Structural and functional investigations on multi-site metallo enzymes of the biological sulfur cycle

Structural and functional investigations on multi-site metallo enzymes of the biological sulfur cycle

Dissertation submitted to

Fachbereich Biologie, Universität Konstanz, Germany

for the degree of

Doctor of Natural Sciences

presented by

Dipl.-Chem. Alexander Schiffer

Konstanz, November 2003

Examiner: Prof. Dr. P.M.H. Kroneck Coexaminer: PD Dr. U. Ermler

Dissertation der Universität Konstanz Datum der mündlichen Prüfung: 16.01.2004 Referenten: Prof. Dr. P. M. H. Kroneck

Priv. Doz. Dr. U. Ermler

für Alicia

Table of contents

ZUSAMMENFASSUNG VI SUMMARY IX

1 INTRODUCTION 1

1.1 BIOGEOCHEMICAL SULFUR CYCLE 1

1.2 EVOLUTIONARY ASPECTS OF DISSIMILATORY SULFATE REDUCTION 1

1.3 PHYLOGENY OF SULFATE-REDUCING BACTERIA 2

1.4 ENZYMES INVOLVED IN DISSIMILATORY SULFATE REDUCTION 3

1.5 ADENYLYLSULFATE (APS) REDUCTASES 4

1.5.1 Assimilatory APS reductase 4 1.5.2 Dissimilatory APS reductase 5

1.6 SULFITE REDUCTASES 6

1.6.1 Assimilatory sulfite reductase 6 1.6.2 Dissimilatory sulfite reductase 7

1.7 HIGH-SPIN IRON CLUSTERS IN BIOLOGICAL SYSTEMS 8

1.8 PROTEIN CRYSTALLOGRAPHY 9

1.9 SCOPE OF THE STUDY AND RESEARCH OBJECTIVES 10

2 MATERIALS AND METHODS 11

2.1 CHEMICALS 11

2.2 PROTEIN BIOCHEMISTRY 12

2.2.1 Organism and cultivation 12 2.2.2 Preparation of cell fractions 12

2.2.3 Purification protocols 12 2.2.3.1 APS reductase of Archaeoglobus fulgidus 12 2.2.3.2 Dissimilatory sulfite reductase of Archaeoglobus fulgidus 13 2.2.4 Analytical methods 13 2.2.4.1 Protein determination 13 2.2.4.2 Determination of iron 14 2.2.4.3 Denaturing polyacrylamide gel electrophoresis 14 2.2.5 Experiments under exclusion of dioxygen 14 2.2.6 Enzymatic activities 15 2.2.6.1 Photometric determination of APS reductase activity 15 2.2.6.2 Photometric determination of sulfite reductase activity 15 2.2.7 Spectroscopic methods 16 2.2.7.1 UV/Vis absorption spectroscopy 16 2.2.7.2 Electron paramagnetic resonance spectroscopy 16

2.2.8 Titrations 17

2.3 PROTEIN CRYSTALLOGRAPHY 17

2.3.1 Theoretical background 17 2.3.1.1 Crystal growth 18

2.3.1.2 Crystals 18

2.3.1.3 X-ray diffraction by crystals 18 2.3.1.4 The electron density function 20 2.3.1.5 The phase problem 21 2.3.2 Protein crystallization 22

2.3.2.1 APS reductase 22

2.3.2.2 Sulfite reductase 22 2.3.3 Substrate complexes of APS reductase 22 2.3.4 Preparation of derivatives of sulfite reductase crystals 22 2.3.5 Cryocrystallography 23

2.3.5.1 APS reductase 23

2.3.5.2 Sulfite reductase 23 2.3.6 Measurement of datasets 23

2.3.6.1 Sulfite reductase 24

2.3.7 Data processing 24

2.3.8 Substructure solution and phase calculations 24 2.3.9 Density modifications 24 2.3.10 Molecular replacement using experimental phases 25 2.3.11 Interpretation of electron density maps 25 2.3.12 Model building and refinement 25 2.3.13 Structure comparison 25 2.3.14 Graphical representation 27

3 RESULTS 29

3.1 APS REDUCTASE FROM ARCHAEOGLOBUS FULGIDUS 29

3.1.1 Crystallization and diffraction analysis 29

3.1.2 Data collection 29

3.1.3 Overall molecular structure 30

3.1.4 The α-subunit 31

3.1.4.1 Fold description 31 3.1.4.2 Comparison to structurally related proteins 32

3.1.5 The β-subunit 36

3.1.6 Structure based enzyme mechanism 38 3.1.6.1 Structures of APS reductase in different states 38 3.1.6.2 APSR-red state 38 3.1.6.3 APSR-sulfite state 39

3.1.6.4 APSR-ox state 40

3.1.6.5 APSR-d-red state 41 3.1.6.6 APSR-amp state 42 3.1.6.7 APSR-aps state 46

3.2 SULFITE REDUCTASE FROM ARCHAEOGLOBUS FULGIDUS 48

3.2.1 Purification 48

3.2.2 Enzyme properties 48 3.2.3 UV/Vis spectroscopy 49 3.2.3.1 Oxido-reduction experiments 49 3.2.3.2 Binding of substrates and products 50 3.2.4 EPR spectroscopy 53 3.2.4.1 Sulfite reductase as isolated 53 3.2.4.2 Oxidized sulfite reductase 60 3.2.4.3 Sulfite reductase with sulfide 68 3.2.5 Crystallization and diffraction analysis 69 3.2.5.1 Data collection 69 3.2.5.2 Structure determination 71 3.2.5.3 Phase calculations 72 3.2.5.4 Electron density modifications 73 3.2.5.5 Arrangement of the cofactors 73

4 DISCUSSION 77

4.1 APS REDUCTASE FROM ARCHAEOGLOBUS FULGIDUS 77 4.1.1 Comparison with structurally related flavin containing enzymes 77 4.1.1.1 Comparison of the α-subunit fold of APS reductase with the flavoprotein

subunit of fumarate reductase 77 4.1.1.2 Comparison of the FAD domain of APS reductase with that of other FAD

dependent reductases. 80 4.1.1.3 Comparison of the active site and substrate binding in APS reductase with

that in other members of the succinate dehydrogenase family 81 4.1.2 Structure based enzyme mechanism 83 4.1.2.1 The reaction of APS reductase 83 4.1.2.2 Catalytic mechanism 84 4.1.2.3 The electron transfer 88

4.2 SULFITE REDUCTASE FROM ARCHAEOGLOBUS FULGIDUS 90 4.2.1 Molecular and catalytic properties of sulfite reductase 90 4.2.2 Spectroscopic properties of sulfite reductase 91

4.2.2.1 High-spin S=5/2 signals 91 4.2.2.2 High-spin S=9/2 signals 91 4.2.2.3 Coupling of redox centers 93 4.2.2.4 Origin of the S=9/2 signals in sulfite reductase 93 4.2.2.5 Redox states and substrate binding 94 4.2.3 Crystallization and structure determination of sulfite reductase 96 4.2.3.1 Crystallization 96 4.2.3.2 Data collection and reduction 96 4.2.3.3 Structure determination 97 4.2.3.4 Cofactors of sulfite reductase 98

5 REFERENCES 101

6 APPENDIX 119

6.1 ABBREVIATIONS 119

6.2 EQUATIONS USED IN X-RAY CRYSTALLOGRAPHY 120

6.3 CURRICULUM VITAE 121

6.4 PUBLICATIONS 122

6.5 CONFERENCE ABSTRACTS 123

7 ACKNOWLEDGEMENTS 124

Zusammenfassung

In dieser Dissertation wurden die strukturellen, funktionellen und spektroskopischen Eigenschaften zweier Schlüsselenzyme der dissimilatorischen Sulfatreduktion untersucht.

1. Kristallstruktur der APS Reduktase aus Archaeoglobus fulgidus

Das Eisen-Schwefel Flavoprotein Adenylylsulfat (Adenosin 5’-phosphosulfat, APS) Reduktase katalysiert die reversible Reduktion von APS zu Sulfit und AMP. Die Struktur der APS Reduktase aus dem hyperthermophilen Organismus Archaeoglobus fulgidus wurde in der Zweielektronen- reduzierten Form mit einer Auflösung von 1.6 Å bestimmt (Proc Natl Acad Sci USA. 2002; 99:1836-1841).

