Workshop Lecture Journal of Inorganic Biochemistry 96 (2003) 3

Structural Genomics

Antonio Rosato, Magnetic Resonance Center, University of Florence, Italy

To realize the true value of the wealth of data provided by genome sequencing data, it is necessary to relate them to the functional properties of the proteins they encode. Since the biological function of a protein is determined by its 3D structure, the systematic determination of proteins’ structures on a genome-wide scale is a crucial step in any (post-)genomic effort, which may (or may not) provide initial hints on the function. This is what is commonly referred to as ‘Structural Genomics’ (or Structural Proteomics). Because of the huge number of systems into question, all the complex steps necessary for structure determination must be optimized, streamlined and, possibly, robotized in order to shrink the time needed to solve each protein structure. This approach is dubbed ‘high-throughput’ (HTP) and is an intrinsic feature of Structural Genomics. What can be the relationship between Biological Inorganic Chemistry and Structural Genomics? A major challenge is that to reconcile the concept of HTP with the care that metalloproteins most often require because of their metal cofactors. The identifi cation of metalloproteins is even not explicitly taken into account in purely Structural Genomics projects, nor is any methodology particularly developed for them. To create true correlations between Biological Inorganic Chemistry and Structural Genomics it is necessary to develop new computational tools (e.g. to identify metalloproteins in databanks, or to correctly model their structures), as well as new methodological approaches to HTP metalloprotein expression/purifi cation and structural characterization. Of course, focus on the metal site, and its interplay with the protein moiety, is of fundamental importance. On the longer term, these developments will be crucial to unravel the complex mechanisms underlying the interactions between metal ions and bio-molecules in living cells, even though they may fail to provide information on the metal(s) actually bound in vivo. References: Bertini and Rosato, PNAS, 100:3601-4, 2003; Banci and Rosato, Acc.Chem.Res, 36:215-221, 2003. Plenary Lectures Journal of Inorganic Biochemistry 96 (2003) 7

Scanning Probe Microscopy and the Electrochemistry of Metalloproteins and Metalloenzymes: Seeing is Believing?

Allen Hill, Oxford University, United Kingdom

The development of bioelectrochemistry over the past quarter of a century has concentrated on the behaviour of metalloproteins and metalloenzymes at electrode surfaces and, of course, the applications to bioanalytical chemistry. However, our understanding of such systems rests on the behaviour of such systems at, or on, electrode surfaces. The advent of methods of applying Scanning Probe Microscopy in water opened up the possibility of studying biological molecules under conditions appropriate to their natural functions and hence one could assume that they were not signifi cantly changed in their structures or functions. Though the fi rst protein examined was the zinc-containing protein, metallothionein, we have concentrated on azurin and cytochrome P450 and its partner proteins, putidaredoxin (Pd)and putidaredoxin reductase (PdR). In particular, we have sought conditions that allow the investigation of structures of the complexes formed between cytochrome P450 and Pd and PdR. With all these expermiments, it was important to show that the proteins were still active when adsorbed on the electrode surface and, as expected, it was usually important to modify the surface with a promoter (or, more recently, a facilitator.) We are now attempting to study the electrochemistry of the proteins under these conditions and to move the proteins into organised arrays in preparation for the use of similar systems as truly molecular sensors. The investigation of the ubiquitous glucose sensor by Scanning Tunneling Microscopy under conditions closely related to those found in devices proved possible.

Some New Aspects of Molecular Engineering of Heme Proteins to Manipulate Their Functions: Ligand Binding, Catalysis and Interprotein Recognition

Isao Morishima, Kyoto University, Japan

We have been concerned with rational redesign of the structures of heme proteins in order to better understand the chemical diversity of natural heme proteins and to generate novel heme proteins of potential utility. Detailed structural analysis of proteins have revealed that proteins structures are constructed by ‘modules’, which are compact structural units and correspond to exons on gene structure. In addition to the conventional point mutation, the approach we have developed is the ‘module’ substitution on the protein structure as the effective rational design step, and is, if necessary, followed by the second step, the point or random mutations for the fi ne-tuning. By inserting or substituting the specifi c module playing an important role in a desired function, we can expect that the starting point might jump to the goal. We have studied molecular engineering of heme proteins to better understand P450cam catalyzed oxygenation reactions with particular focus on putidaredoxin (electrondonor)-induced conformational changes of P450cam, to convert horseradish peroxidase to catalase and to position the oxidizing equivalents in peroxidase to change the reactivity. We have designed and created the calcium binding site in myoglobin in which the calcium binding module of troponin c is implanted into myoglobin and oxygen binding affi nity is modulated by calcium binding. We have also extensively studied the module-substituted chimeric globins to change the protein-protein recognition characteristics. For example, the module-substituted b subunit of hemoglobin or myoblobin having the module M4 from the a-subunit assembled with the b-subunit, indicating that the module M4 plays a crucial role in the specifi c subunit association in globins. Some examples of the chimeric myoglobin exhibiting association properties with other proteins will be presented. Some of the chimeric substitutions among different proteins with no homology have resulted in quite unstable proteins. We have developped the irrational design by the fi ne-tuning using the directed evolution, which allows us to construct novel and stable chimeric proteins between the different kinds of proteins. 8 Journal of Inorganic Biochemistry 96 (2003)

