species from the nitrate reductase are given. The low-pH species (gav = 1.983) is in pH-

46 Results and Discussion

dependent equilibrium with the high-pH species (gav = 1.976). Of a variety of anions tested, only nitrate and nitrite formed complexes with the enzyme (in the low-pH form), giving modified Mo(V) EPR spectra. These complexes, as well as the low-pH form of the free enzyme, showed interaction of molybdenum with a single exchangeable proton [Vincent et al., 1978]. The 98Mo(V) EPR signal of TH (pH 6.5) has a rhombic lineshape, with g-values at 1.997, 1.981, 1.963 similar to the two forms of nitrate reductase from E. coli (Table 3.4). The effect of the pH on the Mo(V) EPR signal of nitrate reductase can be assigned to the Asp residue (Asp-222) located close to the Mo center [Gonzalez et al., 2006]. From the crystal structures of nitrate reductase [Jormakka et al., 2004] and TH [Messerschmidt et al., 2004] the distances between the Asp oxygen and molybdenum are 2.1 Å (NarGHI) and 3.6 Å (TH), respectively. The possibility of an interaction between the Mo center and Asp-174 in TH has been supposed (Fig 3.5 B).

Table 3.4: pH dependence of the EPR parameters of 98Mo (V) transhydroxylase

Enzyme gz gy gx gav. gzz – gxx gzz – gyy / gzz – gxx anisotropy rhombicity TH 98Mo 1) 1.997 1.981 1.963 1.980 0.034 0.47 pH 6.5 TH 98Mo 2) 1.989 1.982 1.968 1.980 0.021 0.33 pH 7.5 TH 98Mo 3) 1.986 1.980 1.965 1.981 0.021 0.29 pH 8.5 For computer simulation the experimental g-value are used (Fig. 3.5) -4 -1 -4 -1 -4 -1 -4 -1 1) linewidth: az 6.5 x 10 cm , ay 4.5 x 10 cm , ax 3.5 x 10 cm ; A x,y,z = 0.5 x 10 cm -4 -1 -4 -1 -4 -1 2) linewidth: az 10 x 10 cm , ay 7 x 10 cm , ax 6.5 x 10 cm -4 -1 -4 -1 -4 -1 3) linewidth: az 3.3 x 10 cm , ay 3.3 x 10 cm , ax 4.5 x 10 cm

For the TH the effect of anions on the interconversion between high-pH and low-pH forms of the enzyme still needs to be investigated in detail. The pH effect on the EPR spectrum in connection with the binding of chloride was also observed for nitrate reductase from E. coli [George et al., 1985]. Perhaps, the observed changes of the Mo(V) EPR signal of both enzymes are related to anion uptake which is not accompanied by proton transfer but a proton is moving from a weakly coupled to a

47 Results and Discussion

strongly coupled site [Gutteridge et al., 1983]. Probably, there is an anion binding site in TH with relatively low specificity towards small inorganic anions.

Effect of redox agents on Mo (V) signal Effects resulting from addition of various reducing or oxidizing agents, such Na- 95 95,97 98 dithionite or K3[Fe(CN)6] to “as isolated” Mo-TH, Mo-TH and Mo-TH was determined. Addition of reductant (7-10 equivalents Na-dithionite/mol TH) to samples of 95Mo-TH, and 95,97Mo-TH resulted in a decrease in intensity of the Mo(V) signal. During reductive titration the line shape of the EPR spectra did change significantly. The new ‘resting’ 95,97Mo(V) signal was obtained with g-values at 1.988, 1.974, 1.964 (Fig. 3.6, spectrum number 4). Large excess of dithionite (10 reduction equivalents / mol TH) did not yield fully reduced Mo centers, and the EPR spectrum integrated to 20% of signal intensity of “as isolated” enzyme. In this spectrum an additional weak signal tentatively assigned to + a [4Fe-4S] cluster could be seen. Oxidative titration (20 equivalents K3[Fe(CN)6] / mol TH) did not show any change in the line shape, the intensity of the Mo(V) decreasing in 10%. In most other familiarly enzymes, the oxidation state of Mo(V) changes to Mo(IV) or Mo(VI) upon application of one equivalent of reductant or oxidant. It seems interesting, that TH in opposite shows very stabile Mo(V) signal.

EPR monitored potentiometric titrations were used to determine the midpoint reduction potentials of the redox centres of TH. 95,97Mo (V) signals developed over the potential range -10 mV to -120 mV, and reached approximately 50% of maximum magnitude at around -83 mV. The low redox potential of the 95,97Mo centre confirms the experimental findings to reduce this metal site.

48 Results and Discussion

A g-Value B 2.11 2.08 2.05 2.02 1.99 1.96 1.93 1.90 1.88 1.85

1. oxidized

2. relative line intensity line relative

3. *

reduced

4. * - 83 mV +/- 5 mV

relative line intensity line relative

3100 3200 3300 3400 3500 3600 -200 -150 -100 -50 0 50 100 150 Magnetic Field [G] E°' / mV vs SHE

Figure 3.6: EPR monitored redox titration of 95,97Mo-TH from P. acidigallici in the presence of mediators, (A) Comparison of EPR spectra of TH “as isolated” (spectrum number 2) oxidized with ferricyanide (spectrum number 1) and with dithionite reduced enzyme (spectrum number 3, 4); (B) redox titration of Mo(V) to indicate the potential for the highest field resonance determined with the double integral of the signal at g = 1.984. Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature

95,97 The midpoint potentials (Em) of the Mo(V) centers in TH determined by EPR are much more negative by comparison to Em values reported for dimethylsulfoxide 5+/4+ 5+/6+ reductase from Rhodobacter capsulatus, Em(Mo ) = +141 mV, and Em(Mo ) = +200 mV respectively [Bennett et al., 1994]. Note that the potential of Mo(V) centers of NarGHI from E. coli, with g parameters similar to those of TH, was also found to be positive. Among the enzymes of the DMSO reductase family, negative potentials of Mo(V) centers were found for dimethyl sulfoxide reductase (DmsABC) from E. coli in the range from 0 to -138 mV (pH 7.0), and for periplasmic nitrate reductase (NapA) from E. coli in the range from +100 to -100 mV (pH 8.0) [Heffron et al., 2001; Jepson et al., 2007].

49 Results and Discussion

The reduction of the EPR-detectable Mo(V) center in TH, with g- values at 1.997, 1.984, 1.961, to a diamagnetic Mo(IV) state was not achieved with dithionite as a reductant in the presence of redox mediators.

g-Value 2.15 2.11 2.08 2.05 2.011.98 1.95 1.92 1.89 1.86

1.

oxidazed

2. x 5

intensity line relative

reduced 3.

gz gy gx gav. anisotropy rhombicity 98 TH Mo 4.

Ox (1.) 1.988 1.982 1.965 1.978 0.023 0.26

As isol. (2.) 1.989 1.981 1.969 1.980 0.020 0.40 3100 3200 3300 3400 3500 3600 Magnetic Field [G] Red (4.) 1.983 1.970 1.957 1.970 0.026 0.50

Figure 3.7: EPR monitored redox titration of 98Mo -Transhydroxylase from P. acidigallici. Comparison of EPR spectra of TH “as isolated” form (spectrum number 2) oxidized with ferricyanide (spectrum number 1) and reduced with dithionite (spectrum number 3,4). The table shows EPR parameters of spectra 1, 2 and 4. Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature.

From EPR monitored redox titrations of 98Mo(V)-TH a change of the line shape was observed. After addition of 12 reduction equivalents, the EPR spectrum gave the following data: gz = 1.983, gy = 1.970, gx = 1.957, (Fig. 3.7, spectrum 3,4). The new signal 98Mo(V) indicated the high rhombicity comparison to other studies in this work Mo(V) signal of transhydroxylase. Although, oxidized TH (30 equivalent

50 Results and Discussion

ferricyanide/mol TH) gave a Mo(V) EPR signal with g-value at 1.988, 1.982, 1.965 (Fig 3.7, spectrum 1). Comparison of spin hamiltonian parameters for 98Mo and 95,97Mo signal show that after oxidation the 98Mo(V) signal indicate broader line widths than other Mo(V) signals. This effect of redox again on Mo(V) signal has not been described in literature so far.

95Mo(V) Hyperfine interaction For mononuclear Mo(V) sites (S=1/2, 4d1 system) 6 hyperfine lines are expected from the relationship 2I+1, with 95Mo I = 5/2. Because of the low natural abundance of 95,97Mo with I = 5/2 (25%) these hyperfine lines are usually hard to unravel in non- enriched biological samples. Analysis of the hyperfine structure in the Mo-95 spectrum obtained at microwave power of 0.63 mW provides the following data: gz = 2.041, gy = 1.972, gx = 1.899 (gav=1.97) with Az = 49G, Ay = 45G, Ax = 43G (Aav = 45G). The EPR spectrum of the same sample obtained at microwave power of 2.0 mW showed the signal at gz = 1.985, gy =

1.981, gx = 1.938, (gav=1.968) with Az = 48G, Ay = 31G, Ax = 50G, (Aav = 43G).

The most obvious nucleus to cause such a splitting would be a proton and this was proved by experiments in which the enzyme was dissolved in D2O instead of H2O. The doublet splitting observed at the signal 95Mo(V), disappeared only partially in the

EPR spectrum of TH exchanged with buffered D2O solution (Fig 3.8). Results indicated that the Mo splitting was influenced by several factors. Also the amino acids found in the metal’s environment had to be considered.

51 Results and Discussion

A B

g y g z g g z y g x g x

H O 2

D O 2 relative line intensity line relative

relative line intensity

3100 3200 3300 3400 3500 3600 3700 3100 3200 3300 3400 3500 3600 3700 Magnetic Field [G] Magnetic Field [G]

Figure 3.8: Mo (V) electron paramagnetic resonance (EPR) spectra of TH enriched with 95Mo. (A) TH in

Hepes/H2O, pH 7.5 (black line) and Hepes/ D2O, pD 7.5 (gray line) obtained at microwave power of 2.0 mW; (B) spectra of this same sample at microwave power of 0.63 mW. Instrument settings were 9.34 GHz microwave frequency, 10 G modulation amplitude, 10 K temperature

In the EPR spectrum of TH in D2O buffer the line shape of the gx and gz resonances remained unchanged in contrast to the gy area, where the shoulder disappeared upon deuteration, indicating that this shoulder must arise from proton hyperfine coupling. The anisotropic coupling was estimated from the line- broadening observed in TH in

H2O buffer, compared to that observed for the samples in D2O buffer. The reduction of line width arise from the smaller value of gnβn for deuterium (0.857440 µB) versus hydrogen (2.79285 µB) [McDevitt et al., 2002]. 95 The power decreasing change, observed in the Mo EPR spectrum (Fig 3.8 B, in H2O) was compared to the measurement of isotope enriched 95Mo(V)-cysteine complexes at room temperature with gav = 1.975 and Aav = 35G [Huang et al., 1970]. The broad component in the g region 2.08-1.87 suggested the presence of unresolved splitting(s) a no exchangeable proton with solvent, what shows signal upon D2O exchange [George et al., 1996]. The simulation of the 95Mo(V) signal was not successively in this work.