Die α-Untereinheit der APS Reduktase war strukturell sehr ähnlich zur Flavoprotein- Untereinheit der Fumarat Reduktase Familie. Es wurde deshalb vorgeschlagen, dass sich die α-Untereinheiten aus einem gemeinsamen der archaealen APS Reduktase ähnlichen Vorläufer entwickelt haben.

Die strukturelle Ähnlichkeit spiegelt sich nicht in der Sequenz-Ähnlichkeit wieder. Der Sequenz-Vergleich zeigte, dass es nur eine einzige konservierte Aminosäure in der gesamten FAD-Bindedomäne gab. Obwohl die gleiche Aminosäure Histidin eine zentrale aber unterschiedliche Rolle in der Katalyse spielt, war dieses Histidin nicht konserviert. Der Bereich des Proteins, der für die FAD Bindung zuständig ist, wurde bereits in der großen Familie strukturell charakterisierter FAD abhängiger Reduktasen beobachtet (Flavins and Flavoproteins 14th ed. (2002), pp. 69-75).

Die beiden für die APS Reduktion benötigten Elektronen werden von der Oberfläche des Proteins über zwei [4Fe-4S] Zentren I und II zum FAD übertragen. Der ungewöhnlich große Unterschied der Redox Potentiale dieser beiden Zentren (Zentrum I -60 und Zentrum II -500 mV) konnte durch die Wechselwirkungen der Proteinumgebung mit den Zentren erklärt werden.

2. Aus der 3D- Struktur hergeleiteter Katalyse-Mechanismus der APS Reduktase

Um den Reaktions-Mechanismus der APS Reductase aufzuklären, wurden verschiedene Zustände des Enzyms entlang der Reaktionskoordinate strukturell charakterisiert. Ein FAD- Sulfit Addukt wurde gefunden, nach Inkubation der Kristalle mit APS. Dies ist ein Indiz dafür dass das Enzym im Kristall funktionsfähig war. Der Kanal zum Aktivzentrum, der durch eine Gruppe hydrophober Aminosäuren stabilisiert wurde, bildete die Substratbindestelle. Die

Bindung des Substrats APS hatte eine gespannte Konformation des FAD im aktiven Zentrum zur Folge. Die Reaktion wurde durch den nukleophilen Angriff des Flavin N5 Atoms am Schwefel des APS eingeleitet. Zur S-O Bindungsspaltung bzw. S-N Bindungsausbildung war nur eine Bewegung des Schwefels um 1 Å hin zum FAD notwendig. His A398 und Arg A265 waren für die Stabilisierung der zusätzlichen Ladungen des entstehenden FAD-Sulfit Adduktes und des AMPs wichtig. In diesem Zustand wurde eine kleine weitreichende Konformationsänderung des Proteins gefunden, die vermutlich das Redoxpotential des [4Fe- 4S] Zentrums I und den Elektronenfluss zum FAD beeinflusste. Die Protonierung des Sulfit O3 Atoms durch aktivierte Wassermoleküle erleichterte den vor der Reduktion des FAD letzten Schritt im Reaktionszyklus, die Spaltung des FAD-Sulfit Adduktes.

3. Biochemische und spektroskopische Charakterisierung der Sulfit Reduktase aus Archaeoglobus fulgidus

Die dissimilatorische Sulfit Reduktase aus dem hyperthermophilen Organismus A. fulgidus wurde unter striktem Luftsauerstoffausschluss in N2/H2 Atmosphäre isoliert und gereinigt.

Das aktive Enzym enthielt eine α-Untereinheit (51 kDa) und eine β-Untereinheit (45 kDa), die ein α2β2-Heterotetramer bildeten. Die Eisenbestimmung durch ICP-MS ergab einen Eisengehalt von 12-14 Eisen pro α2β2-Heterotetramer. Die Sulfit Reduktase wurde in einem gemischten Redox Zustand isoliert, bei dem das Sirohäm-[4Fe-4S] Zentrum oxidiert und mindestens eines der insgesamt drei Eisen-Schwefel Zentren im reduzierten Zustand vorlag.

In diesem Zustand wurde mit UV/Vis Spektroskopie sowohl Substrat- als auch Produktbindung an das Enzym nachgewiesen.

High-Spin Fe(III) EPR Signale wurden im oxidierten Zustand und im Zustand wie isoliert des Enzyms beobachtet. Im oxidierten Zustand wurde ein einzelnes S=9/2 Signal mit g-Werten von 17.5 und 9.7 beobachtet. Es wurde hauptsächlich ein einzelnes S=5/2 Signal des Sirohäm- [4Fe-4S] Zentrums mit g-Werten von 6.7 und 5.1 gefunden. Außerdem war ein S=1/2 Signal (gx=1.978, gy=2.007, gz=2.03) vorhanden. Low-Spin Häm Signale wurden im EPR Spektrum der Sulfit Reduktase nicht gefunden. Die EPR Spektren wurden simuliert und die Nullfeldaufspaltungen wurden bestimmt. Der Wert, der durch die Erniedrigung der Besetzungszahlen des | ±1/2 > Dubletts bei thermischer Anregung bestimmt wurde, war in der Größenordnung von 4 cm^-1 sowohl für das S=9/2 System mit E/D=0.154 als auch für das S=5/2 Signal mit E/D=0.036. Die Simulation des S=9/2 Systems ergab eine Nullfeldaufspaltung von nur 2 cm^-1. Die Halbsättigungs-Leistung (P1/2) der High-Spin Signale bei 6 K lag bei ca. 1 mW Mikrowellen-Leistung.

4. Kristallisation und Röntgenstrukturanalyse der Sulfit Reduktase

Die Sulfit Reduktase aus A. fulgidus wurde unter Ausschluss von Luftsauerstoff mit Hilfe der Methode des hängenden Tropfens kristallisiert. Die Kristallisation wurde bei 18°C mit PEG 4000 als Präzipitanz durchgeführt. Die grün-braunen Kristalle gehörten der Raumgruppe P21

an und hatten die Einheitszellenparameter a= 94.8, b= 69.4, c= 148.3 Å und β= 106.9°. Die asymmetrische Einheit enthielt zwei αβ-Einheiten. Die Kristalle streuten bis 2.5 Å und eigneten sich zur Röntgenkristallstrukturbestimmung.

Die Auswertung des anomalen Streuverhaltens an der Eisen-Absorptionskante ergab, dass der α2β2-Heterotetramer der Sulfit Reduktase sechs Eisen-Schwefel Zentren und zwei Häm-Eisen enthielt. Vier der Zentren bestanden aus vier Eisen Atomen und weitere zwei Zentren enthielten drei oder vermutlich vier Eisen Atome. In einer αβ-Einheit war die Entfernung zwischen dem Häm-Eisen und dem nächstgelegenen Eisen-Schwefel Zentrum 3.5-4.0 Å. Von diesem Zentrum aus waren die beiden anderen Zentren 15 bzw. 38 Å entfernt. Die Entfernung zwischen Zentren die durch nicht-kristallographische Symmetrie ineinander Überführbar waren, war in der Größenordnung von 30 Å.

Summary

In this Ph.D. thesis the structural, functional and spectroscopic properties of two key enzymes of dissimilatory sulfate reduction were investigated.

1. Crystal structure of APS reductase from Archaeoglobus fulgidus

The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5’-phosphosulfate, APS) reductase catalyzes reversibly the reduction of APS to sulfite and AMP. The structure of APS reductase from the hyperthermophilic A. fulgidus in the two-electron reduced state was reported at 1.6 Å resolution (Proc Natl Acad Sci USA. 2002; 99:1836-41).

The α-subunit of APS reductase had a high structural similarity to the flavoprotein subunit of the fumarate reductase family. Therefore it was proposed that the α-subunits originated from a common ancestor resembling archaeal APS reductase.

The structural similarity was not reflected in the sequence similarity as the alignment showed only a single conserved amino acid in the FAD-binding domain. In addition, there was no conservation of catalytic residues although the amino acid histidine always played a crucial but different role in catalysis. Moreover, the fold of the domain involved in FAD binding was observed in a large family of structurally characterized FAD dependent reductases (Flavins and Flavoproteins 14th ed. (2002), pp. 69-75).

