Metalloproteins containing iron and tungsten: biocatalytic links between organic and inorganic redox chemistry Metalloproteins containing iron and tungsten: biocatalytic links between organic and inorganic redox chemistry Proefschrift ter verkrijging van de graad van doctor aan de Technische Universiteit Delft, op gezag van de Rector Magnificus prof. dr. ir. J.T. Fokkema, voorzitter van het College voor Promoties, in het openbaar te verdedigen op maandag 28 oktober 2002 om 16:00 uur door Peter-Leon HAGEDOORN ingenieur in de landbouw- en milieuwetenschappen geboren te Amersfoort Dit proefschrift is goedgekeurd door de promotor: Prof. dr. W.R. Hagen Samenstelling promotiecommissie: Rector Magnificus voorzitter Prof. dr. W.R. Hagen Technische Universiteit Delft, promotor Prof. dr. S. de Vries Technische Universiteit Delft Prof. dr. J.G. Kuenen Technische Universiteit Delft Prof. dr. C.D. Garner University of Nottingham, UK Prof. dr. ir. A.J.M Stams Wageningen Universiteit Prof. dr. C. Veeger Wageningen Universiteit Dr S.P.J. Albracht Universiteit van Amsterdam The studies presented in this thesis were performed at the Kluyver Department of Biotechnology, Delft University of Technology. This research has been financially supported by the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO) under project number 700-28-102. Published and distributed by: DUP Science DUP Science is and imprint of Delft University Press P.O. Box 98 2600 MG Delft The Netherlands Telephone: +31 15 27 85 678 Telefax: +31 15 27 85 706 E-mail: [email protected] ISBN 90-407-2349-4 Keywords: metalloprotein, redox chemistry, tungsten enzyme Copyright © 2002 by P.L. Hagedoorn All rights reserved. No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, inclu- ding photocopying, recording or by any information storage and retrieval system, without written permission from the publisher: Delft University Press. Printed in the Netherlands Contents Chapter 1 1 General introduction Chapter 2 23 Hyperthermophilic redox chemistry: A re-evaluation Chapter 3 35 Pyrococcus furiosus glyceraldehyde-3-phosphate oxidoreductase has comparable WVI/V and WV/IV reduction potentials and unusual [4Fe-4S] EPR properties Chapter 4 51 Steady-state kinetics and tungsten co-ordination of the glycolytic enzyme glyceraldehyde 3-phosphate oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus Chapter 5 73 Redox characteristics of the tungsten DMSO reductase of Rhodobacter capsulatus Chapter 6 85 Electroanalytical determination of tungsten and molybdenum in proteins v Chapter 7 101 The effect of substrate, dihydrobiopterin and dopamine on the EPR spectroscopic properties and the midpoint potential of the catalytic iron in recombinant human phenylalanine hydroxylase Chapter 8 121 Spectroscopic characterization and ligand binding properties of chlorite dismutase from the chlorate respiring bacterial strain GR-1 References 139 Summary 165 Samenvatting 169 Curriculum Vitae 173 List of abbreviations 175 List of publications 177 Nawoord 179 vi Chapter 1 General introduction 1 Chapter 1 The redox chemistry of iron and tungsten in biology Important chemical reactions in the main metabolic pathways of living organisms involve reduction and oxidation reactions, i.e. redox reactions. Biological redox chemistry is limited by the oxidation and reduction potentials of the solvent that is always present in biological systems: water. These potentials are +830 mV for the oxidation of water to molecular oxygen and –420 mV for the reduction of protons to molecular hydrogen at 25ºC, pH 7.0, and atmospheric pressure. However in an environment shielded from the solvent inside a protein, an overpotential of a few hundred millivolts above and below these limits is possible. Nature frequently uses transition metal centers or clusters as redox catalysts, because of their ability to take up and donate electrons. This thesis is concerned with a set of proteins that, taken together, offer a wide view into metal based redox biochemistry. Both redox enzymes and electron transfer proteins are considered. As can be seen in table 1 these proteins cover almost the whole biological redox potential range. Table 1. Redox properties of the metalloproteins studied in this thesis. Protein Redox Em vs. NHE (mV) Chapter(s) center(s) Electron transfer proteins Rubredoxin Fe(Cys)4 +40 2 Ferredoxin [4Fe-4S] -350 2 Tungsten enzymes Dimethylsulfoxide reductase W-pterin -134 and –194 5 Glyceraldehyde-3-phosphate W-pterin -450 and –650 3-4, 6 oxidoreductase [4Fe-4S] -320 Iron enzymes Phenylalanine hydroxylase Non heme Fe +200 7 Chlorite dismutase Heme iron +10 8 All but one of these enzymes and proteins contain iron as a redox active center, however in different forms. And two of the enzymes contain a tungsten center, which is a rare element in biochemistry. Note that all the enzymes listed in table 1 catalyze 2 electron (or 4 electron in the case of chlorite dismutase) redox chemistry. In 2 General introduction these enzymes