52 Results and Discussion

OH /OH 2 O Ser S S Mo V S S Figure 3.9: Proposed model of TH molybdenum active site

The proposed structure of TH molybdenum active site in +5 oxidation state indicated that a single Mo-O bond arises from Mo-OH2 or Mo-OH ligands. To exactly determine the coordinated ligands, the EXAFS study would be recommended.

Fe-S centers In earlier EPR studies two different types of EPR signals had been detected in TH from P. acidigallici which were assigned to Fe-S centers [Reichenbecher et al., 1999]. Later, the X-ray structure of TH clearly revealed the existence of three [4Fe-4S] centers in the ß-subunit of the αβ-heterodimer [Messerschmidt et al., 2004]. In this study two different signals could be resolved upon addition of dithionite to the enzyme “as isolated”, a major species with gz = 2.08, gy = 1.94, gx = 1.88, and a minor species with gz = 2.05, gy

= 1.95, gx = 1.88, in agreement with earlier results [Kisker et al., 1999].

2.05 1.95 2.08 1.94

1.88 12 g=2,08 g=2,052 10

a 8

P0,5 = 23,98 mW b 6

4

Relative signal intensity 2 c P0,5 = 2,56 mW

d 0 0.00 2.00 4.00 6.00 8.00 10.00 √ P [mW]

3100 3200 3300 3400 3500 3600 3700 Magnetic Field [G]

Figure 3.10: EPR spectra of Fe-S centers in Transhydroxylase from Pelobacter acidigallici. (A) (a) spectrum of TH (10 mg/ml in 50 mM TEA, 200 mM NaCl, pH 7.5) after addition of sodium dithionite

53 Results and Discussion

under anoxic conditions; (b) combined simulations (c) and (d) with weight 2:3; (c) simulation with gx=1.879, gy=1.954, gz=2.052, d simulation with gx = 1.880, gy = 1.940, gz = 2.08; (B) Microwave power saturation study at a range from 0.063 - 50.41 mW as a relation between microwave power and signal intensity at g =2.08 and g = 2.052. Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature.

The presence of two different types of [4Fe-4S] centers was confirmed by a power saturation study at 10K: the major species gave a P1/2 of 23.98 mW vs 2.56 mW for the minor center (Fig. 3.10 B). The values for gz, gy, gx and P1/2 calculated for both Fe-S signals were in good agreement with earlier results [Sommer, 1995].

Depending on the preparation the EPR signals of the Fe-S centers varied slightly with regard to their apparent g-values. A comparison of the Fe-S EPR parameters of TH with those of other Fe-S proteins revealed significant differences (Table 3.6).

Table 3.6: Spin Hamiltonian Parameters of paramagnetic Fe-S sites in Transhydroxylase from P. acidigallici and other Fe-S proteins

Enzyme species gx gy gz gav. rhombicity

TH 95,97Mo Fe-S 2.086 1.938 1.867 1.963 0.676

TH 95Mo Fe-S I 2.080 1.940 1.880 1.967 0.7 Fe-S II 2.052 1.952 1.880 1.961 0.58 TH 98Mo Fe-S I 2.080 1.938 1.875 1.964 0.69 Fe-S II 2.050 1.955 1.880 1.962 0.56 DMSOR 1) Fe-S 2.010 1.930 1.866 1.935 0.56

NarGH 2) Fe-S I 2.049 1.947 1.870 1.955 0.57 Fe-S II 2.010 1.885 1.871 1.922 0.89 Aconitase3) Fe-S 2.06 1.93 1.86 1.95 0.65

1) DMSOR: dimethyl sulfoxide reductase, [Cammack et al., 1990]; 2) NarGH: nitrate reductase, [Guigliarelli et al., 1992]; 3) [Beinert et al., 1996]

In the preparation of TH obtained from a culture grown on 95Mo molybdate, an intense signal of unknown origin with apparent g-values at 6.218 and 5.420 was observed (Fig. 3.11). A similar signal had been reported for nitrate reductase from E. coli and has been

54 Results and Discussion

tentatively attributed to a [4Fe-4S] center [Rothery et al., 2004]. EPR studies by Rothery performed on a variant of the enzyme lacking the molybdenum site revealed the presence of a 5th paramagnetic Fe-S center (g-values at 5.556 and 5.023) that had not been observed before [Gonzalez et al., 2006].

Figure 3.11: EPR spectrum of “as isolated” 95 Mo-TH (14 mg/ml in 50 mM TEA, 200 mM NaCl, pH 7.5) obtained new signal in low magnetic field. Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature.

EPR signals under turnover conditions TH catalyzes the conversion of pyrogallol to phloroglucinol in a reaction without apparent electron transfer. Hereby, 1,2,3,5-tetrahydroxybenzene, or other oxygen-donor molecules, serve as co-substrates [Reichenbecher et al., 1999].The proposed mechanism includes the formation of a diphenylether intermediate, as earlier proposed by Hille et al., (1999) [Messerschmidt et al., 2004]. Most recently, the chemistry of the tranhydroxylase reaction including the formation and decay of the proposed diphenylether compound has been investigated in more detail [Paizs et al., 2007].

55 Results and Discussion

A B

Figure 3.12: Crystal structure of Transhydroxylase from P. acidigallici: (A) TH with substrate pyrogallol bound (pdb, 1VLE) [Messerschmidt et al., 2004]; (B) model of TH active site with bound 1,2,3,5- tetrahydroxybenzene

Upon incubation of as isolated TH with pyrogallol, in the absence of dioxygen, the Mo(V) EPR signal with g-values at 1.997, 1.984, 1.961 decreased in intensity but did not change its lineshape (Fig. 3.13).

A B

Ser 175

H O S S O Mo H O O V S S

relative lineintensity

3100 3200 3300 3400 3500 3600 Magnetic Field [G]

Figure 3.13: EPR spectra of 95,97Mo-TH. (A) TH “as isolated” (15 mg/ml in 50 mM TEA, 200 mM NaCl, pH 7.5), gray line, and after 1h incubation with pyrogallol (5 mM) under exclusion of dioxygen, black line. Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature. under exclusion of dioxygen.(B) The right panel shows the bond between pyrogallol and the active site of TH as it was determinated by X-ray structure of the pyrogallol- TH complex [Messerschmidt et al., 2004].

56 Results and Discussion

In the X-ray structure of the pyrogallol-TH substrate complex, one hydroxyl group is coordinated to the molybdenum site (Fig. 3.12 A and Fig. 3.13 B, Mo-O bond 2.35 Å). Most likely, the pyrogallol displaces a water or OH group at the Mo center.

Incubation of TH with the co-substrate 1,2,3,5-tetrahydroxybenzene led to a decrease of the Mo(V) signal intensity. The spectrum additional showed weak signals with g-values at gx=2.008 and 1.94 attributed to reduced [4Fe-4S] centers (Fig. 3.14).

A B Ser 175

H O S S O Mo H O O V S Fe-S Fe-S S

O H relative line intensity

3100 3200 3300 3400 3500 3600 Magnetic Field [G]

Figure 3.14: EPR spectra of 95,97Mo-TH. (A) TH “as isolated” (15 mg/ml in 50 mM TEA, 200 mM NaCl, pH 7.5) gray line, and after 1h incubation with 1,2,3,5-tetrahydroxybenzene (5 mM) under exclusion of dioxygen, black line. Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature. (B) The right panel shows the proposed binding site of 1,2,3,5-tetrahydroxybenzene in the active site of TH.

The incubation of 98Mo-TH with 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether (5 mM) led to a decrease in intensity of the molybdenum EPR signal. This decrease, however, was minor (5%) compared to the decrease of intensity of the 95,97Mo(V) signal upon incubation with pyrogallol [Messerschmidt et al., 2004; Paizs et al., 2007]. In all cases studied, neither pyrogallol nor 1,2,3,5-tetrahydroxybenzene, or the diphenylether (2,4,6,3’,4’,5’-hexahydroxydiphenyl ether) intermediate led to a complete disappearance of the Mo(V) signal.

57 Results and Discussion

A B

Ser 175 S O OH S Mo V HO O S S

OH relative line intensity O

HO OH 3100 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 Magnetic Field [G]

Figure 3.15: EPR spectra of 98Mo-TH. (A) TH “as isolated” (15 mg/ml in 50 mM TEA, 200 mM NaCl, pH 7.5) gray line, after 1h incubation with 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether (5 mM) under exclusion of dioxygen, black line. Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature. (B) The right panel shows the proposed binding site of 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether in the active site of TH.

In the EPR spectrum of 4,4'-Difluorodiphenyl ether with 98Mo-TH a rhombic signal of Mo(V) with g-values at 1.99, 1.98, 1.96, was observed.

A B

Ser 175 S O F S H Mo V O S S

O relative line intensity F

3100 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 Magnetic Field [G]

Figure 3.16: EPR spectra of 98Mo-TH. (A) TH “as isolated” (15 mg/ml in 50 mM TEA, 200 mM NaCl, pH 7.5) gray line, and after 1h incubation with 4,4'-difluorodiphenyl ether (4 mM) under exclusion of dioxygen, black line. Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature. (B) The right panel shows the proposed binding site of 4,4'-Difluorodiphenyl ether in the active site of TH.

58 Results and Discussion

The EPR parameters very similar to those observed in TH 98Mo at pH 6.5, described in this work previously (see page 47).

Incubation experiments showed that the Fe-S centers of reduced TH coulde be fully oxidized by the enzyme’s substrate and intermediate - pyrogallol and 2,4,6,3’,4’,5’- hexahydroxydiphenyl ether, respectively (Fig. 3.17). No significant decrease of the Fe-S signal intensity was observed after addition of 1,2,3,5- tetrahydroxybenzene to the enzyme.

A B

Fe-S Fe-S

relative line intensity relative lineintensity

3100 3200 3300 3400 3500 3600 3700 3100 3200 3300 3400 3500 3600

Magnetic Field [G] Magnetic Field [G]

Figure 3.17: The effect on reduced Fe-S center of TH upon incubation with the substrate (pyrogallol) or intermediate (2,4,6,3’,4’,5’-hexahydroxydiphenyl ether). (A) [4Fe-4S]+ signal of 95,97Mo-TH (gray line) and after addition of 5 mM pyrogallol (black line); (B) [4Fe-4S]+ signal of partly reduced 98Mo-TH (gray line) and after addition of 5 mM 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether (black line). Instrument settings were 9.34 GHz microwave frequency, 2.007 mW microwave power, 10 G modulation amplitude, 10K temperature.

3.1.4. HPLC with 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether The reactivity of TH from P. acidigallici with the putative reaction intermediate (2,4,6,3’,4’,5’-hexahydroxydiphenyl ether, Prof. Retey syntesis) was investigated by HPLC. In the as isolated state, TH reacted only very slowly (3h incubation, Kpi buffer

59 Results and Discussion

pH 7.0, under exclusion of dioxygen) on the other hand, the dithionite-reduced enzyme reacted slightly faster, with a maximum activity of 1,25 U/mg vs native activity 4.15 U/mg. Under the experimental conditions, the formation of the co-substrate 1,2,3,5- tetrahydroxybenzene was not observed.