2. Structure based catalytic mechanism of APS reductase

To elucidate the reaction cycle of APS reductase various states of the enzyme along the reaction coordinate were structurally characterized. A FAD-sulfite adduct was detected after soaking the crystals with APS indicating functionally intact enzyme in the crystal state. The active site channel that was stabilized by a hydrophobic cluster of residues constituted the substrate-binding site. APS binding resulted in a strained conformation of the active site FAD.

The reaction was initiated by the nucleophillic attack of the N5 atom of the flavin on the sulfur of APS. The S-O bond was cleaved with the shift of the sulfur 1 Å towards the FAD and the S-N bond was formed. His A398 and Arg A265 were essential for compensating the additional negative charges of the generated FAD-sulfite adduct and AMP. In this state a small long-range conformational change was observed that probably influenced the redox potential of cluster I and the electron flow to the FAD. After leaving of the AMP only the FAD-sulfite adduct had to be cleaved. This was promoted by protonation of the sulfite O3 atom by activated water molecules.

3. Biochemical and spectroscopic characterization of sulfite reductase from Archaeoglobus fulgidus

Dissimilatory sulfite reductase from the hyperthermophilic archaeon A. fulgidus was isolated and purified under strict exclusion of dioxygen in a N2/H2 (95/5 %) atmosphere. The enzyme was active and composed of an α-subunit (51 kDa) and a β-subunit (45 kDa) arranged as α2β2- heterotetramer. Iron determination by ICP-MS indicated the presence of 12-14 irons per α2β2. Sulfite reductase was isolated in a mixed redox state with the siroheme-[4Fe-4S] center oxidized and at least one out of three iron-sulfur clusters reduced. In this state binding of substrate and product to sulfite reductase was observed by UV/Vis spectroscopy.

High-spin Fe(III) signals were observed by EPR spectroscopy in the oxidized state and in the as isolated state of the enzyme. In the oxidized state, a single S=9/2 signal with g-values of 17.5 and 9.7 was observed. A single dominating S=5/2 signal with g-values of 6.7 and 5.1 from the high-spin siroheme-[4Fe-4S] cofactor was found. In addition, one major S=1/2 signal was present. There were no signals deriving from low-spin heme. The EPR spectra were simulated and the zero-field splittings were determined. The value from thermal depopulation was in the order of 4 cm^-1 for the S=9/2 system with E/D=0.154 as well as for the S=5/2 system with E/D=0.0036. Simulation of the S=9/2 system yielded in a zero-field splitting of only 2 cm^-1. The high-spin Fe(III) signals saturated at around 1 mW microwave power (P1/2) at 6 K.

4. Crystallization and X-ray analysis of sulfite reductase

Sulfite reductase from A. fulgidus was crystallized under exclusion of dioxygen using the hanging drop vapor diffusion method. The crystallization was carried out at 18°C using PEG 4000 as precipitant. The green-brown crystals grew in the space group P21 with unit cell parameters a= 94.8, b= 69.4, c= 148.3 Å and β= 106.9°. The asymmetric unit contained two αβ-units. The crystals diffracted beyond 2.5 Å resolution and were suitable for X-ray structure analysis.

Analysis of the anomalous scattering at the iron absorption edge revealed the presence of six iron-sulfur clusters and two heme iron centers. Four of the clusters contained four irons and two clusters contained three or probably four irons. In one αβ-unit the distance between the heme iron and the closest cluster was 3.5-4.0 Å. From this cluster the other two were 15 and 38 Å away. The distances between non-crystallographically related clusters were in the order of 30 Å.

1 Introduction

1.1 Biogeochemical sulfur cycle

Sulfur is about 1000 times less abundant in nature than oxygen. The three most abundant forms are elemental sulfur (S⁰), sulfate and sulfide (Hollemann & Wiberg, 1985). The reduction of sulfate to sulfide and the oxidation of reduced inorganic sulfur compounds are widespread biological processes in our environment. Close to 75 % of the sulfur in the earth crust is converted in the biogeochemical sulfur cycle. Microorganisms play a central role in this process. Plants can also reduce sulfate for the purpose of biosynthesis of amino acids and cofactors (Brunold, 2000). Animals as well as plants can oxidize reduced sulfur compounds to sulfate (Peck & Bramlett, 1982).

The reduction of sulfate to sulfide is divided in two processes: the reduction of sulfate for biosynthesis of amino acids and cofactors and the reduction of sulfate to sulfide in sulfate respiration (Postgate, 1984). The assimilatory process in plants and bacteria is used to provide the reduced sulfur compounds.

The dissimilatory sulfate reduction is used for energy conservation by strict anaerobic sulfate reducing bacteria and archaea. The redox equivalents that are generated by the oxidation of organic compounds are transferred to sulfate as terminal electron acceptor (‘sulfate respiration’).

The ability to use sulfate as a terminal electron acceptor for energy conservation is characteristic of several bacterial lineages and one hyperthermophilic genus of archaea. These organisms include gram-positive (Desulfotomaculum) and gram-negative (Desulfovibrio, Desulfobulbus, Desulfobacter, Desulfobacterium, Desulfococcus and Desulfosarcina) sulfate reducing eubacteria (Deuereux et al., 1989) and the sulfate reducing archaeon Archaeoglobus fulgidus (Stetter et al., 1987).

1.2 Evolutionary aspects of dissimilatory sulfate reduction

Several data from recent studies suggest that the ability to reduce sulfate was developed early during prokaryotic evolution. As life may have originated in hot environments (Achenbach- Richter et al., 1987; Wächtershäuser, 1988), the occurrences of sulfate-reducing prokaryotes among hyperthermophilic archaea (Archaeoglobus fulgidus, Archaeoglobus profundus, Archaeoglobus veneficus; the latter organism however is unable to reduce sulfate but forms H2S from thiosulfate or sulfite (Dahl & Trüper, 2001) and deep-branching thermophilic

bacteria (Thermodesulfovibrio yellowstonii, Thermodesulfobacterium commune) indicate an early origin of this process (Wagner et al., 1998; Hipp et al., 1997). Isotopic data suggest that dissimilatory sulfate reduction began 2.8 to 3.1 billion years ago (Schidlowski et al., 1983;

Schidlowski, 1986; Postgate, 1984) but acquired global significance only after sulfate concentrations had considerably increased in the Precambrian oceans approximately 2.35 billion years ago (Cameron, 1982). The isotopic data are reasonably consistent with a recent estimate of the time of domain divergence, approximately 3.1 to 3.6 billion years ago, based on sequence comparisons of a large number of different proteins (Feng et al., 1997, Feng &

Doolitle, 1997). The results of a comparative sequence analysis of dissimilatory sulfite reductase genes, a key enzyme involved in sulfate reduction (Steuber & Kroneck, 1998), show that their inferred evolutionary relationships are nearly identical to those inferred on the basis of 16S rRNA (Wagner et al., 1998).

1.3 Phylogeny of sulfate-reducing bacteria

Sulfate-reducing bacteria constitute a diverse group of prokaryotes that contribute to a variety of essential functions in many anaerobic environments. In addition to their obvious importance to the sulfur cycle, sulfate-reducing bacteria are important regulators of a variety of processes in wetland soils, including organic matter turnover, biodegradation of chlorinated aromatic pollutants in anaerobic soils and sediments, and mercury methylation (Postgate, 1984). Sulfate-reducing bacteria may be divided into four distinct groups: gram-negative mesophilic sulfate-reducing bacteria; gram-positive spore forming sulfate-reducing bacteria;

thermophilic bacterial sulfate-reducing bacteria; and thermophilic archaeal sulfate-reducing bacteria (Dahl et al., 1994). All of these groups are characterized by their use of sulfate as terminal electron acceptor during anaerobic respiration.