the metal has to be connected to the organic world, in which most redox reactions are 2 electron reactions. Molybdenum and tungsten are excellent metals for 2 electron redox chemistry, but iron is more suitable for 1 electron reactions (as is the case for the electron transfer proteins). Nature has dealt with the limitation of iron by stabilizing higher oxidation states of iron, i.e. compound I and II which are further introduced below. Libraries can be filled with the literature on the biochemistry of iron. Therefore, only a concise introduction will be presented to the different biological forms of iron that are relevant for this thesis. Furthermore, a brief introduction will be given to tungsten enzymes and their family ties with the more common molybdenum enzymes. Heme iron containing enzymes Introduction Probably the most abundant and most studied iron containing proteins are the heme proteins. Several heme cofactors exist, all derivatives of a tetrapyrrole ring system. These cofactors coordinate the metal by the four nitrogens forming a planar coordination environment. Although several heme derivatives have been found in nature, iron-protoporphyrin IX, or heme b, is commonly found in heme enzymes (Figure 1). Often heme iron is used to bind small inorganic compounds (e.g. di- oxygen), to perform oxygen chemistry, or to take up or donate electrons. These basic functionalities afford many different biological functions such as oxygen transport, CO sensing, peroxidations, NO synthesis. Heme proteins have been excellent objects for spectroscopic studies. Allowed π-π* transitions of the heme moiety result in intense electronic absorption bands, which have been named α and β in the range 450-700 nm and γ (or Soret band) in the range 390-450 nm. The γ band is usually ten times more intense than the α and β bands. 3 Chapter 1 N N Fe N N O HO O OH Figure 1. Protoporphyrin IX. Table 2 gives a summary of Fe(III/II) midpoint potentials of different heme proteins with different proximal ligands of the iron site. Clearly, heme iron is a versatile redox catalyst whose redox potential can be modulated by a proximal amino acid residue which coordinates the iron center. However, as can be seen in table 2, heme proteins with a histidine proximal ligand can have FeIII/FeII midpoint potentials from –200 to +50 mV. This broad potential range can be explained by taking the hydrogen bonding of other amino acid residues to the proximal histidine (i.e. the electronegativity of the local environment of the proximal histidine) into account [1]. The proximal histidine in peroxidases has a hydrogen bond to a nearby aspartate residue, while in the globins the proximal histidine has a hydrogen bond to a peptide backbone oxygen. As a consequence the proximal histidine in the globins has a less electronegative environment than in peroxidases. Mutational studies on peroxidases have shown a qualitative correlation where the Fe(III/II) midpoint potential decreases as the negative charge on the proximal ligand increases [2, 3]. The FeIII/FeII couple may not always be relevant for catalysis. Ferric heme is often proposed as the starting oxidation state. For catalases and peroxidases two transient reaction intermediates which are one and two electron oxidized with respect to the ferric state, have been described and named compound II and I, respectively. Compound II has been found to be an oxo-ferryl species and compound I and oxo-ferryl porphyrin π-cation radical. However, since the high valent states of iron are generally transient, the midpoint 4 General introduction potential of the FeIV/FeIII couple is much more difficult to obtain than that of the FeIII/FeII couple. Table 2. Redox properties of protoporphyrin IX containing proteins. Heme protein Proximal Em Em Em Ref. Ligand (FeIII/FeII) (C-II/FeIII) (C-I/FeIII) Myoglobin His +50 [4] Myeloperoxidase His +21 +1100 [5, 6] Chlorite dismutase His -23 to -21 [7, 8] Lignin peroxidase His -142 +1400 [9] Cytochrome c His -194 +740 [10, 11] peroxidase Cytochrome P450 Cys ~-200 [12] Horseradish His -250 +869 +898 [13, 14] peroxidase Catalase Tyr <-500 [15] Chlorite dismutase To date chlorite dismutase has only been isolated from three bacterial species: strain GR-1, strain CKB, and Ideonella dechloratans [7, 16, 17]. However chlorite dismutase activity has been found in whole cell suspensions of several other chlorate respiring bacteria as well [17]. The enzyme catalyzes the reduction of chlorite to chloride, while producing molecular oxygen. A more appropriate name for this enzyme is chloride:oxygen oxidoreductase. The biological function of the enzyme is to detoxify chlorite, which is the product of the respiration on chlorate. Chlorite dismutase has been found to be a homotetramer of 32 kDa subunits containing one iron protoporphyrin IX per subunit. Although it has a common heme cofactor, the spectroscopic features and redox properties of chlorite dismutase are unusual for a heme enzyme (chapter 8).