Figure 3.18: Possible electron transfer pathways of TH showing the involvement of Arg 740. The distances between N-pterin and N-Arg 740, 3.15 Å, and between N-Arg 740 and Fe-S cluster, 6.61 Å, are indicated.

Somewhat surprising, addition of the co-substrate 1,2,3,5-tetrahydroxybenzene led to a partial reduction of the Fe-S center with g-values at gx=2.008 and 1.94 on the ß-subunit of TH. Possibly, there exists an electron transfer pathway from the Mo(MGD)2 center via residue Arg 740 to the first Fe-S center nearby. The same electron pathway in the catalytic subunit of NarGHI from E. coli was described by Moura et al., 2004.

60 Results and Discussion

3.2. Acetylene hydratase of Pelobacter acetylenicus

3.2.1. Cultivation of Pelobacter acetylenicus Large amounts of cells of P. acetylenicus could be harvested following the protocol developed earlier by Abt (2001), which allowed to prepare the quantities of active acetylene hydratase in high yield and purity needed for the crystallization experiments and spectroscopic studies described in this thesis. Acetylene was introduced successively to the gas phase of the cultures of P. acetylenicus in 20-liter glass bottles, filled with 18-liters of medium [Schink, 1985].

0.9

0.8

0.7

0.6

0.5

578 nm 0.4 OD 0.3

0.2

0.1 0 102030405060708090100110120130 time [h]

Figure 3.19: Growth curves of P. acetylenicus. 18-l batch culture; C2H2 as sole energy and carbon source; 183 183 2- freshwater medium containing either tungstate or molybdate: ■ AH W (isotope enriched WO4 ); ◧ n.a 2- n.a 2- AH W (from natural abundance WO4 ); ● AH Mo (from natural abundance MoO4 )

During growth, the pH of the medium was maintained at pH 6.7 - 7.0 with 2 M Na2CO3.

Cells were harvested after 3 days (Fig. 3.19, A578 = 0.5).

61 Results and Discussion

3.2.2. Purification of Acetylene hydratase under exclusion of dioxygen AH containing either tungsten (W-AH) or molybdenum (Mo-AH) was purified to homogeneity according to SDS-PAGE in a five-step procedure in a Coy anaerobe chamber, filled with 94% N2 / 6% H2 (Table 3.7).

Table 3.7: Purification of AH of P. acetylenicus with tungstate n.aW (A) and 183W (B). Purification steps of protein from 15 g cells (wet weight) were documented by SDS-PAGE (12.5%). Activity: 1 U = 1 µmol acetylene · min-1. A

Purification step Protein Yield Specific activity kDa [mg] [%] [U· mg-1] 1 2 3 4 5 6 Crude extract (CE) 734 100 1.3 -73 Ammonium sulfate (AS) 300 41 2.3

Resource 30Q (R30Q) 78 10 5.7

Resource 15Q (R15Q) 23 3.1 10.0

Superdex (S200 ) 9 1.2 15.5

Lane 1: CE; Lane 2: AS; Lane 3: R30Q; Lane4: molecular weight markers; Lane 5: R15Q; Lane 6: S200

B

Purification step Protein Yield Specific activity kDa [mg] [%] [U· mg-1] 1 2 3 4 5 Crude extract (CE) 380 100 0.8 -73

Ammonium sulfate (AS) 259 68 1.1

Resource 15Q (R15Q) 24 6.3 6.8

Superdex (S200 ) 6 1.6 11.3

Lane 1: molecular weight markers; Lane 2: CE; Lane 3: AS; Lane4: R30Q; Lane 5: S200

62 Results and Discussion

Table 3.8: Purification of AH of P. acetylenicus with molybdate n.aMo. Purification steps of protein from 22 g cells (wet weight) were documented by SDS-PAGE (12.5%). Activity: 1 U = 1 µmol acetylene · min-1.

Purification step Protein Yield Specific activity kDa [mg] [%] [U· mg-1] 1 2 3 4 5 6 7

Crude extract (CE) 841 100 0.46 -73

Ammonium sulfate (AS) 370 44 0.82

Resource 30Q (R30Q) 250 30 0.9

Resource 15Q (R15Q) 54 6.4 1.14

Superdex (S200 ) 35 4.1 1.6

Lane 1: CE; Lane 2: AS; Lane 3: molecular weight markers; Lane 4: R30Q; Lane 5 R15Q; Lane 7: S200

Depending on the batch, the yield of pure AH amounted to 1.7 mg per 1 g cells. The activity of AH was determined at pH 7.5. Note that the pH optimum of the activity is around pH 6.5 which would increase the AH activity measured by approximately 30% [Rosner et al., 1995].

3.2.3. Spectroscopic characterization of the metal sites

3.2.3.1. Circular Dichroism The room-temperature CD spectrum of as isolated W-AH showed a negative band at 435 nm and positive shoulders at 320 and 540 nm, compared to Mo-AH with two negative band 385 nm and 625 nm, and positive shoulders at 320, 460 and 540 nm (Fig. 3.2). The same preparations were also used in the subsequently descibed EPR experiments (3.2.3.2). The positive shoulders around 320 nm in both W-AH and Mo-AH most likely can be assigned to a [4Fe-4S] cluster as reported for Ferredoxins (Fd I, Fd II) from Chlorobium tepidum, [Yoon et al., 2001].

63 Results and Discussion

A B 5 a 100 a 4 80 ) 3 -1 60 b ) dmol

-1 40

b ∗

2 2

*cm 20 cm

-1

∗

(M 1 0 (deg

Δε

−3 -20 0 -40

-1 [Θ]∗10 -60

-2 -80 190 200 210 220 230 240 250 260 270 280 300 350 400 450 500 550 600 650 Wavelength (nm) Wavelength (nm)

Figure 3.20: Room temperature CD spectra of “as isolated” acetylene hydratase from P. acetylenicus. (A) Visible CD spectrum of Mo-AH (a: gray line) and W-AH (b: black line); (B) Far-ultraviolet CD spectra of same samples as in (A). 10 mM Tris pH 7.6, enzyme concentration: 2 mg/ml (A) and 0.4 mg/ml (B), under exclusion of dioxygen.

Comparison of the CD spectra of Mo-AH with that of the glycerol inhibited Mo(V) form of DMSO reductase from Rhodobacter sphaeroides shows some similarities in the wavelength range 300-500 nm [Finnegan et al., 1993]. The CD spectra of the Mo-pterin prosthetic group has been reported for assimilatory nitrate reductase from Chlorella vulgaris in the wavelength range 410-650 nm and a positive shoulder at 333 nm [Kay et al., 1988]. These bands between ~550-700 nm are assigned as dithiolene-to-Mo(V) charge transitions (CT) [Benson et al., 1992].

The far-ultraviolet CD spectra revealed minor differences in the calculated secondary structural elements of W-AH and Mo-AH (Fig. 3.2 B): α helices (11.30, 14.30 %); antiparallel β sheets (34.10, 29.30 %); parallel β sheets (5.80, 6.10 %); β turns (18.60, 18.40 %). The higher content of α helices and lower content of β sheets in Mo- AH indicated a slightly different conformation of the protein.

64 Results and Discussion

3.2.3.2. EPR Spectroscopy Upon addition of sodium dithionite to a solution of W-AH, 183W-AH and Mo-AH, respectively, under the exclusion of dioxygen, an intense EPR signal at gav = 1.966±0.001 at 10K appeared as described earlier by Meckenstock et al., (1999), (Table 3.9). The rhombic line shape and the g values can be assigned to the presence of a low potential ferredoxin type [4Fe-4S] cluster [Cammack et al., 1985].

Table 3.9: EPR parameter of the [4Fe-4S]-center in acetylene hydratase from P. acetylenicus after reduction with 1.25 mM Na2S2O4. Samples (10mg/ml) were in 50 mM Tris/HCl 200 mM NaCl pH 7.5; spectra were recorded at 9.34 GHz, temperature 10K; modulation frequency 100 kHz; modulation amplitude 10 G; microwave power 2.007 mW

Enzyme sample gz gy gx gav W-AH nat.ab. 2.046 1.935 1.918 1.966 183W- AH 2.048 1.935 1.920 1.967 Mo-AH nat.ab. 2.045 1.933 1.917 1.965

W-AH as isolated under the exclusion of dioxygen did not show any EPR signals in contrast to Mo-AH which showed a weak EPR signal at gav = 1.997 which was assigned to Mo(V) carrying a coordinated hydroxo-group in addition to the two MGD ligands (Table 3.10, Fig. 3.21 B).

A B

2.023 1.99 2.044 2.012

1.978 1.995

EPR Signal [a.u.] EPR Signal [a.u.] Signal EPR

2.049 2.014

3100 3150 3200 3250 3300 3350 3400 3450 3500 3550 3100 3200 3300 3400 3500 3600 Magnetic Field [G] Magnetic Field [G]

65 Results and Discussion

3- nat.ab Figure 3.21: EPR spectra of W(V)-AH and Mo(V)-AH. (A) Enzyme plus [Fe(CN)6] : W-AH (gray line) and 183W enriched AH (black line); (B) Mo-AH “as isolated”, from two molybdate cultivation. EPR conditions: microwave frequency, 9.4 GHz; modulation frequency, 100 kHz; modulation amplitude, 10 G; microwave power, 2.0 mW, 10K

3- 183 Upon addition of [Fe(CN)6] to W-AH (nat. abun- and W-AH), in the absence of dioxygen, a new EPR signal at gav = 2.017 and gav = 2.025 (Table 3.10) was observed resulting from a W(V) center [Meckenstock et al., 1999]. The g-values of the EPR signal of W(V) are clearly higher than those of the signal observed in “as isolated” Mo-AH, i.e. W(V) signal at gav = 2.02 vs Mo(V) at gav = 1.99. The two signals differ also markedly in linewidth (Table 3.10).

Table 3.10: Representative EPR parameters for various tungsten and molybdenum enzymes

Enzyme gz gy gx gav. gzz – gxx gzz – gyy / gzz – gxx anisotropy rhombicity AH W 2.044 2.012 1.995 2.017 0.049 0.65

AH 183W 2.048 2.014 2.014 2.025 0.034 1

AH Mo 2.023 1.99 1.978 1.997 0.045 0.73

a W-AOR 2.03 2.01 2.002 2.014 0.028 0.71

b W-FDH I 2.049 2.012 1.964 2.008 0.085 0.43

c Mo-Psr 2.026 1.99 1.99 2.002 0.036 1

d Mo-Nap 2.005 1.996 1.989 1.997 0.016 0.59 a aldehyde oxidoreductase from C. formicoaceticum [Huber et al., 1994]; b tungsten-substituted molybdenum formyl-methanofuran dehydrogenase I from M. wolfei [Schmitz et al., 1992]; c molybdenum polysulfide reductase from W. succinogenes [Prisner et al., 2003]; d molybdenum periplasmic nitrate reductase from E. coli [Jepson et al., 2007]

An EPR spectrum most similar to that of W-AH was reported for aldehyde oxidoreductase (W-AOR) from C. formicoaceticum. In this case, the tungsten site

66 Results and Discussion

carries one oxo and one sulfide group [Huber et al., 1994]. Nitrate reductase from

E. coli contains one Cys residue and one OH/OH2 coordinated to molybdenum. Within the members of the DMSO reductase family, it shows the highest similarity to W-AH as confirmed by the X-ray structure [Jepson et al., 2007]. To answer the questions about the architecture of the mediate oxidized form of AH we proposed the structure model shown in Fig. 3.22.