Gram-negative sulfate-reducing bacteria are located within the delta subdivision of the Proteobacteria. At some point in their evolutionary history, the delta subdivision diverged from other Proteobacteria from a common ancestral phototroph. The Desulfovibrionaceae, including the genera Desulfovibrio and Desulfomicrobium have been proposed within the δ- Proteobacteria. The most well characterized species in the group of bacterial thermophilic sulfate-reducing bacteria are Thermodesulfovibrio yellowstonii and Thermodesulfobacterium commune. They possess optimal growth temperatures lower than those of archaeal sulfate- reducing bacteria but higher than those described for other sulfate-reducing bacteria. The group of archaeal thermophilic sulfate-reducing bacteria (Archaeoglobus fulgidus, Archaeoglobus profundus, Archaeoglobus veneficus, the latter organism however is unable to

reduce sulfate but forms H2S from thiosulfate or sulfite, substrates that can also serve as electron acceptors to the other two species (Dahl & Trüper, 2001) exhibits optimal growth temperatures above 80°C. Today, A. fulgidus is thought to have evolved from methanogenic ancestors (Castro et al., 2000). The members of the genus Archaeoglobus are closely related to the Methanosarcinales and represent a missing link between methanogens and sulfur- metabolizing archaea. In contrast with methanogens, A. fulgidus does not produce methane, as it is devoid of coenzyme M, coenzyme B, coenzyme F430 (Hansen, 1994), and methyl- coenzyme M reductase genes (Brüggemann et al., 2000). Note that this organism contains other methanogenic cofactors such as methanofuran, methanopterin, and coenzyme F420

(Adams, 1993).

1.4 Enzymes involved in dissimilatory sulfate reduction

+

H2 2H⁺

2e^-

SO42- APS

HSO3-

HS-

Figure 1.1: Electron transfer pathways in Desulfovibrio sp. Electrons are delivered from the periplasmic [Ni,Fe] hydrogenase to cytochrome c3 or the membrane-bound Hmc (Fritz, 1999) and Hdr complexes. Cytochrome c3 delivers six electrons to the membrane-bound sulfite reductase (Steuber &

Kroneck, 1998), whereas the Hmc and Hdr complexes shuttle two electrons to APS reductase via a thiol-disulfide exchange mechanism. The reduction of the disulfide might be coupled to energy conservation.

Dissimilatory sulfate reduction includes the reduction of sulfate to sulfur or sulfide and involves three key enzymes, localized in the cytoplasm or at the cytoplasmic aspect of the inner membrane: ATP-sulfurylase, adenosine 5’-phosphosulfate (APS) reductase and

dissimilatory sulfite reductase (LeGall & Fauque, 1988). Because of its low redox potential (E°’ = -516 mV), sulfate cannot be directly reduced by H2 or organic acids (Thauer et al., 1977). Sulfate has to be activated to adenosine 5’-phosphosulfate (APS) in a reaction catalyzed by ATP-sulfurylase (Dahl et al., 1990), whereby the redox potential (APS/AMP + HSO3-) is shifted to E°’ = -60 mV (Thauer et al., 1977). The formation of APS is endergonic and probably driven by the subsequent hydrolysis of pyrophosphate and the favorable APS reduction. Therefore, the activation of sulfate to APS is assumed to consume two ATP equivalents (Peck, 1959). The enzyme APS reductase catalyzes the reduction of APS to sulfite and AMP. The natural electron donor for APS reductase is still unknown. As a final step, the dissimilatory sulfite reductase finally catalyzes the six-electron reduction of sulfite to sulfide (Dahl & Trüper, 2001). The mechanism how a proton gradient is generated in sulfate- reducing bacteria is still unclear.

The electrons for sulfate reduction are provided by an electron transport chain consisting of periplasmic hydrogenases (H2/2H⁺, E°’ = -414 mV), several cytochromes, and other membrane-bound and cytoplasmic redox enzymes (Odom & Peck, 1981). Oxidation of hydrogen in the periplasmic space and electron transfer across the cytoplasmic membrane liberates two protons. From H⁺/H2 ratios greater than two, Fitz and Cypionka (Fitz &

Cypionka, 1991; Fitz & Cypionka, 1989; Cypionka & Pfennig, 1986) concluded that in Desulfovibrio sp. the proton gradient is generated by proton translocation and vectorial electron transport. Recent sequence data support the idea that the Hmc complex generates a proton motive force. This transmembrane complex found in Desulfovibrio sp. comprises subunits with high sequence homology to archaeal heterodisulfide reductase, which is coupled to proton translocation in methanogenic archaea (Deppenmeier et al., 1990; 1991; 1996;

Peinemann et al., 1990). All redox enzymes in sulfate-reducing organisms contain iron-sulfur centers as prosthetic groups.

1.5 Adenylylsulfate (APS) reductases

1.5.1 Assimilatory APS reductase

Archaea, bacteria, fungi, and plants reduce sulfate to sulfide, but they do so for different purposes. The sulfate assimilation pathways serve for the synthesis of sulfur compounds necessary for growth and development. In all organisms, sulfate assimilation begins with the enzyme ATP sulfurylase that catalyzes the adenylation of sulfate to adenosine 5’- phosphosulfate (APS), which is then reduced by adenosine 5’-phosphosulfate reductase to sulfite and AMP in plants and some bacteria. In other bacteria and fungi, APS is further

phosphorylated at the 3’-position by APS kinase forming 3’-phosphoadenosine 5’- phosphosulfate (PAPS) before either being reduced by PAPS reductase (CysH) in a thioredoxin-dependent reaction to sulfite or being used for sulfatation. Ferredoxin-dependent sulfite reductase completes the reduction of sulfite to sulfide. Cysteine is formed when sulfide reacts with O-acetylserine mediated by O-acetylserine thiol-lyase (Bick & Leustek, 1998).

Plant APS reductase is unique in that it is able to use reduced glutathione at physiological concentrations as a source of electrons. Glutathione is thus the most likely physiological electron donor for APS reduction. By contrast, PAPS reductase requires thioredoxin or glutaredoxin as reductant. The glutathione-dependency of plant APS reductase is probably mediated through a carboxyl terminal domain that functions as a glutaredoxin, which is lacking in the bacterial and fungal enzymes (Weber et al., 2000).

Plant APS reductase from two species, Arabidopsis thaliana and Catharanthus roseus, overexpressed in E. coli was described as lacking prosthetic groups or cofactors. However, the enzyme was isolated from Lemna minor as a yellow protein indicating the presence of a cofactor, possibly FAD and/or iron-sulfur centers (Suter et al., 2000).

1.5.2 Dissimilatory APS reductase

Adenosine 5’-phosphosulfate (APS) reductase of sulfate-reducing prokaryotes is a αβ- heterooligomer, which contains FAD and iron-sulfur clusters. It catalyzes the two-electron reduction of APS to sulfite and AMP (Lampreia et al., 1994):

APS + 2e^- → AMP + HSO3- E°’(APS/AMP+HSO3-) = -60 mV (Thauer et al., 1977)

The molecular parameters of APS reductase, such as molecular mass, subunit composition, and cofactor stoichiometry, have been a matter of debate for a long time. Lampreia (Lampreia et al., 1994) proposed an α2β2-subunit composition with one FAD and two [4Fe-4S]

prosthetic groups per αβ-heterodimer (α ≈ 70 kDa, β ≈ 23 kDa). Speich (Speich et al., 1994) proposed an α2β-subunit composition with one FAD located at the interface of two α-subunits (73.3 kDa), and a [4Fe-4S] as well as a [3Fe-4S] center located on the β-subunit (17.1 kDa).

In contrast, Verhagen (Verhagen et al., 1994) reported that APS reductase contains a single iron-sulfur center per αβ-heterodimer, which was proposed to consist of more than four iron atoms, arranged in a novel, non-cuboidal structure. Those authors proposed a α2β2-subunit composition. Analysis of the genes encoding the α- and β-subunits of the APS reductase from the sulfate-reducing archaeon A. fulgidus (Speich et al., 1994), and the sulfate-reducing

bacterium D. vulgaris revealed a putative FAD-binding domain on the α-subunit and iron- sulfur binding motifs on the β-subunit. The N-terminal part of the β-subunit is highly homologous to 2[4Fe-4S] ferredoxins. It contains eight conserved cysteinyl residues, with four of them arranged in a conventional Cys-x¹-x²-Cys-x³-x⁴-Cys... Pro-Cys (xⁿ = variable amino acid) binding motif. The other four cysteinyl residues are arranged in a modified Cys- x¹-x²-Cys-x³-...-x⁹-Cys...Cys-Pro motif, where five additional residues are inserted (Hipp et al., 1997).

The catalytic mechanism of APS reductases has not been studied in detail so far. Micheals (Micheals et al., 1970) observed the formation of a sulfite-adduct at the N(5) position of the isoalloxazine ring of FAD, and proposed it as an intermediate during catalysis. However, his data is not very strong because flavin N(5)-sulfite adducts have been described as a characteristic feature of numerous flavin-dependent oxidases (Müller & Massey, 1969;

Massey et al., 1969) that catalyze the reduction of molecular dioxygen.