Chromium in Biology – Toxicology and Nutritional Aspects

Peter A Lay, University of Sydney, Australia

Chromium is probably the most controversial of the transition metal ions in terms of its toxicity and nutritional value.1,2 Cr(VI) compounds constitute one of the fi rst classes of chemicals to be implicated as human carcinogens, whereas Cr(III) is considered as one of the least-toxic transition metals and its use as a dietary supplement in nutrition and diabetes treatment is both very controversial and the basis of a multi-million dollar industry.1 Apart from occupational health issues, the controversy over industrial uses of Cr(VI) has also escalated as a result of recent massive lawsuits in the USA involving environmental pollution that has adversely affected local populations.2 The literature on both the mechanisms of chromium-induced cancers and the use of Cr(III) as dietary supplements is full of diverse opinions and confl icting reports.1 The divergent opinions can, in part, be attributed to the complexity of Cr redox and substitution chemistry in vitro and in vivo and, in part, to the diverse scientifi c backgrounds from which different research groups tackle the problems posed by Cr bioinorganic chemistry. This talk will concentrate on new developments in Cr carcinogenicity and nutritional supplements and on the need to investigate such complex bioinorganic questions using a multi-disciplinary approach. This is required to provide real insights into understanding the bioinorganic chemistry of Cr (and other metals) in biology and medicine. Acknowledgments: We are grateful for fi nancial support from the Australian Research Council, National Health and Medical Research Council, the Australian Synchrotron Research Program and the Access to Major Facilities Program. We are also grateful for beamtime at the Advanced Photon Source and the Stanford Synchrotron Research Laboratory. 1 Codd, R.; Dillon, C. T.; Levina, A.; Lay, P. A. Coord. Chem. Rev. 2001, 216-217, 537-582. 2 Levina, A.; Codd, R.; Dillon, C. T.; Lay, P. A. Prog. Inorg. Chem. 2003, 51, 145-250.

Radicals and Metal Centers as Spectroscopic Probes for Structure and Function of Photosynthetic Reaction Centers

Wolfgang Lubitz, Max-Planck-Institut für Strahlenchemie, Mullheim, Germany

The reaction centers (RCs) of photosynthetic organisms are highly complex multisubunit integral membrane proteins with several cofactors that are actively involved in the conversion of sun light into chemical energy. In oxygenic photosynthesis two photosystems (PS I and PS II) exist, which have recently been crystallized with 2.5 ≈ and 3.8 ≈ resolution, respectively [1,2]. The major pigment chlorophyll a is involved in light harvesting and excitation transfer in the antenna, and acts both as electron donor and intermediate electron acceptor in the charge separation process. The stable electron acceptors are quinone molecules coupled to a non-heme iron in PS II and three iron-sulfur clusters in PS I. Light-induced charge separation in PS II leads to oxidation of water and the release of protons and molecular oxygen. The locus of water splitting is a tetranuclear manganese/calcium cluster directly bound in the PS II core protein. In spite of intensive studies this process is currently not well understood and functional bioinorganic model systems are non-existing. In this lecture spectroscopic experiments performed on PS I and PS II are presented and related to the now available structural data. Particular emphasis is placed on advanced electron magnetic resonance techniques applied to the paramagnetic species generated during the photoprocesses, on the spectroscopy of related model systems and on the theoretical interpretation of the data [3]. [1] P. Jordan, P. Fromme, H. T. Witt, O. Klukas,W. Saenger, N. Kraufl, Nature, 411, 909, 2001. [2] A. Zouni. H.-T. Witt, J. Kern, P. Fromme, N. Kraufl, W. Saenger, P. Orth, Nature, 409, 739, 2001; N. Kamiya, J.-R. Shen, PNAS, 100, 98, 2003. [3] W. Lubitz, F. Lendzian, R. Bittl, Acc. Chem. Res. 35, 313, 2002; W. Lubitz, Phys. Chem. Chem. Phys., 4, 5539, 2003; K.-O. Schäfer, R. Bittl, F. Lendzian, V. Barynin, T. WeyherMüller, K. Wieghardt, W. Lubitz, J. Phys. Chem. B, 107, 1242, 2003. Journal of Inorganic Biochemistry 96 (2003) 9

The Many Highways for Intracellular Traffi cking of Metals

Valeria C Culotta, Johns Hopkins University, United States Edward Luk, Johns Hopkins University, United States