HOH OH O MGD 1 S S-Cys MGD 1 S S-Cys MGD 1 S S-Cys W W W S S IV S V S S VI S S S S

MGD 2 MGD 2 MGD 2

Figure 3.22: Proposed models of the W active site structure of acetylene hydratase in the oxidation states IV, V, and VI, [Musgrave et al., 1999; Enemark et al., 2004].

3.2.3.3. Reaction with Acetylene and Derivatives The incubation of as isolated W-AH with acetylene did not produce any significant EPR signal either from tungsten or from the Fe-S center. To achieve activity, W-AH had to be reduced by dithionite, or Ti(III)-citrate. Incubation of reduced W-AH (1h, 18°C, 10 equivalents of dithionite) led to substantial changes both of the UV/Vis and the EPR spectrum. The signal from the [4Fe-4S] cluster disappeared accompanied by a decrease in absorbance around 320 nm (Fig. 3.23). With propargyl alcohol, HC≡C-CH2OH, a similar but less pronounced effect was observed.

67 Results and Discussion

A B

g-Value 0.8 0.40 2.14 2.10 2.07 2.03 2.00 1.97 1.94 1.91 1.88

0.7 0.35

0.30 0.6

0.25

0.5 0.20

0.4 0.15

0.10 290 300 310 320 330 340 350 360 370 380 Absorbance 0.3 EPR Signal [a.u.] Signal EPR

0.2

0.1

0.0 200 300 400 500 600 700 800 3100 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 Wavelength [nm] Magnetic Field [G]

Figure 3.23: Reaction of dithionite-reduced W-AH with C2H2. (A) UV/Vis spectra of W-AHred (solid line); reaction with C2H2, three step addition of 2 ml of acetylene, measurement time 5 min (dashed and dotted line); (B) EPR spectra of W-AHred (black line); reaction with C2H2 (gray line), 250 µl reduced sample incubated with 10 ml of acetylene. EPR conditions: microwave frequency 9.4 GHz; modulation frequency 100 kHz; modulation amplitude 10 G; microwave power 2.0 mW, temperature 10K. All experiments prepared under N2/H2 atmosphere.

3- W-AH as isolated, after incubation with the oxidant [Fe(CN)6] (2.8 mM oxidant, 30 min, 18°C) also reacted with acetylene and propargyl alcohol, as indicated by the appearance of a new EPR signal centered at g = 2.008 (Fig. 3.24). This new EPR signal had not been seen before for W-AH and related tungsten enzymes. The results of these EPR monitored studies with acetylene, propargyl alcohol, and other potential substrates have been compiled in Table 3.11

68 Results and Discussion

A B 2.044 2.012 1.995 2.049 2.014 2.040 2.013 1.996

2.008

2.009

EPR Signal [a.u.] Signal EPR EPR Signal [a.u.] Signal EPR

3150 3200 3250 3300 3350 3400 3450 3150 3200 3250 3300 3350 3400 3450 Magnetic Field [G] Magnetic Field [G]

183 Figure 3.24: EPR spectra of W(V)-AH complex with the C2H2 and propargyl alcohol. (A) W(V) species (gray, solid line) in complex with acetylene (black line) where the gray dashed line show mediate state of the titration; (B) W(V) species (gray, solid line) in complex with propargyl alcohol (black line) where the gray dashed line show mediate state of the titration. The acetylene and propargyl alcohol were with AH 1h incubated. EPR conditions: microwave frequency 9.4 GHz; modulation frequency 100 kHz; modulation amplitude 10 G; microwave power 2.0 mW, temperature 10K

Table 3.11: EPR monitored reaction of W-AH and Mo-AH with different substrates.

Specific activity EPR Compound Enzyme Form [U· mg -1] W-AH Reduced 15.5 Reaction (Fig.3.23) Acetylene Oxidized - new EPR signal (Fig.3.24) Mo-AH Reduced 1.6 - W-AH Reduced 14.8 Reaction similar to Fig 3.23 Acetylene in DMSO Mo-AH Reduced 2.1 - Reduced 2.5 Reaction similar to Fig 3.23 Propargyl alcohol W-AH Oxidized - new EPR signal (Fig.3.24) As isolated 0 New EPR signal (10K) Ethylene Mo-AH Reduced 0 - Oxidized - -

NO (papaNOate) Mo-AH As isolated - New signal

69 Results and Discussion

3.2.4. Metal content of Acetylene hydratase In order to correlate activity and spectroscopic properties of the various preparations of AH, their W/Mo and Fe content was determined by ICP-MS (Table 3.12).

Table 3.12:Metal analysis by ICP-MS of acetylene hydratase from different preparations. Mo-AH from molybdate cultivation; W-AH from tungstate cultivation; Acetylene hydratase analyzed was 95 2- take from tungstate cultivation by Krieger (1997) and from tungsten or molybdate ( MoO4 ) cultivation by Abt (2000), respectively. Enzyme concentration: about 2.0 mg ml-1

AH-Sample Fe Mo W Fe/M Specific activity

mol mol mol [U mg-1] in 30°C

Mo-AH 2.74 0.51 0.003 5.4 1.6

This work

Mo-AH 3.11 0.51 0.01 6.1 ----

Abt (2001) W-AH 3.64 0.00 0.34 10.7 16.5 This work W-AH 3.52 0.00 1.13 3.1 22.0 Abt (2001) W-AH 3.61 0.00 0.38 9.5 12.56 Krieger (1997)

Clearly, in all preparations both the tungsten and the molybdenum site were much less occupied compared to iron in the [4Fe-4S] cluster. Especially the low occupancy of the tungsten site will become important for the following analysis and discussion of the X- ray structure of W-AH.

3.2.5. Reaction with [14C]- acetylene Both W-AH and Mo-AH (“as isolated” and reduced with dithionite) were incubated with [14C]-acetylene to investigate the affinity of AH towards its substrate acetylene, and to localize potential binding sites. This technique has been developed in the laboratory of Prof. O. Meyer (Bayreuth) and has been already successfully applied to

70 Results and Discussion

ammonia monooxygenase (Dr. Ingo Schmidt and PhD student Stefan Gilch, private communication; VAAM 2006). Incorporation of 14C from [14C]-acetylene into the polypeptide chain of both W-AH and Mo-AH was observed. Hereby, both enzymes (as isolated) showed a similar uptake of the 14C label (Fig. 3.25). In the case of the reduced enzymes, Mo-AH was more active compared to the W-AH with regard to labeling with 14C.

A M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8 B

73 - -73 kDa kDa

Figure 3.25: Electrophoretic analysis of the 14C-labelled acetylene hydratatase from P.acetylenicus. (A) Coomasie blue-stained SDS-Page 12% of AH; (B) Flurometric image of [14C]-acetylene-labeled AH taken from the SDS-PAGE shown in A; Lanes 1: Mo-AH “as isolated”; Lanes 2: Mo-AH reduced with dithionite; Lanes 3: W-AH “as isolated”; Lanes 4: W-AH reduced with dithionite; Lanes 5: W-AH “as isolated” incubated with [14C]-acetylene; Lanes 6: reduced with dithionite W-AH and incubated with [14C]-acetylene; Lanes 7: Mo-AH “as isolated” incubated with [14C]-acetylene; Lanes 8: reduced with dithionite Mo-AH and incubated with [14C]-acetylene. M: molecular weight marker.

3.2.6. Crystallization of W-AH and three-dimensional structure The quality of the first diffraction data of W-AH crystals was not sufficient enough to obtain suitable electron density maps which would allow proper model building [Einsle et al., 2005; Niessen, 2004]. In addition, under those conditions no crystals suitable for X-ray analysis could be obtained.

71 Results and Discussion

3.2.6.1. Crystallization P. acetylenicus acetylene hydratase was crystallized using sitting drop vapour diffusion and a reservoir solution of 0.1 M sodium cacodylate at pH 6.5, 0.3 M magnesium acetate, 21% polyethylene glycol 8000 and 0.04 M sodium azide. Crystals formed under strict exclusion of oxygen over a period of 1-3 weeks at 20°C temperature (Fig. 3.26). 2 µl of protein solution (10 mg/ml in 5mM HEPES/NaOH pH 7.5) reduced by sodium dithionite (a final concentration 5 mM) were mixed with 2.2 µl of precipitant solution. Crystals were transferred into a buffer containing the mother liquor with 15% (v/v) of 2- methyl-2,5-pentanediol as a cryoprotectant and flash-cooled in liquid nitrogen.

Figure 3.26: Crystals of acetylene hydratase and diffraction image of AH crystal. The image was recorded on a MarCCD detector on beamline BW6 at DESY, Hamburg with a rotation of 0.5° during exposure and limiting resolution of 1.26 Å at an X-ray wavelength of 1.05 Å.

The crystals were light yellow-brownish colored, plate shaped and belonged to space group C2 with a unit cell dimension of a=120.8 Å, b=72.0 Å, c=106.8 Å (Fig. 3.26). Assuming a molecular weight of 83 kDa with one monomer per asymmetric unit and 3 -1 the packing densities VM of 2.35 Å Da the a solvent content corresponded to 47.59 %.

72 Results and Discussion

3.2.6.2. Structure determination X-ray data were collected at beam lines BW6 (native data set and high redundancy SAD data set) and X11 (native data set) at DESY in Hamburg, Germany.

As a prerequisite for SAD data collection, an X-ray fluorescence scan was carried out to determine the exact position of the iron K-edge of AH (Fig. 3.27).

A B

Figure 3.27: The X-ray fluorescence measurements of the AH crystal used to choose exact energetic location of the absorption edge for SAD experiment. X-ray fluorescence spectra are recorded for scaling purposes at λ =1.05 Å (A) and of the K-Fe (red line) absorption edge at λ = 1.738 Å (B)

For these scans, the wavelength for SAD data collection was set at λ = 1.738 Å to maximize the anomalous contribution of the iron atoms. A second, remote, data set was collected at λ = 1.050 Å. The native structure was solved by the single anomalous diffraction technique using data set (rotation angle 360° and oscillation range a 0.5°) of the Fe absorption edge with 1.95 Å resolution. At the energy of the iron K-edge, the anomalous signal of tungsten corresponded to approximately 6.8 e-, such that this atom appeared as the strongest peak in an anomalous difference Patterson map. Heavy metal sites were located with the program SHELXD [Schneider et al., 2002], which produced five clear solutions, corresponding to the four iron atoms of the [4Fe-4S] cluster and the tungsten atom. The phase for model building was calculated with the SHARP program [De la Fortelle et al., 1997]. To facilitate model building, a bulk solvent correction was carried out with SOLOMON [Abrahams et al., 1996]. This led to an electron density map of sufficient quality to build automatically a 61% of the model of AH using

73 Results and Discussion

RESOLVE [Terwilliger et al., 2004]. All subsequent manual rebuilding steps were carried out using COOT [Emsley et al., 2004]. The model was refined using REFMAC5 [Murshudov et al., 1997].