1.6 Sulfite reductases

1.6.1 Assimilatory sulfite reductase

Assimilatory sulfite reductases in bacteria, fungi, algae and plants provide the reduced sulfur (oxidation state –2) necessary for incorporation into biomolecules required by themselves and other higher organisms (Cole & Ferguson, 1988; Murphy & Siegel, 1973). Sulfite reductase generates sulfide from sulfite for subsequent cysteine biosynthesis in the terminal step of the 3’-phosphoadenylyl sulfate (PAPS) pathway.

The E. coli assimilatory sulfite reductase (E.C. 1.8.1.2) is an oligomer of eight 66-kDa flavoprotein (SirFP) and four 64-kDa hemoprotein (SirHP) subunits. In vivo, SirFP transfers electrons from NADPH to SirHP. Each SirFP has one FAD and one FMN binding site the SirFP octamer binds only four FAD and four FMN cofactors (Ostrowski et al., 1989). Isolated SirHP, when provided with suitable electron donors can reduce SO32- to HS^- and NO2- to NH4+ without releasing intermediates (Siegel & Davis, 1974). SirHP accommodates an electron at the siroheme with a redox potential of -340 mV and at the Fe4S4 cluster with an

'

E0 of –405 mV (Siegel et al., 1982; Jannick & Siegel, 1982). Reduction of SirHP enhances substrate binding and dissociation rates 10⁵ times, suggesting a link between cofactor electronic states and protein conformation (Janick et al., 1983). The crystal structure revealed how the protein utilizes underlying twofold symmetry to associate cofactors and enhance their

reactivity for catalysis. The saddle-shaped siroheme shares a cysteine thiolate ligand with the Fe4S4 cluster and ligates the substrate sulfite (Crane et al., 1995).

1.6.2 Dissimilatory sulfite reductase

Dissimilatory sulfite reductases or desulfoviridins catalyze the six-electron reduction of sulfite to sulfide in sulfate respiration (LeGall & Fauque, 1988):

HSO3- + 6e^- + 6H⁺ → HS^- + 3H2O E°’ (HSO3-/HS^-) = -116 mV (Odom & Peck, 1981)

This enzyme has been described as α2β2γmδn-multimers with α ≈ 50 kDa, β ≈ 45 kDa, γ ≈ 11 kDa, δ ≈ 8 kDa, and a total molecular mass of approximately 200 kDa (Steuber & Kroneck, 1998; Steuber et al., 1995). Dissimilatory sulfite reductase has been isolated from D. vulgaris (Lee et al., 1973), D. gigas (Lee et al., 1971), D. baculatus (Moura et al., 1988) and A.

fulgidus (Dahl et al., 1993). The γ- and δ-subunits are only loosely bound in some organisms and even completely absent in the A. fulgidus enzyme. The structure of the γ-subunit from Pyrobaculum aerophilium as well as the δ subunit from D. vulgaris has been characterized.

The γ-subunit from Pyrobaculum aerophilium reveals a novel fold with an orthogonal helix bundle with a β hairpin resembling the helix-turn-helix motif involved in DNA-binding. A flexible seven residue c-terminal arm with a c-terminal cysteine is suggested to be involved in interaction with the α2β2-tetramer (Cort et al., 2001). The δ-subunit from D. vulgaris contains a winged helix motif suggesting that it is involved in DNA binding (Mizuno et al., 2003). It has been suggested that it binds sulfate or sulfite (Karkhoff-Schweizer et al., 1995) and indeed 5 sulfates are found in the crystal structure, but previous studies had already ruled out physiological binding of sulfate or sulfite (Hittel & Voordouw, 2000).

Found throughout the three domains of living organisms, many of these enzymes employ a siroheme that is located right next to an iron-sulfur cluster (Crane et al., 1995; 1997a; 1997b).

EPR signals at high g-values are found in dissimilatory sulfite reductases with the highest apparent g-values at g = 17 and g = 9, which are proposed to result from an S = 9/2 system.

Those EPR signals at high g-values are found in dissimilatory sulfite reductases and were assigned to a novel type of iron-sulfur cluster (Pierik & Hagen, 1991; Marritt & Hagen, 1996).

1.7 High-spin iron clusters in biological systems

The most commonly found iron clusters in biology are low-spin (S=1/2) or diamagnetic systems depending on the redox state. They are classified according to the number of irons and their redox state. Apart from these ‘classical’ iron sulfur clusters there are clusters with unusual properties, which can be divided, into two groups: clusters with more than four iron ions and clusters with one to four irons but unusual spin in the ground state.

On a [2Fe-2S] cluster of a 2Fe ferredoxin from Clostridium pasteurianum one of the cysteine ligands has been mutated to serine with the surprising result of a [2Fe-2S]⁺ cluster with an S=9/2 valence-delocalized ground state (Grouse et al., 1995;Achim et al., 1996). This is of interest as this is the first report of existence of a fragment is used to describe the magnetic properties of [4Fe-4S], [3Fe-4S] and [8Fe-7S] clusters.

This fragment is also used to rationalize the properties in the [3Fe-4S]^- situation where an S=5/2 ground state is observed. A valence delocalized S=9/2 [2Fe-2S]⁺ fragment couples antiferromagnetically with a valence localized S=2 Fe²⁺ site.

Nitrogenase is the protein with the most unusual metal cofactors known. It contains three types of iron clusters.

The Fe protein of nitrogenase from Azotobacter Vinelandii has a regular [4Fe-4S] cluster that can be reduced to the all ferrous state (Watt & Reddy, 1994) (cluster charge=0) and has an integer spin S=4 ground state (Angove et al., 1997; Yoo et al., 1999). In contrast to other [4Fe-4S] clusters it was proposed that this cluster is capable of two-electron transfer that might be needed for the six-electron reduction of molecular nitrogen to ammonia.

The active site of nitrogen reduction is the Fe Mo cofactor, which is a [Mo-7Fe-8S-N] cluster (Einsle et al., 2002). It has been extracted and characterized as an S=3/2 system (Rawlings et al., 1978; Burgess et al., 1980).

The third cluster in nitrogenase is the P-cluster it is used for the electron transfer from the Fe protein to the Fe Mo cofactor active site. It has two high spin ground states depending on the redox state. It is diamagnetic in the reduced state, in the one-electron oxidized state it is most probably S=3 and the oxidized state has a spin admixed S=1/2 S=7/2 ground state (Chan, 1999).

1.8 Protein crystallography

The first protein structures solved to almost atomic resolution by three-dimensional Fourier synthesis of X-ray diffraction patterns of single crystals were the ones of hemoglobin (Perutz et al., 1960) and myoglobin (Kendrew et al., 1960). Seven years earlier, fiber diffraction experiments were used to unravel the three-dimensional structure of deoxyribose nucleic acid (Watson & Crick, 1953).

Since then, protein crystallography has developed into a well-established and reliable technique with a wide range of possible applications. Driven by rapid progress in molecular biology and biochemistry and the advance of computer hard- and software in the last 20 years, this has led to a total number of more than 20939 protein structures deposited in the Protein Data Bank by the end of 2003. More than 18100 of those structures were solved by X-ray diffraction and around 2830 by multidimensional nuclear magnetic resonance spectroscopy (NMR). This ratio emphasizes the central role of protein crystallography in the field of structural biology.

Structure solution by NMR complements protein crystallography in several ways. It provides information on protein dynamics that cannot be obtained from the rather rigid environment of a crystal lattice and it is also not dependent on the availability of protein crystals.

1.9 Scope of the study and research objectives

Subject of the presented dissertation were the structural and functional studies on key enzymes of the dissimilatory sulfate reduction. Adenylylsulfate (APS) reductase and sulfite reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus were used as model systems.

The iron-sulfur flavoenzyme APS reductase was already biochemically and spectroscopically extensively characterized and the overall structure was recently established (Schiffer, 2000;

Fritz, Roth, Schiffer et al., 2002). The investigations were then directed to explore the enzymatic mechanism of the multi-step reaction on an atomic level. Accordingly, the enzyme from A. fulgidus had to be purified and crystallized under exclusion of dioxygen. Afterwards the crystals were soaked with diverse reagents in order to structurally characterize the enzyme in different redox states, in complex with substrate and products and in intermediate states.