A B

Figure 3.28: Representative electron densities. (A) Experimental density of AH after SAD phasing and remote with 61% the model, (B) The same region of the protein with the final, refined 2FO – FC electron density contoured at 1.5 σ. This pictures show characteristic by AH the loop ranging with privileged amino acid residues 335’-336’.

After building of the model the structure was completed and refined to a final value with a crystallographic R-factor (Rfree) of 0.160 (0.199) in a resolution range of 1.26 Å. The model consists of 730 amino acid residues, 880 water molecules, two MGD cofactor molecules and one [4Fe-4S] cluster. Additionally, two MPD molecules, one acetate molecule and sodium ion from the crystallization buffer were detected in the crystal structure of AH.

74 Results and Discussion

Table 3.13: Data collection and refinement statistics

Data sets SAD Remote 1 Remote 2

Wavelength, Å 1.738 1.050 1.050 Space group C2 C2 C2 a = 120.9 Å; a = 120.8 Å; a = 121.0 Å; b = 72.1 Å; b = 72.0 Å; b = 72.2 Å; Unit cell dimensions c = 106.9 Å; c = 106.8 Å; c = 106.8 Å; β= 124.3° β= 124.3° β= 124.3° 50.0 – 1.95 50.0 – 1.26 50.0 – 1.10 Resolution limits, Å (2.05 – 1.95) (1.35 – 1.26) (1.20-1.10) Completeness, % 93.4 (88.9) 95.5 (92.5) 95.2 (92.2) I/ (I) 29.2 (13.3) 19.62 (1.91) 13.8 (1.3) 0.0797 a Rmerge 0.053 (0.146) 0.036 (0.333) (0.429) 0.0473 b Rp.i.m. 0.021 (0.057) 0.037 (0.332) (0.253) Multiplicity 7.04 (6.58) 1.85 (1.65) 3.64 (3.51) Phasing power (anomalous) 1.400 — — c

d RCullis 0.717 — — Figure of merit 0.390 — — Correlation (SOLOMON) 0.723 — — e Rwork — 0.160 0.18

Rfree — 0.199 0.20 Overall correlation — 0.972 0.971 coefficient rmsd in bond distances, Å — 0.010 0.007 rmsd in bond angles, ° — 1.419 1.225 a R = I − I I ; b R , [Weiss et al., 1997]; c anomalous phasing merge ∑hkl ∑hkl p.i.m power = 2 F " E where F '' is the anomalous contribution amplitude; ∑n H ∑n H d R = F ± F − F F ± F for centric reflactions and isomorphous cullis ∑hkl PH P H (calc) ∑hkl PH P contributions; e R = F − F F ; work ∑hkl ()obs calc ∑hkl obs

75 Results and Discussion

The high resolution data set (1.1 Å) was key to develop a refined picture of the active site of W-AH. Earlier, the relation between resolution and calculated density was described for the oxo ligand and Moco cofactor [Dobbek et al., 2002]. Accordingly, from the high resolution structure of AH, valuable information about the type of hydroxo/water ligand at the active site could be derived, which helped to postulate a first catalytic mechanism for W-AH. Furthermore, the fine quality of the crystal data helped to identify two forms of the enzyme: apo-AH and W-AH. As discussed earlier, all preparations of AH were relatively low with regard to the occupancy of the tungsten site, which can be clearly seen in the crystal data. Consequently, better protocols have to be developed for the purification of AH to produce enzyme with fully occupied tungsten, or molybdenum, sites.

3.2.6.3. Description of the structure

Overall structure

Acetylene hydratase is a monomer enzyme with approximate dimensions of ~ 69Å x 72 Å x 56 Å. Located at the N-terminus a cubane type [4Fe-4S] cluster and two MGD cofactors as ligands for tungsten atom can be found. The polypeptide is organized into four domains. Domain I (blue) comprises residues 4 – 60; domain II (yellow) comprises residues 65 - 136 and 393 – 542; domain III (brown) comprises residues 137- 327 and domain IV (green) comprises residues 590 – 730 (Fig. 3.29). This arrangement is similar to that observed in the structure of other enzymes in members of the DMSO reductase family. The global arrangement of the four domains shows a clear twofold pseudo-symmetry [Dias et al., 1999].

76 Results and Discussion

A B

Figure 3.29: Overall structure of AH. Cartoon representation shows four domains arrangement (I-IV in wheat, olive, red and green, respectively) typically found in members of the DMSO reductase family. The region coloured gray is the region differently arrangement in comparison to all members.

The [4Fe-4S] cluster identified by EPR is located in domain I close to the N terminus of the protein and coordinated by Cys9, Cys12, Cys16 and Cys46. This domain is formed by three β strands and two small helices. The α/β fold of the subsequent domains (II and III) displays a topology similar to domains found by nitrate reductase and all relevant proteins containing the typical NAD-binding fold. The C-terminal domain IV is dominated by a six-stranded β-barrel structure. The two MGD cofactors make multiple hydrogen-bonding interactions with residues from domain II, III, and IV of the protein.

77 Results and Discussion

Key:

= extended strand, = turn, = alpha helix, = 310 helix,

PDB Domain I Domain II Domain III Domain II Domain IV 2e7z (A) ____Ss−Sh −−−−ssHs− Hhhs− shShShHsh−−−−− hhShhsh−−−−hhhhSh−hSh −Shshhhhs−−−−−−− shhshshSsshhs 2iv2 (X) ____Ss−Sh −−−−− Hs− Hhhs− hhShShHhh−−−−−− hShhs−−−−− hhhSh−hSh −Ssshhh−−−−−−−−−−− shshSsssh- 2nap (A) ____Ss−Sh −−−− hHs− Hshh− shhShShHsh−−−−−− hShsh−−−−−− hhSh−hSh −Shsshhh−−−−−−−−− sshhshSsshs- 1h0h (K) ____Ss−Shh−−−− sHsssHhhh− shhShShHhh−−−− hhShshh−−−− hhhSh−hShh Shsshhhsshh−− hhhssshhshSsshs- 1ogy (G) ____Ss−Sh −−−−− Hs− Hshhh− shhShShHsh−−−− hhShhs−−−−− hhhSh−hSh −Shsshhhh−−−−−−− hhsshshSsshs- 1vld (U) Ss−SsshhshhsHs− Hshhhh shhShShHhh−−−−−− hShhhhh−−− shssSh−hSh −Shshshhhs−−−−−− hhhshshSsshs-

Figure 3.30: Sequence and secondary structure of P. acetylenicus AH. Comparison of secondary structure of AH with the members of DMSO reductase family. Multiple alignment of Acetylene hydratase (2e7z) with Formate dehydrogenase H from E. coli (2iv2), dissimilatory Nitrate reductase from D. desulfuricans (2nap), W-Formate dehydrogenase from D. gigas (1h0h), Nitrate reductase R. sphaeroides (1ogy) and Transhydroxylase from P. acidigallici (1vld) are shown

78 Results and Discussion

The secondary structure of AH had 46% helical (40 helices; 338 residues) and 15% beta sheet (27 strands; 115 residues) character. The enzyme was compared to other structurally characterized molybdenum enzymes such as formate dehydrogenase, nitrate reductase. The results of a multiple alignment of AH with structural homologous enzymes showed 12 highly similar secondary structure elements (Fig. 3.30). The alignment gives a 54% identity of structurally equivalenced residues between AH and nitrate reductase (2nap) and indicates 46% structural identity of AH to formate dehydrogenase (2vi2). From a superposition of the structures an rms deviation Cα atoms of 2.342 Å and 2.508 Å was obtained for the AH aligned to nitrate reductase or formate dehydrogenase, respectively (Fig. 3.31). Additionally, the comparison of the amino acid sequences of both enzymes show only 25% (Fdh) and 23% (NapA), respectively, identity to AH.

A B

Figure 3.31: Superimposition of AH (green) and NapA (2nap: blue) and Fdh (2iv2: aqua blue); backbones (A) and cartoons (B) in an orientation where the active site channel of AH is visible. A view (A) show superimposition of ligands: bis-MGD cofactors (wheat), [4Fe-4S] (wheat for sulfur and yellow for Fe atom), and molybdenum or tungsten atom (dark red). Structure Alignment Results, http://www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver

79 Results and Discussion

The overall arrangement of AH metal sites is similar to those observed in Fdh, NapA and other members of the DMSO reductase family, where one of MGD molecules has an elongated conformation in close proximity to the [4Fe-4S] cluster. Main differences between the AH and NR or Fdh are observed at the rearranged region of AH between residues 327 and 393, as shown at the Figure 3.31 and Figure 3.34 in gray colors.

Arrangement of metal sites The active site of acetylene hydratase is clearly the tungsten ion coordinated by the sulfur atoms of dithiolene moieties of two MGD cofactors and this coordination is completed by cysteine residue (Cys-141). The same architecture of the active site was found in the dissimilatory nitrate reductase from D. desulfuricans and R. sphaeroides [Arnoux et al., 2003; Dias et al., 1999]. The coordination sphere around W ion includes at sixth position an oxygen ligand. In the reduced form the W-O bond length is 2.04 Å (Fig. 3.32).

A B

HOH C 2.04 MGD 2 S S-Cys 2.40 2.32 2.28 W 2.34 S IV S 2.52 S

MGD 1

Figure 3.32: The tungsten center of acetylene hydratase from P. acetylenicus. (A) View of W-bis-MGD center, (B) representation of the 2_Fo_Fc_ electron density map contoured at 1.5σ, (C) schematic representation of the active site structure with the atom distances.

80 Results and Discussion

Reaction of AH with K3[Fe(CN)6] resulted in an oxo ligand bound to the metal in a distance of 1.7 Å, whereas the rest of the structure remaines essentially unchanged. The lengths of the W-S bonds range from 2.28 – 2.52 Å (see Figure 3.32).

The tungsten and molybdenum centers of the enzymes belonging to the DMSO reductase family is approximated by a square pyramidal or trigonal prismatic geometry [Sigel et al., 2002]. In AH, a slight rotation of the MGD 1 (P-MGD in literature) cofactor yields a geometry that more closely resembles octahedral, or trigonal antiprismatic.

Examination of a |Fo – Fc| electron density map confirmed the position of a bound oxygen to the metal (Fig 3.32 B). The W-O bond distance derived from the crystal structure of other enzymes in this class is ambiguous, the values expected for a hydroxo ligand amount to 1.9 - 2.1 Å and for a coordinated water to 2.0 - 2.3 Å, depending on structure resolution. In AH, the high resolution data set (1.26 Å and 1.1 Å) required that the oxygen come from water molecule as from hydroxyl group, in reduced state of the enzymes. The distance of 2.004 Å was indicated by the high resolution structure (at

1.28 Å) for Mo-OH2 bond of aldehyde oxidoreductase [Rebelo et al., 2001]. Additionally, the density-functional theory calculation for W-O bond indicated to bonds the oxygen as oxo ligand in the oxidized W6+ state and favors water ligand in reduced W4+ state of metal site in AH. The electron density map was calculated from high resolution data set with the 40% occupancy of the metal site, the tungsten amount in AH was determined by ICP mass spectroscopy. The tungsten atom is located at the bottom of the deep cavity 11 Å away from the closest iron atom of the cubane type [4Fe–4S] cluster (Fig. 3.33 B). These findings are in analogy to what is observed in the other molybdopterin-containing enzymes (NapA and Fdh) where electron-transfer pathway occurs through the bonds. However in AH redox chemistry does not take place. In other DMSO reductase family proteins the Fe-S center makes a close interaction with one of the MGD moieties with the help of a water molecule. This water molecule is positioned between the conserved Lys47 (by NapA) and pterin ring of MGD 2 (Q-MGD in literature) and considered to be essential for electron transfer [Jepson et al., 2007].

81 Results and Discussion

A B

Figure 3.33: A view of [4Fe-4S] cluster and W-bis-MGD cofactor in the W-AH structure showing the distance between both metal sites and key amino acid residues. (A) Electrostatic surface representation of the α-helices (residues 180 - 192) with positive charged in blue and negative in red, (B) View of Fe-S center sphere with conserved Lys 48 at 3.0 Å distance to MGD 2 and conserved Asp 13 residue as such structural and/or functional role.

The same structural motif was observed in the W-AH structure, however the water molecule was not found in the space between the iron-sulfur cluster and the pterin moiety, such functionality is obviously not required in the enzyme (Fig. 3.33 B). On the other hand, close to the [4Fe-4S] cluster in AH an α-helix belonging to domain III was observed (Fig. 3.33 A). This helix is oriented to point directly at the [4Fe-4S] cluster of domain I, with the positively polarized end of its helix dipole, and contributes to the low redox potential of -410 ± 20 mV observed for this center [Meckenstock et al., 1999].

The substrate channel In the AH the region between domains II and III (residues 327-393) forms the loop region which is completely different by comparison to the other known DMSO reductase family enzymes. The loop position is shifted by more than 15 Å towards the protein surface.

82 Results and Discussion

A B

Figure 3.34: Surface representation of the domain arrangement and schematic view of active site access. (A) View of AH the four-domain structure which is typically found in members of the DMSO reductase family. (B) Active site access for all members has a pathway along the red cone with the exception of AH which use the black cone. A schematic view was prepared by Prof. O. Einsle.

In formate dehydrogenase and nitrate reductase this loop separates the Mo/W center from the [4Fe-4S] cluster, and its displacement in AH opens up a new face of the protein surface. The superimposed structures of AH and Fdh, NapA showed β strand belong to aligned proteins Fdh and NapA in enter of the AH funnel. The tungsten active site is accessible from the surface through the funnel-like cavity (a structural feature also observed by molybdopterin-containing enzymes). All members of DMSO reductase family have an active site access between domains II and III in contrary to AH where the access is provided from a completly different angle at the intersection of domains I, II and III to the active site (Fig. 3.34 B). Additionally, there were no separate pathways for substrate and product. In AH a substrate channel approximately 17 Å deep and 6-8 Å wide, is situated at a position close to the N-terminal domain (domain I) that harbors the [4Fe-4S] cluster in opposite to all members enzymes. Probably this channel position has been in close correlation to the function of Fe-S center in AH, as they is no electron transfer involved during catalysis. Cys 12 liganded to the [4Fe-4S] cluster interacts with its neighboring

83 Results and Discussion

residue Asp 13 in such a way that a short hydrogen bond of 2.4 Å is formed between Asp 13 and the water molecule bound to the W ion (Fig. 3.35).

A B

Figure 3.35: A view of the active site channel. (A) Cartoon and (B) surface presentation of the hydrophobic residue ring with the cofactors looking into the active site channel of acetylene hydratase of P. acetylenicus. Electrostatic surface potential colored in blue for a positive and in red for negative potential (cutoff +10 and -10, respectively). The distances between hydrophobic residues and tungsten ion are in range of 4.41-6.20 Å and distance between Asp-13 and water coordinated to tungsten ion 2.4 Å

The substrate access funnel ends in a ring of hydrophobic residues, made up by Ile 14, Ile 113, Ile 142, Trp 179, Trp 293 and Trp 472 (Fig. 3.35). These residues with the tungsten atom and the coordinated water molecule combine to create a small binding cavity with dimensions that make it a perfect mold for binding the substrate-acetylene. The electrostatic potential surfaces of AH shown in Figure 3.36 suggests a strongly acidic protein. A negative electrostatic potential around and inside of the active site channel indicates preference for cationic intermediates or product, whereas the substrate

C2H2 is uncharged. A negative potential inside the channel was confirmed by density- functional theory calculations, which also predicted that residue Asp13 remained fully protonated.

84 Results and Discussion

Figure 3.36: Electrostatic surface representations of the tungsten containing AH structure. The surface viewing of the tungsten funnel face of the AH structure and ration at 90° and 180° around the y axial of the structure. Electrostatic surface potential colored in blue for a positive and in red for negative potential (cutoff +10 and -10, respectively).

Factors that affect the reduction potentials of [4Fe-4S] clusters include electrostatic surface potential, with more negative surface potential predicted to make the cluster more difficult to reduce [Schindelin, et al., 1996; McAlpine, et al., 1998], which was observed in nitrate reductase enzymes (recombinant R. sphaeroides NapA E = -250 mV) [Jepson et al., 2007]. The electrostatic surface of AH similar to the surface of R. sphaeroides NapA (1ogy) and requires the most negative surface potential of all members of these family.

3.2.6.4. Binding of substrate and related compounds In most cases soaking of small molecules into crystals of W-AH – obtained in the presence of reductant - did not alter their diffraction properties significantly, with the exception of acetylene dissolved in acetone. In summary, no conditions could be worked out so far to detect acetylene, or related compounds, in the crystals of W-AH. Even with propargyl alcohol which had been used successfully to localize its binding site in nitrogenase [Lee et al., 2004] no binding to W-AH could be achieved in the crystalline state.

85 Results and Discussion

Table 3.14: Substrate and derivates used in crystal soaking experiment of W-AH with determination of X-ray structure

Substrate Substrate Soaking time Resolution Completes [%] concentration [min] Å Acetylene/acetone 10% in drop 5-10 1.7 96.8 (Saturated solution) Acetylene/DMSO 15% in drop 10-20 1.9 99.9 (Saturated solution)

Methylcarboxy acetylene 5mM 15 1.6 90.0 NO (PapaNOate) 5mM 10 1.6 99.6 Propargyl alcohol 15% in drop 10 1.7 96.2

Acetylene (C2H2) Gas pressurization 1.6 99.3%

Acetylene (C2H2)* Gas pressurization 1.6

N2/H2 (94% / 6%) Gas pressurization 2.3 92.2 Xe Gas pressurization 1.7 96.3

* AH oxidized with K3[Fe(CN)6]

Through shape complementarity, the residues of the hydrophobic ring thus are a key determinant for the substrate specificity of the enzyme. The selectivity of the substrate through the channel was clearly shown in the native structure of W-AH or in the structure with Xe.

A B

Figure 3.37: A view of MPD and Xe binding site in acetylene hydratase of P. acetylenicus. (A) View of the active site channel with two MPD molecules, which cames from cryo-buffer; (B) View of the Xe binding site far away of the channel 86 Results and Discussion

In the native structure the two MPD molecules are found in the channel, but none in the small substrate cavity. After the pressurization experiment with Xe, the crystal structure showed the Xe atom far away from the channel and active centers.

3.2.7. Towards the reaction mechanism of Acetylene hydratase The three-dimensional structure of acetylene hydratase presented in this thesis provides valuable information to identify the protein residues essential for substrate binding, and to postulate a structure-based reaction mechanism of this rather unusual tungsten enzyme. In organometallic chemistry, the hydration of alkynes is catalyzed by mercuric ion, 2+ Hg through the formation of a cyclic mercurinium ion. In a consecutive step, water attacks the most substituted carbon atom of this cyclic intermediate followed by the formation of a mercuric enol which then rearranges to the corresponding ketone. The type of reaction is not achieved by acetylene hydratase. The hydration of acetylene to acetaldehyde catalyzed by W-AH requires the presence of water and a strong reductant [Meckenstock et al., 1999; Rosner et al., 1995]. In the structure of W-AH the tungsten center is most likely in the W(IV) oxidation state. It is coordinated to five sulfur atoms – four from dithiolene sulfur, and one from cysteine – and as a sixth ligand functions a water molecule or hydroxo group which can become activated through interaction with the nearby residue Asp 13. Additionally, the structure reveals in small binding cavity sixteen well-defined water molecules in close neighborhood to the active site. On the basis of the three-dimensional structure of W-AH, and general chemical considerations, the following mechanisms for the formation of acetaldehyde from acetylene can be brought forward:

1) Electrophilic additions in chemistry – acetylene is a nucleophile. In the first step of the reaction the electrophilic H+ add to acetylene and vinylic cation would be formed.

In the enzyme, a tungsten liganded H2O molecule would gain a partially positive net charge through the proximity to the protonated Asp 13, making it an electrophile that in

87 Results and Discussion

turn could directly attack the triple bond in a Markovnikov-type addition reaction with a vinyl cation intermediate, as shown in Reaction 1 [Yurkanis-Bruice, 2004].

⊕ Θ HOH + HC≡CH → H2C=C H + OH →H2C=CHOH ' H3C−CHO Electrophile Nucleophile Vinyl cation Vinyl alcohol Acetaldehyde Reaction 1: Electrophilic addition of water to acetylene with generation of intermediate vinyl cation

Docking of an acetylene molecule at this position is possible and gives an excellent fit, positioning the two carbon atoms of the substrate exactly above the water molecule coordinated to tungsten.

2) Nucleophilic additions in chemistry - the reaction of acetylene with alkoxides in alcoholic solution to yield vinyl ether (vinyl anion). This reaction requires conditions of high temperature and high pressure. In the enzyme, the hydroxo ligand could be coordinated to tungsten and constitute a strong nucleophile to react with acetylene. The next step of this reaction could be the formation of a vinyl anion as sufficient basicity to deprotonate Asp 13 and form the enol, vinyl alcohol, as shown in Reaction 2. Another water molecule could then bind to tungsten and get deprotonated by the basic Asp 13, thereby regenerating the hydroxo ligand for the next reaction cycle.

Θ Θ ⊕ OH + HC≡CH → HOHC=C H + H →H2C=CHOH ' H3C−CHO Strong Nucleophile Vinyl anion Vinyl alcohol Acetaldehyde

Reaction 2: Nucleophilic addition of hydroxo group to acetylene with generation of intermediate vinyl anion

A definitive distinction between both mechanisms will require further studies. Both mechanisms nevertheless require the modified architecture of the enzyme with a relocated substrate access pathway as well as the ring of hydrophobic residues to guide and orient the substrate so that the reaction can take place. A molecule of acetylene modeled into its putative binding site would be situated directly above the activated water or hydroxo ligand and would be held in place by hydrophobic interactions with

88 Results and Discussion

the surrounding residues. The initial product of acetylene hydration would be the enol, which would spontaneously tautomerize to acetaldehyde. With the product thus bound in the active site, the hydrophobic constriction might present a barrier for the access of water from the side of the substrate channel, the final step required to replenish the coordination of the tungsten atom and complete the reaction cycle. This arrangement offers an elegant solution to the problem of a possible product inhibition by association of the enol or aldehyde to tungsten.