Subsequently a dataset of each state was collected at high resolution using synchrotron radiation. The derived structures were then refined to extract also minute differences between the states.

The siroheme containing enzyme dissimilatory sulfite reductase is of extraordinary biochemical and biophysical interest but so far no detailed structural information is available.

Consequently, a purification procedure for the enzyme from A. fulgidus was worked out. Both purification and crystallization experiments were performed under strict exclusion of molecular oxygen. Assuming that X-ray suitable crystals could be obtained an X-ray structure analysis was intended. In parallel, the purified sulfite reductase was characterized with biochemical and spectroscopic (UV/Vis and EPR) techniques. Again the ultimate goal was to understand the reaction mechanism and the unusual electronic properties of the enzyme on an atomic level.

2 Materials and Methods

2.1 Chemicals

All chemicals were obtained in p.a. quality and were used without further purification.

Buffers

TRIS (trishydroxymethylaminomethane), BICINE (N,N-bis-(2-hydroxyethyl)-glycine), Roth;

K2HPO4, NaH2PO4, Merck; KH2PO4, Na-citrate dihydrate, Riedel-de-Haën.

Chromatographic resins

Q Sepharose Fast Flow, Resource Q, Amersham Pharmacia Biotech; Chelex^® 100 Chelating Ion Exchange Resin, Bio-Rad.

Dyes

Coomassie Brilliant Blue G250, methyl viologen, Serva; flavin adenine dinucleotide (FAD), Fluka.

Gas

N2, N2/CO2 (80:20 v/v), H2, N2/H2 (94:6 v/v) Sauerstoffwerk Friedrichshafen; Argon 4.8, Argon 4.9, Helium 4.6, Messer Griesheim.

General chemicals

Zn-acetate, Na2SO4, Na2S·xH2O (35 % Na2S), CaCl2·2H2O, acetone, 70 % trichloroacetic acid, methanesulfonic acid, acetonitrile, 37.5 % HCl, Merck; MgCl2·6H2O, NaCl, NaOH, NH4Cl, CuSO4·5H2O, FeCl3·6H2O, Riedel-de-Haën; Na-ethylmercurythiosalicylate, Serva.

Proteins and enzymes

Low molecular mass standards (PAGE), BioRad; gelfiltration molecular mass markers, Sigma; desoxyribonuclease I, Fluka.

Reagents

Bicinchoninic acid solution (BCA), Sigma.

5-Deaza-10-methyl-3-sulfopropyl-isoalloxazine potassium salt (5-deazaflavin) was synthesized and kindly provided by K. Sulger, Universität Konstanz.

Crystal screen solutions were obtained from Hampton Research (USA).

JB screen solutions were obtained from Jena Bioscience (Germany).

Adenosine 5'-phosphosulfate (APS) was obtained from Sigma or was synthesized and kindly provided by Dr. T. Büchert, Universität Konstanz.

β-Methyleno-adenosine 5'-phosphosulfate (βmAPS) was obtained from JenaBioScience GmbH (Germany).

2.2 Protein biochemistry

2.2.1 Organism and cultivation

The cultivation of Archaeoglobus fulgidus (DSM 4304T) was carried out as previously described (Stetter et al., 1987) by H. Huber, Universität Regensburg.

2.2.2 Preparation of cell fractions

All manipulations were carried out in an anaerobic chamber (95 % N2, 5 % H2; Coy) in the absence of dioxygen.

Frozen cells were brought into the anaerobic chamber, suspended in 1-2 volumes of 20 mM potassium phosphate buffer, pH 7.0 containing a few crystals of desoxyribonuclease I and 5 mM MgCl2·6H2O and filled into a French press (Aminco). Cells were broken by one passage in a French press (138 MPa; Aminco). The cell lysate was collected in a N2/H2

containing glass bottle sealed with a rubber septum. The cell lysate was brought again into the anaerobic chamber and filled into dioxygen-free centrifuge tubes. The lysate was centrifuged for 120 min at 100,000 g (4°C) giving the soluble fraction as supernatant, containing both periplasmic and cytoplasmic proteins. The black pellet was referred to as membrane fraction.

2.2.3 Purification protocols

In order to minimize protein denaturation by thermal-induced unfolding or by protease activity, each purification was performed within 48 h.

2.2.3.1 APS reductase of Archaeoglobus fulgidus

APS reductase was isolated in the absence of dioxygen on a FPLC system (Amersham Pharmacia Biotech). All chromatographic steps were performed at 18°C in an anaerobic chamber (95 % N2, 5 % H2; Coy).

After centrifugation, the soluble fraction was applied to a Q Sepharose Fast Flow column (5.0

× 5.0 cm; Amersham Pharmacia Biotech) equilibrated with 20 mM potassium phosphate buffer, pH 7.0. APS reductase was eluted in a linear gradient (0-1.0 M KCl) at about 0.10 M KCl. Fractions containing APS reductase were combined and desalted by ultrafiltration (cut- off 30 kDa; Amicon) with subsequent dilution with 20 mM potassium phosphate buffer, pH 7.0, 5 % (v/v) glycerol. The desalted protein was loaded onto a Resource Q15 column (1.6 cm × 14 cm; Amersham Pharmacia Biotech) equilibrated with 20 mM potassium phosphate, pH 7.0, 5 % (v/v) glycerol. A linear gradient (0-0.5 M KCl) led to elution of APS reductase at 0.15 M KCl. Fractions containing APS reductase were combined. The combined

fractions were concentrated by ultrafiltration (cut-off 30 kDa; Amicon) and loaded onto a Superdex 200 HiLoad 26/60 gelfiltration column (2.6 × 60 cm; Amersham Pharmacia Biotech) equilibrated with 50 mM potassium phosphate buffer, pH 7.0, 150 mM KCl, 5 % (v/v) glycerol.

2.2.3.2 Dissimilatory sulfite reductase of Archaeoglobus fulgidus

Sulfite reductase was isolated in the absence of dioxygen on a FPLC system (Amersham Pharmacia Biotech). All chromatographic steps were performed at 18°C in an anaerobic chamber (95 % N2, 5 % H2; Coy).

After centrifugation, the soluble fraction was applied to a Q Sepharose Fast Flow column (1.6

× 10.0 cm; Amersham Pharmacia Biotech) equilibrated with 20 mM potassium phosphate buffer, pH 7.0. Sulfite reductase was eluted in a linear gradient (0-1.0 M KCl) at about 0.54 M KCl. Fractions containing sulfite reductase were combined and desalted by ultrafiltration (cut-off 30 kDa; Amicon) with subsequent dilution with 20 mM potassium phosphate buffer, pH 7.0, 5 % (v/v) glycerol. The desalted protein was loaded onto a Resource Q15 column (1.0 cm × 13 cm; Amersham Pharmacia Biotech) equilibrated with 20 mM potassium phosphate buffer, pH 7.0, 5 % (v/v) glycerol. A linear gradient (0-1 M KCl) led to elution of sulfite reductase at about 0.27 M KCl. Fractions containing sulfite reductase were combined. The combined fractions were concentrated by ultrafiltration (cut- off 30 kDa; Amicon) and loaded onto a Superdex 200 HiLoad 26/60 gelfiltration column (2.6 × 60 cm; Amersham Pharmacia Biotech) equilibrated with 50 mM potassium phosphate buffer, pH 7.0, 150 mM NaCl, 5 % (v/v) glycerol.

2.2.4 Analytical methods

2.2.4.1 Protein determination

Protein was determined by the bicinchoninic acid method according to Smith (Smith et al., 1985). 100 µl unknown sample (5-20 µg protein) and 1 ml 50:1 (v/v) BCA / 4 % (w/v) CuSO4·5H2O were mixed and incubated for 25 min at 60°C. After incubation, the samples were cooled on ice, mixed and centrifuged for 5 min at 9,500 g. The absorbance at 562 nm was measured on a HP 8452 A diode array spectrophotometer (Hewlett Packard) and the concentration calculated using a calibration curve (5-20 µg BSA).

The microbiuret method (Goa, 1953) was performed with the following modifications: 700 µl unknown sample (100-400 µg protein) were precipitated by subsequent addition of 0.0125 % (w/v) Na-deoxycholate and 5.8 % (w/v) trichloroacetic acid (Bensadoun & Weinstein 1976).