89

4. Conclusion

Conclusion

Molybdenum and tungsten are important constituents of numerous essential enzymes found in biological systems. With the exception of nitrogenase where molybdenum is coordinated to a complex iron-sulfur cluster, both molybdenum and tungsten form mononuclear sites with either one or two dithiolene ligands (molybdopterin) bound to the active site metal center. The work presented in this thesis has been centered on the characterization of two novel bacterial enzymes of the DMSO reductase family: the Mo- Fe-S enzyme Pyrogallol-Phloroglucinol Transhydroxylase (Mo-TH), and the W-Fe-S enzyme Acetylene Hydratase (W-AH). Both enzymes depend on sophisticated redox- active metal sulfur centers, and catalyze chemically rather unusual reactions, without the net transfer of redox equivalents. The aim of this study was to elucidate functional and structural characteristics of Mo-TH and W-AH, to get a deeper understanding of their unusual chemistry.

4.1. Pyrogallol-Phloroglucinol Transhydroxylase (Mo-TH) from Pelobacter acidigallici

Transhydroxylase (Mo-TH) was isolated from the anaerobic bacterium Pelobacter acidigallici. The 3D structure of Mo-TH, with molybdenum most likely being in the reduced M(IV) state, revealed a Mo(MGD)2 center at the active site (MGD = molybdopterin guanine dinucleotide) with four dithiolene sulfur atoms coordinated to molybdenum [Messerschmidt et al., 2004]. Such a coordination site is typical for the members of the DMSO reductase family. In addition, molybdenum of TH is bound to serine oxygen, and acetate completes the trigonal pyramidal site. The molybdenum center of TH – 95,97Mo, 95Mo and 98Mo isotopes have been inserted into the enzyme - has been characterized in the Mo(V) oxidation state applying EPR spectroscopy (X band, variable temperature) under different conditions of pH, redox potential, and chloride concentration. 95 The EPR spectra of Mo(V)-TH revealed a splitting of Azz= 48G, Ayy= 31G, Axx = 50G which was not completely eliminated in D2O. Two different EPR signals were detected

93 Conclusion

depending on pH which had also been reported for nitrate reductase from E. coli

(NarGHI). At pH 6.5 the EPR signal at 1.996, 1.982, 1.963 showed a splitting of Azz= 98 26G, Ayy= 30G, Axx = 28G (best resolved in Mo(V)-TH) which disappeared at pH 8.5.

Note that close to the metal in the active site (Mo-OAsp = 3.49 Å, and Mo-NHis, 3.00 Å), there are two amino acid residues which might function as an acid (Asp 174) or as a base (His 144) depending on pH. Most likely, the molybdenum site carries a hydroxo group during catalysis. This comes from a comparison of the EPR signal of Mo-TH with that of nitrate reductase (NarGHI) from E. coli, where nitrate binds in a monodentate fashion to molybdenum [George et al., 1989]. Chloride anion also binds to molybdenum in the Mo(V) state according to splittings in the EPR spectrum. Binding of small anions such as acetate has been also documented in the X-ray structure of Mo-TH. For dimethylsulfide dehydrogenase from

R. sulfidophilum equilibrium between a Mo-OH and Mo-H2O site was proposed depending on pH [McDevitt et al., 2002]. Most likely, such an equilibrium between two Mo(V)-TH species will exist in solution. Our results show that in TH the situation is slightly different; at low pH the enzymes contains the protonated Mo-OH form and high pH the non-protonated or Mo-OH2 form. Protonated and non-protonated forms of the molybdenum site had been proposed earlier for nitrate reductase from E. coli [Vincent et al., 1978]. 18 Transhydroxylation experiments in H2 O and the crystal structure of the pyrogallol-TH supposed reaction scheme for TH indicated formation of 2,4,6,3’,4’,5’- hexahydroxydiphenyl ether as intermediate (supposed by Retey) (Fig 4.1). The chemical course of the transhydroxylase reaction indicated that there is no oxygen transfered from water to pyrogallol and that the hydroxyl groups are transferred only between phenolic substrates.

94 Conclusion

Tyr 404

NH Mo (IV) His 144 N OH Pyrogallol HO HO * OH HO O OH OH HO HO O Asp 174 Diphenylether HO OH HO OH OH OH

TRANSHYDROXYLASE OH OH

HO OH HO OH * OH

1,2,3,5-Tetrahydroxybenzene Phloroglucinol

Figure 4.1: Proposed reaction scheme for TH based on the 3D structure and chemical studies [Messerschmidt et al., 2004; Reichenbecher et al., 1999]

Note that the EPR signal of the Mo(V) center does not change its shape and g-values during catalysis, only the intensity changes. It was speculated that during catalysis the molybdenum ion is found in its reduced form Mo(IV), silence for EPR. According to the crystal structure the substrate pyrogallol is bound to molybdenum – most likely Mo(IV) – via a hydroxyl group, with a Mo-O distance of 2.31 Å (in literature, Mo-O distance by

H2O ligand is between 2.0-2.6 Å, [Dobbek et al., 2002; Jepson et al., 2007]). Two possible modes of oxygen transfer could be envisaged at pH 7:

(i) Mo-OH + HOR ' Mo-OR + H2O, − ii) Mo-OH2 + OR ' Mo-OR + H2O with HOR being Pyrogallol, and −OR its deprotonated form.

In reaction (i) the interaction with the base His 144 is not required during substrate binding, whereas in reaction (ii) His 144 will be needed to protonate the pyrogallol anion. Surprisingly, soaking of crystals of Mo-TH – molybdenum being most likely in the Mo(IV) state – with the inhibitor 1,2,4-trihydroxybenzene led to conversion of the

95 Conclusion

inhibitor to 1,2,4,5-tetrahydroxybenzene, as shown in X-ray structure (Messerschmidt et al., 2004). In the EPR spectra recorded for the reaction of Mo-TH (“as isolated”) and the inhibitor 1,2,4-trihydroxybenzene [Sommer, 1995], and of Mo-TH (“as isolated”) with 4,4’-Difluordiphenyl ether (this work) a splitting of approximately 20 Gauss was detected which might result from a hydroxyl proton bound to the Mo(V) center. The incubation of Mo-TH (as isolated) with pyrogallol, or with the putative reaction intermediate 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether [Messerschmidt et al., 2004; Paizs et al., 2007] led to a minor decrease of the EPR signal of the Mo(V) center with gav= 1.98. Upon reaction of Mo-TH (as isolated) with 1,2,3,5-tetrahydroxybenzene EPR signals of reduced [4Fe-4S] centers became visible. In contrast, the reaction of Mo-TH (as isolated) with pyrogallol, or the inhibitor 1,2,4-trihydroxybenzene, did not yield any EPR active Fe-S sites. According to the structure based reaction mechanism proposed for Mo-TH [Messerschmidt et al., 2004] 1,2,4,5-tetrahydroxybenzene becomes released in the final step. Probably, the [4Fe-4S] cluster located in the ß-subunit close to the Mo(MGD)2 has a regulatory effect – mediated by arginine Arg-740 on the redox properties of the molybdenum center.

4.2. Acetylene hydratase from Pelobacter acetylenicus

Acetylene hydratase (AH) was isolated from the anaerobic δ-bacterium P. acetylenicus and was characterized biochemically and spectroscopically as a tungsten-[4Fe-4S] enzyme (W-AH). In addition, a molybdenum isoenzyme (Mo-AH) could be collected from P. acetylenicus grown on a medium enriched with molybdate. Relating to amino acid sequence AH was assigned as a member of the DMSO reductase family (Abt, 2001). The activity of Mo-AH was approximately 10-fold lower compared to W-AH [Meckenstock et al., 1999]. Results from experiments with [14C]-acetylene, however,

96 Conclusion

suggest a tighter binding of acetylene to Mo-AH compared to binding of acetylene to W- AH.

The reaction of W-AH with dithionite led to a rhombic EPR signal with gav = 1.966 ± 0.001 indicative of a low-potential ferredoxin-type [4Fe-4S]+ cluster. UV-Vis and EPR studies showed that acetylene, as well as propargyl alcohol, could reoxidize [4Fe-4S] center. Upon reaction of either acetylene or propargyl alcohol with W-AH which had been incubated with the oxidant ferricyanide, a new EPR signal of yet unknown origin appeared at gav= 2.008.

So far, the X-ray structure of Mo-AH has not been solved yet. But W-AH has been successfully crystallized most recently in the absence of dioxygen, with a reductant such as dithionite or Ti(III)-citrate added to the crystallization buffer. The crystals of W-AH diffracted to a resolution of 1.26 Å, new crystals even to 1.1 Å. The active site of W-AH is composed of a tungsten center coordinated to four dithiolene sulfur atoms from two molybdopterin cofactors (MGD), as found in all members of the DMSO reductase family. In addition, fifth sulfur from cysteine Cys-141 is bound to tungsten. The coordination sphere of tungsten which most likely is in the W(IV) state, is completed by a water/hydroxo group, with W-O bond length of 2.04 Å. The oxidized enzyme (using

K3[Fe(CN)6] as oxidant) shows a shortened W-O distance of 1.7 Å indicative of an oxo ligand. The active site passese an octahedral or trigonal antiprismatic geometry also in the W(IV) and the W(VI) state.

W-AH has a well defined substrate channel. The access to the active site of AH is completely rearranged by comparison to other enzymes of DMSO reductase family, known so far. Acetylene can access the W(MGD)2 center through a channel ≈ 17 Å deep and 6-8 Å wide, close to the N-terminal domain that harbors the [4Fe-4S] cluster. The channel ends in a ring of hydrophobic residues. These residues, and the tungsten center with a coordinated H2O/OH and Asp-13 in close position to the oxygen ligand, create a small binding cavity with dimensions that make it a perfect mold for binding acetylene. In W(IV)-AH, the negative electrostatic potential surface of the active site and the unsually high pKa of Asp-13 suggest that the oxygen ligand most likely comes from water, and not from OH-.

97 Conclusion

W-AH catalyzes the hydration of acetylene to acetaldehyde, via the enol tautomer. The reaction is peculiar in the sense that formally redox equivalents are not transferred. Yet, for catalysis a strong reductant is required, and the enzyme depends on rather sophisticated metal centers. Based on the structure of W-AH, a proposed mechanism consisting of three steps, is proposed for the addition of water to the C,C triple bond of acetylene (Fig 4.2).