After centrifugation for 5 min at 9,500 g, the pellet was dissolved in 700 µl 3 % (w/v) NaOH at 45°C for 10 min. After addition of 35 µl biuret reagent and vigorous mixing for 10 s, the samples were incubated for 15 min at room temperature in the dark. The absorbance at 545 nm and 330 nm was measured on a HP 8452 A diode array spectrophotometer (Hewlett Packard) and the concentration calculated using a calibration curve (60-450 µg BSA). The biuret reagent was prepared by adding 0.221 g anhydrous CuSO4 in 6 ml H2O to 3.46g Na-citrate dihydrate / 2.0 g NaCO3 in 12 ml H2O and adjusting the final volume to 20 ml.

2.2.4.2 Determination of iron

Iron determinations by inductively coupled plasma mass spectroscopy (ICP-MS) were performed by Spurenanalytisches Laboratorium Dr. Heinrich Baumann, Maxhütte-Haidhof, Germany.

2.2.4.3 Denaturing polyacrylamide gel electrophoresis

SDS-PAGE was carried out with the Hoefer Mighty Small II SE 250 System (80 × 70 × 0.75 mm; Hoefer Scientific Instruments), or the BioRad MiniProtean 3 System (80 × 70 × 0.75 mm; BioRad) using glycine buffered 12.5 % polyacrylamide gels (Laemmli, 1970) and tricine buffered 16 % acrylamide gels (Schägger & von Jagow, 1987). The molecular mass of the subunits was estimated using Low Range SDS-PAGE Molecular Weight Standards (BioRad).

Gels were stained with Coomassie Brilliant Blue G250 (Zehr et al., 1989) or silver (Rabilloud, 1990).

2.2.5 Experiments under exclusion of dioxygen

Experiments under exclusion of dioxygen were carried in an anaerobic chamber (95 % N2, 5

% H2; Coy) equipped with a Palladium catalyst type K-0242 (0.5 % Pd/Al2O3; ChemPur) to remove traces of dioxygen. The content of dioxygen in the anaerobic chamber was < 1 ppm, which was experimentally confirmed according to Beinert (Beinert et al., 1978). Glass and plastic ware was stored in the anaerobic chamber for at least 24 h prior to use.

Dioxygen from buffers and solutions was removed by 6-8 cycles of degassing and flushing with Argon 4.9 (Messer Griesheim) (Beinert et al., 1978). Traces of dioxygen were removed from Argon 4.9 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.

2.2.6 Enzymatic activities

2.2.6.1 Photometric determination of APS reductase activity

APS reductase activity can be followed photometrically as described (Büchert, 2001). In the reductive reaction (APS + 2e^-→ AMP + HSO3-), reduced methyl viologen served as electron donor.

All steps were performed under exclusion of dioxygen in an anaerobic chamber (95 % N2, 5 % H2; Coy). The following chemicals were added directly to a cuvette (final volume 1 ml):

500 µl 200 mM potassium phosphate buffer, pH 7.6 (final concentration 100 mM); 160 µl 250 mM Na-oxalate (final concentration 40 mM); 150 µl 5 mM methyl viologen (final concentration 0.75 mM); 5 µl 5 mM 5-deazaflavin (final concentration 25 µM); 30 µl 1.58 mM APS (final concentration 47 µM); 105 µl H2O. The cuvette was sealed with a Suba Seal 9 red rubber septum (Sigma) and the solution was thoroughly mixed. APS reductase (0.2 mg/ml) was diluted 1:1 with 12 mg/ml BSA in 100 mM potassium phosphate buffer, pH 7.6. This solution was transferred to a glass vial sealed with a rubber septum. The cuvettes and the vial containing APS reductase were transferred outside the anaerobic chamber just prior to use. Methyl viologen was reduced photochemically by irradiation in a modified slide projector with a thermostatted cell holder. After 60 s of irradiation the absorbance at 732 nm was measured. About 30-35 % of the methyl viologen was reduced and the absorbance at 732 nm was 0.80 ± 0.05. After reduction, the cuvettes were tempered for 5 min (80-85°C).

After the rate without enzyme was recorded, the reaction was started by addition of 50 µl APS reductase (final concentration 53 pM) using a gas-tight syringe (Hamilton). The oxidation of methyl viologen was followed at 732 nm (ε⁷³² =3,150 M^-1·cm^-1) in a Cary 50 conc Spectrophotometer (Varian) with thermostatted cell holder (80-85°C). After the rate determination, the cuvette was opened and the temperature was directly measured in the solution with a resistance thermometer (Digitalthermometer 500; MAWI). The specific activity was calculated as µmol APS reduced per min and mg APS reductase.

2.2.6.2 Photometric determination of sulfite reductase activity

Sulfite reductase activity can be followed photometrically as described (Büchert, 2001). In the reductive reaction (HSO3- + 6e^-→ HS^-), reduced methyl viologen served as electron donor.

All steps were performed under exclusion of dioxygen in an anaerobic chamber (95 % N2, 5 % H2; Coy). The following chemicals were added directly to a cuvette (final volume 1 ml):

200 µl 250 mM potassium phosphate buffer, pH 7.0 (final concentration 50 mM); 160 µl

250 mM Na-oxalate (final concentration 40 mM); 150 µl 5 mM methyl viologen (final concentration 0.75 mM); 20 µl 0.5 mM 5-deazaflavin (final concentration 10 µM); 420 µl H2O. The cuvette was sealed with a Suba Seal 9 red rubber septum (Sigma) and the solution was thoroughly mixed. Sulfite reductase (5-10 mg/ml) was diluted 1:1 with 12 mg/ml BSA in 100 mM potassium phosphate buffer, pH 7.6. This solution was transferred to a glass vial sealed with a rubber septum. The cuvettes and the vials containing sulfite and sulfite reductase were transferred outside the anaerobic chamber just prior to use. Methyl viologen was reduced photochemically by irradiation in a modified slide projector with a thermostatted cell holder. After 90 s of irradiation the absorbance at 732 nm was measured. About 30-35 % of the methyl viologen was reduced and the absorbance at 732 nm was 0.80 ± 0.1. After reduction, the cuvettes were tempered for 5 min (80-85°C). Then 50 µl 60 mM Na2SO3 (final concentration 3 mM) was added using a gas-tight syringe (Hamilton). After the rate without enzyme was recorded, the reaction was started by addition of 50 µl sulfite reductase (final concentration 53 pM) using a gas-tight syringe (Hamilton). The oxidation of methyl viologen was followed at 732 nm (ε⁷³² =3,150 M^-1·cm^-1) in a Cary 50 conc Spectrophotometer (Varian) with thermostatted cell holder. The specific activity was calculated as µmol sulfite reduced per min and mg sulfite reductase.

2.2.7 Spectroscopic methods

2.2.7.1 UV/Vis absorption spectroscopy

UV/Vis absorption spectra were obtained with a Cary 50 conc Spectrophotometer (Varian), with a Perkin Elmer Lambda 16 Spectrophotometer (Perkin Elmer), or with a HP 8452 A Diode Array Spectrophotometer (Hewlett Packard). All spectrophotometers except the HP 8452 A Diode Array Spectrophotometer were equipped with thermostatted cell holders.

Measurements with the HP 8452 A Diode Array Spectrophotometer were performed at room temperature.

2.2.7.2 Electron paramagnetic resonance spectroscopy

X-band EPR spectra were recorded with a Bruker Elexsys 500 with an ER 049X microwave bridge (Bruker). The system was equipped with an Oxford ESR 900 helium cryostat controlled by an ITC 503 temperature controller (Oxford Instruments). The modulation frequency was 100 kHz and the modulation amplitude was typically 0.1-1 mT. The measurements were performed with a Bruker 4122 SHQE cavity in the perpendicular field mode in which the resonance frequency was ≈ 9.38 GHz. The sample tubes were Suprasil

quartz tubes with a diameter of 2-3 mm. The sample volume was 250 µl. The g-values were calculated according to the resonance equation:

h·ν = g·β·H

with h = 6.6262·10^-34 J·s (Planck’s constant) β = 9.274096·10^-24 J·T^-1 (Bohr Magneton) ν = microwave frequency in Hz

H = magnetic field in T.

The simulation of the spectra was performed with the program WEPR (Neese, 1995).