A B

Figure 4.2: Proposed model for acetylene binding in W-AH enzyme. (A) Nucleophilic addition of hydroxo group to acetylene with generation of intermediate vinyl-anion (B) Electrophilic addition of water to acetylene with generation of intermediate vinyl-cation

In the first step a molecule of H2O bound to W ion would gain a partially positive net charge through the proximity of the protonated Asp 13. H2O as electrophile could directly attack the triple bond of acetylene in a Markovnikov-type addition reaction with a vinyl cation intermediate (Fig 4.2) [Yurkanis-Bruice, 2004]. In this scheme, Asp 13 would remain protonated. This can be rationalized considering that the central metal ion is surrounded by five negative charges from its five thiolate ligands, yielding a total charge of +1 in the oxidized versus -1 in the reduced state, complementary to that of the respective ligand found to be stable in the calculations. As a consequence, the active W(IV) state should favor a water ligand and therefore an electrophilic addition mechanism. The mechanism nevertheless requires the modified architecture of the enzyme with a relocated substrate access pathway as well as the ring of hydrophobic residues to guide and orient the substrate such that the reaction can take place. A molecule of acetylene modelled into its putative binding site would be situated directly

98 Conclusion

above the activated water and would be held in place by hydrophobic interactions with the surrounding residues. The initial product of acetylene hydration would be the enol, which would spontaneously tautomerize to acetaldehyde. With the product thus bound in the active site, the hydrophobic constriction might present a barrier for the access of water from the side of the substrate channel, the final step required to replenish the coordination of the tungsten atom and complete the reaction cycle. However, the structure shows that water can instead be recruited from a significant reservoir of at least 16 well-defined water molecules in a vestibule directly adjacent to the active site. This arrangement offers an elegant solution to the problem of a possible product inhibition by association of the enol or aldehyde to tungsten.

Currently, experiments are underways to express W-AH heterologously in E. coli [ten Brink, 2006]. Subsequently, variants of acetylene hydratase will be produced via site directed mutagenesis. As a first target, the specific roles of several amino acid residues at the active site, especially Asp 13, will be investigated to shed further light into the unusual chemistry of acetylene hydratase.

99

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6. Acknowledgement

Mein größter Dank gilt Prof. Dr. Peter M. H. Kroneck, Universität Konstanz, für die vertrauensvolle Überlassung des interessanten und spannenden Themas sowie für seine ausgezeichnete Betreuung und engagierte Unterstützung bei der vorliegenden Arbeit. Die Förderung der Zusammenarbeit mit anderen Laboren sowie die Ermöglichung des Besuches von (inter)nationalen Tagungen und Kursen waren für mich eine exzellente Möglichkeit meinen Horizont zu erweitern.

Prof. Dr. Bernhard Schink danke ich für die Unterstützung dieser Arbeit und für die anregenden Gespräche.

Prof. Dr. Oliver Einsle danke ich für die Übernahme des Koreferats dieser Arbeit, die interessanten Diskussionen, die große Hilfe bei der AH Kristallstrukturanalyse. Der AG Einsle, im Besonderen Maren und Susana, danke ich für informative Gespräche und eine sehr gute Arbeitsatmosphäre.

Prof. Dr. Albrecht Messerschmidt danke ich für die Möglichkeit, in der Abteilung Strukturforschung am Max-Planck-Institut für Biochemie in Martinsried Kristallisationsexperimente durchführen zu können.

Dr. Ingo Schmidt und Stefan Gilch danke ich für die Möglichkeit und Unterstützung bei der Durchführung von radioaktiven Experimenten und den herzlichen Aufenthalt in Bayreuth.

Den jetzigen Mitgliedern der AG Kroneck: Klaus Sulger, Sonja Fraas, Felix ten Brink, Michael Koch, Günter Fritz und ehemaligen Mitgliedern der AG: Alma Katharina Steinbach, Thorsten Ostendorp, Marc Rudolf, Alexander Schiffer, Holger Nießen, danke

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ich für eine wunderbare Zeit, eine sehr nette Atmosphäre, informative Diskussionen und große Hilfsbereitschaft.

Meinen Vertiefungskursstudenten: Felix ten Brink, Eva Zeiser danke ich für ihre gute wissenschaftliche Arbeit, Wissbegierde und weiterführende Ideen.

Meinen Freunden sei für die schöne Zeit in Konstanz, lange Telefonate, in Erinnerung bleibende Treffen und ehrliche Freundschaft gedankt.

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7. Appendix

7.1. Abbreviations

Å Ångström; 1 Å = 10-10 m ADH Alcohol dehydrogenase BCA bicinchoninic acid BSA bovine serum albumin Da Dalton; 1 Da = 1 g·mol-1 DNA deoxyribonucleic acid DNase I deoxyribonuclease I E°´ standard reduction potential, pH 7.0, 25° C EDTA Ethylenediaminetetraacetic acid Tetrasodium salt dihydrate EPR electron paramagnetic resonance FPLC fast protein liquid chromatography h hour/hours HEPES N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid HPLC high performance/pressure liquid chromatography ICP-MS inductively coupled plasma-mass spectroscopy KPi Potassium phosphate MAD multiple-wavelength anomalous dispersion MES 2-(N-morpholino)ethanesulfonic acid min minute/minutes MGD Molybdopterin-guanine-dinucleotide MPD 2-methyl-2,4-pentanediol NAD+/NADH β-nicotinamide adenine dinucleotide (oxidized/reduced) PEG polyethylene glycol PVDF Polyvinylidene difluoride

109 Appendix

rms root-mean-square SAD single-wavelength anomalous dispersion SDS/PAGE sodium dodecylsulfate polyacrylamide gelelectrophoresis TRIS α,α,α-trishydroxymethyl aminomethane TEA Triethanolamine = 2,2',2''-Nitrilotriethanol = Tris(2-hydroxyethyl) amine UV/Vis ultraviolet/visible v/v volume per volume w/v weight per volume

Enzyme Names

AH Acetylene hydratase BSO/BSOR Biotin sulfoxide/Biotin sulfoxide reductase DMSO/DMSOR Dimethyl sulfoxide/ Dimethyl sulfoxide reductase FDH-H Formate dehydrogenase H FDH-N Formate dehydrogenase N FDH-W Tungsten-containing formate dehydrogenase NAP Periplasmic nitrate reductase NAR GHI Membrane-bound nitrate reductase TH Transhydroxylase TMAO/TMAOR Trimethylamine-N-oxide/Trimethylamine-N-oxide reductase

110 Appendix

Amino Acids

A Ala alanine M Met methionine B Asx Asn or Asp N Asn asparagine C Cys cysteine P Pro proline D Asp aspartate Q Gln glutamine E Glu glutamate R Arg arginine F Phe phenylalanine S Ser serine G Gly glycine T Thr threonine H His histidine V Val valine I Ile isoleucine W Trp tryptophan K Lys lysine Y Tyr tyrosine L Leu leucine Z Glx Gln or Glu

Nucleic Acid Bases

A adenine S G/C strong G guanine W A/T weak C cytosine B G/C/T not A T thymine D A/G/T not C

111 Appendix

7.2. Curriculum vitae

Personal data Born September 30th 1979, Zdzieszowice, Poland Sex Female Nationality Polish-German Marital status Single

Education 1986 – 1994 Primary School, Zdzieszowice, Poland 1994 – 1998 Grammar School, Kedzierzyn-Kozle, Poland

University studies 1998/10 to 2003/06 Student of the Faculty of Chemistry at the University of Wroclaw, Poland specializing in Environmental Chemistry 2002/10 to 2003/06 Master Thesis in the Department of Chemistry at the University of Wroclaw under supervision of Prof. Dr. Teresa Kowalik-Jankowska in the Research Group of Prof. Dr. Henryk Kozlowski: „Complexes of ion metals with α- hydroxymethylmethionine and tripeptides containing blocked α-hydroxymethylserine”, graduation as Master of Science since 2003/10 PhD thesis in the Department of Biology, University of Konstanz Germany, under supervision of Prof. Dr. P.M.H. Kroneck: „Novel molybdopterin and iron-sulfur containing enzymes”

Memberships since 2003/12 Gesellschaft für Biochemie und Molekularbiologie e.V.

112 Appendix

Professional Training and Activities

2002/06 to 2002/09 Work at the Umweltforschungszentrum Leipzig-Halle GmbH in the Leonardo da Vinci II project, Magdeburg, Germany 2005/03 CW-EPR & XSophe Training Course, BRUKER BIOSPIN, Rheinstetten/Karlsruhe, Germany

Publications

Oliver Einsle, Holger Niessen, Dietmar J.Abt, Grazyna Seiffert, Bernhard Schink, Robert Huber, Albrecht Messerschmidt and Peter M.H.Kroneck, ”Crystallization and preliminary X-ray analysis of the tungsten-dependent acetylene hydratase from Pelobacter acetylenicus “Acta Cryst. 2005 F61, 299 – 301

Grazyna B. Seiffert, Dietmar J.Abt, Felix TenBrink, Bernhard Schink, Henner Brinkmann, Axel Meyer, and Peter M.H.Kroneck, “Bacterial Acetylene Hydratase: Structural and Functional Properties of the W,FeS Enzyme from Pelobacter acetylenicus and Variants” FEBS J. 2006, manuscript in preparation

Einsle, O., Grazyna B. Seiffert, Sosa-Torres, M.E., *Kroneck, P.M.H. “Bakterien sind die besten Chemiker“, BIOSPEKTRUM, 2006, 12, 346-349

Grazyna B. Seiffert, Ullmann, G.M., Messerschmidt, A., Schink, B., Kroneck, P.M.H., Einsle, O. “Structure of the non-redox-active tungsten/[4Fe-4S] enzyme acetylene hydratase”, Proc. Natl. Acad. Sci. (USA), 2007, 104, 3073-3077

113 Appendix

Contributions to Scientific Conferences (talks/posters)

2004/03 VAAM Jahrestagung 2004, Braunschweig, Germany “Structural aspects of the Mo-enzyme pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici”

2004/10 The 6th Workshop on Applications of EPR in Biology and Medicine, Krakow, Poland “Novel bacterial Mo and W enzymes: MoFeS-transhydroxylase and WFeS-acetylene hydratase”

2005/04 Workshop of the Deutsche Forschungsgemeinschaft (DFG) „Radicals in enzymatic catalysis“, Schloss Rauischholzhausen, Germany “MoFeS Transhydroxylase (TH) & WFeS Acetylene Hydratase (AH). Structural and Spectroscopic Studies”

2005/09 Xth International Symposium on Bioinorganic Chemistry „Challenge for a new generation”, Szklarska Poreba, Poland “Spectroscopic and Structural properties of novel bacterial Molybdo- and Tungstoenzymes: MoFeS-transhydroxylase and WFeS- acetylene hydratase”

2005/10 VAAM Jahrestagung 2005, Göttingen, Germany “Spectroscopic and Structural properties of novel bacterial Mo and W enzymes: MoFeS-transhydroxylase and WFeS-acetylene hydratase”

2006/03 VAAM Jahrestagung 2006, Jena, Germany “Spectroscopic and structural properties of the MoFeS-enzyme pyrogallol phloroglucinol transhydroxylase from Pelobacter acidigallici”

2006/07 EUROBIC 8, Aveiro, Portugal “Spectroscopic and Structural properties of novel bacterial Mo and W enzymes: MoFeS-transhydroxylase and WFeS-acetylene hydratase”

2007/04 VAAM Jahrestagung 2007, Osnabrück, Germany “Molybdenum- and tungsten-dependent hydroxylases from anaerobic bacteria”

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