2.2.8 Titrations

Titrations with reductants, oxidants or substrates were carried out in a modified Thunberg cuvette with two rubber septa. The reactant was added in steps of 2-10 µl using a gas-tight syringe (Hamilton). A spectrum was recorded immediately after addition and after incubation with the reactant. The cuvette was filled and the syringe was pierced through the septa in an anaerobic chamber (95 % N2, 5 % H2; Coy).

2.3 Protein crystallography

A detailed discussion of protein crystallography was beyond the scope of this work and can be found in relevant textbooks (Blundell & Johnson, 1994; Drenth, 1994; Massa, 1994;

McRee, 1993) and in Meth. Enzymol. 276.

2.3.1 Theoretical background

The maximum attainable resolution of any microscopic technique is limited by the applied wavelength. The radiation needed to analyze atomic distances (e.g. 1.54 Å for a carbon- carbon σ-bond) lies within the spectral range of X-rays. However, while light or electron microscopy uses lenses to merge the waves diffracted by an object into an enlarged image, there are no such lenses available for X-rays. Max von Laue realized in 1912, that the three- dimensionally ordered lattice arrangement of a crystal will cause interference of the diffracted photons resulting in discrete maxima whose intensity can be measured in an appropriate experimental setup.

2.3.1.1 Crystal growth

The process of crystal formation is in principle thermodynamically favored, driven by the gain of entropy through the loss of the proteins' ordered hydratation shell. A solution of the protein is slowly brought into a state of supersaturation – usually by evaporation of water – until ordered crystals are formed. Mechanistically, the crystallization process can be divided into two stages, seed formation and crystal growth. Supersaturation of the system – defined as the difference of the chemical potentials of solution and crystal – is a prerequisite for both stages. Seed formation occurs in equilibrium of formation and dissolving of small aggregates, determined by the free energy ∆G. It will have a maximum at a critical radius, meaning that aggregates with a radius smaller than the critical radiuswill redissolve, while for those bigger than the critical radiusfurther crystal growth means a decrease of ∆G. The critical cluster size for protein crystals is between 10 and 200 molecules.

2.3.1.2 Crystals

A crystal can be regarded as a three-dimensional repetition of a single building block, the unit cell. Within the unit cell, a crystal can contain further symmetry elements, dividing it into several asymmetric units, which form the most basic structural element within the crystal. The geometry of the unit cell together with the possible symmetry operations defines the space group of the crystal. Although there are 230 space groups in seven crystal systems (triclinic, monoclinic, orthorhombic, tetragonal, trigonal, hexagonal and cubic), only 65 are enantiomorphic and are thus feasible for chiral molecules such as proteins. Identification of the correct space group is essential for correct indexing of diffraction patterns and therefore the first step of understanding a crystal structure.

2.3.1.3 X-ray diffraction by crystals

Upon interaction with the atoms in a crystal, the oscillating electrical field of an X-ray photon induces an oscillation of equal frequency in the electron hull of the atom. The electrons act as oscillating dipoles emitting secondary radiation of the same frequency as the incident radiation, but with a phase difference of 180°. In this elastic or coherent diffraction, the phase shifts between single waves originating from any point of finite electron density sum up to a total intensity of the secondary radiation of zero (destructive interference), except if the path difference between the waves is an integer multiple of their wavelength (constructive interference). Given the correct orientation of the crystal, this condition is fulfilled for corresponding positions in all unit cells. Diffraction of X-rays on the real lattice of a crystal thus creates another three-dimensional lattice of diffraction maxima. As the geometric

properties of this lattice are inverse to those of the real crystal, it is referred to as the reciprocal lattice. A convenient way to describe diffraction by a crystal lattice is to imagine every single diffraction spot to be a reflection of the incident beam on an imaginary lattice plane, which is identified by the Miller indices (h,k,l). The normal vectorsSr

of those lattice planes then build up the reciprocal lattice, their length reflecting the reciprocal distance of the planes.

θ

d

2d sin θ

Figure 2.1: Bragg’s law: Two waves that are reflected by two adjacent lattice planes with distance d have a difference in path length that is equal to 2d sin θ, as it can easily be derived from the scheme.

A prerequisite for constructive interference is, that this difference in path is an integer multiple of the wavelength used: 2d sin θ = nλ (Bragg’s law).

Regarding elastic diffraction on a set of lattice planes with distance d, constructive interference will occur at an angle θ, if the path difference between the diffracted waves is an integer multiple of the wavelength λ. This relation between reflection angle and lattice plane distance is known as Bragg’s law:

2 dhkl sin θ = n λ

The Ewald sphere is a tool for constructing reciprocal lattice points on the basis of Bragg’s law. It is a sphere of radius 1/λ with the crystal in its center. The point where the incident beam sr₀enters the sphere and the origin O of the reciprocal lattice are on opposite sides of the center. Bragg’s law is fulfilled for every reciprocal lattice point that lies on the Ewald sphere.

A rotation of the crystal rotates the reciprocal lattice in the same way, allowing different reciprocal lattice points to intersect with the sphere. For the given orientation of the crystal, those points are the ones that can be recorded on an X-ray detector.

C

θ d 1/λ

S O

Figure 2.2: The Ewald construction. In reciprocal space, the crystal (C) is placed in the center of a sphere (here, in two dimensions, a circle) with radius 1/λ, called the Ewald sphere. The origin of the reciprocal lattice, i.e. reflection (0 0 0), is placed in (O). The reciprocal lattice (grey dots) will rotate as the crystal does and only those reciprocal lattice points that intersect with the Ewald sphere will be in diffraction condition and will be recorded on an image plate detector in real space.

As every recorded diffraction spot represents one lattice plane (h,k,l), the measurement of the positions of the spots is sufficient to deduce the geometry of the crystal and in most cases also the space group, as additional symmetry elements can manifest in the form of systematic extinctions of reflections. The result of a data collection on a crystal will primarily be the knowledge about space group and unit cell dimensions, and – based on this – an intensity measurement I(h,k,l) for every reflection (h,k,l).

2.3.1.4 The electron density function

The goal of a crystallographic experiment is to calculate the distribution of electron density in the asymmetric unit of the crystal in order to be able to place an atomic model of the crystallized molecule therein.

The scattering of all atoms in the asymmetric unit is the sum of all atomic scattering factors, taking in account individual phase shifts. For every single reflection (h,k,l) this summation leads to a structure factor F(h,k,l):

( )

∑ ( (

) )

i

i i hx ky lz

f l

k h

F , , exp 2π

The reciprocal lattice is the Fourier transform of the electron distribution in the crystal, split up in the form of the structure factors. This means that the electron density ρ(x,y,z) for every point in real space can be calculated as a Fourier summation over all structure factors:

( )

∑ ( ) ( (

) )

l k h

lz ky hx i l

k h V F

z y x

, ,

2 exp , 1 ,

,

, π

ρ

2.3.1.5 The phase problem

Approaching from the side of the diffraction experiment, each structure factor F(h,k,l) also represents a reflection by one lattice plane. It is described by a wave function with amplitude and phase angle. The structure factor amplitude can be obtained experimentally, as it is in principle the square root of the measured intensity.

While the structure factor amplitude can be derived from the measured intensity, information about the phase angle is lost. Without correct phase angles, the calculation of an interpretable electron density is impossible, a dilemma commonly referred to as the phase problem of crystallography. Four approaches to overcome this problem are applicable today:

• Molecular Replacement (MR)

• Multiple Isomorphous Replacement (MIR)

• Multiple-wavelength Anomalous Dispersion (MAD)

• Direct Methods

The method of Molecular Replacement depends on the availability of a sufficiently homologous model structure, which is oriented by Patterson search techniques and then used for initial phase calculations (Hoppe, 1957; Huber, 1965; Rossmann & Blow, 1962). Without any previous knowledge of the structure, multiple isomorphous replacement is still the most commonly used method. Herein it is attempted to place heavy atoms on specific sites in the protein – either by soaking or by cocrystallization – and to identify their positions by comparing the data collected from a native crystal with that of a derived one (Green et al., 1954). MAD depends on precisely tunable synchrotron radiation, which has only been available for the last years, but is becoming more and more standard (Hendrickson et al., 1988).

Direct methods are the common way to determine phases in small molecule crystallography, but due to the high number of atoms per asymmetric unit and the limited resolution that is obtained from most protein crystals, this approach has rarely been successful for large biomolecules. Most recently the number of protein structures solved by direct methods is increasing and the development is promising, but small molecule size, high resolution and good data quality are still a prerequisite.