Metal ions such as manganese and copper are potentially toxic to living cells and as such, organisms have evolved with a number of detoxifi cation and sequestration mechanisms that prevent intracellular metals from accumulating in the free ionic form. However, these ions also serve as essential co-factors for various metalloenzymes that are housed in diverse cellular locations. How then, can metals that are not freely available, still fi nd their way home to the active site of their cognate metalloenzyme? This is accomplished through an ‘underground network’ of intracellular metal traffi cking pathways that guide the ion safely to the metalloenzymes. The best understood of such pathways are those that separately deliver copper to enzymes in the mitochondria, in the secretory pathway and in the cytosol. By comparison, virtually nothing was known regarding the pathways for traffi cking manganese ions. We have recently undertaken a genetics approach in bakers yeast to defi ning manganese transporters and potential manganese chaperones that escort the metal to the secretory pathway for activation of manganese requiring sugar transferases and to the matrix of the mitochondria for activating manganese superoxide dismutase. The site and mechanism of action of these various factors for manganese traffi cking will be described. It is noteworthy that all these players for manganese have homologues in humans as well. Hence, the network of highways for metal traffi cking are indeed conserved.

Molybdenum and Tungsten in Biology

José JG Moura, REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidadé Nova de Lisbon, Portugal

Molybdenum and Tungsten (Group 6 elements) are the only 4d and 5d metals with known biological function.1 Their importance for biology has been recognized since 75 and 25 years, respectively. Molybdenum is used by a wide range of organisms (from archae, bacteria, fungi, plants to animals including humans) and more than 50 enzymes are known. Only a few organisms use Tungsten, mainly (but not exclusively) thermophyles. We will review, in this lecture, mononuclear Molybdenum and Tungsten pterin containing enzymes that cover different functions such as Aldehyde oxidoreductase, Formate dehydrogenase and Nitrate reductase. An interplay of spectroscopic and crystallography data to functional aspects will be the main stream for discussion. Novel arrangements of molybdenum sites in biology will be also presented, opening the participation of this metal to new performances. 1 Metal Ions in Biological Systems, Volume 39, Edited by Astrid Sigel and Helmut Sigel, Marcel Dekker Inc., N.Y., 2002 10 Journal of Inorganic Biochemistry 96 (2003)

Microbial iron mining in the deep blue sea

Alison Butler, University of California, Santa Barbara, United States

The transition metal composition of the oceans differs dramatically from that of terrestrial environments, and recent studies are beginning to unravel some of the mysteries associated with the unusual metal composition. While molybdenum and vanadium are the two most abundant transition metal ions in surface ocean water, iron levels, are surprisingly low. Yet, iron is an essential micronutrient for marine microbial growth and the low iron levels have been shown to limit microbial growth over vast regions of the world’s oceans. To acquire iron many aerobic bacteria secrete siderophores, which are low molecular weight iron(III)-binding compounds. We are discovering that marine microorganisms acquire iron through new siderophores and new siderophore-mediated and other processes. Many of the siderophores are photoreactive, which is a distinguishing characteristic that likely plays a signifi cant role in the biogeochemical cycling of iron in the ocean. Self- assembling amphiphilic peptides also comprise a growing class of these siderophores, which again likely plays a signifi cant role in the iron acquisition process. Acknowledgment: Support from NIH GM38130 and the NSF/DOE EMSI CHE-9810248 (CEBIC) is gratefully acknowledged.

Oxygen Activation at Nonheme Iron: The Oxoiron(IV) Landscape

Lawrence Que, Jr, University of Minnesota, United States

Iron(IV)-oxo species are commonly invoked in the oxygen activation mechanisms of both heme and nonheme iron enzymes. While oxoiron(IV) porphyrin species have long been established as intermediates in the catalytic cycles of heme enzymes like peroxidases and cytochrome P450, only for nonheme diiron enzymes have high-valent intermedaites been observed: a diiron(IV) species with an Fe2(O)2 diamond core for methane hydroxylating intermediate Q of methane mnonooxygenase and an iron(III)iron(IV) intermediate X responsible for tyrosyl radical formation in Class I ribonucleotide reductases. Thus far, there is only indirect evidence for the participation of iron(IV)-oxo intermediates in mononuclear nonheme iron enzymes such as isopenicillin N synthase, pterin-dependent phenylalanine and tyrosine hydroxylases, and 2-oxoglutarate- dependent prolyl hydroxylase and clavaminate synthase. We have recently obtained the fi rst examples of well characterized synthetic nonheme oxoiron(IV) species, both mononuclear and dinuclear, from the reactions of iron(II) or iron(III) precursors with peroxides and other oxidants. Shown in the fi gure below is the fi rst crystal structure of one such mononuclear complex. This exploration has uncovered a fascinating oxoiron(IV) reaction landscape with many peaks and valleys, representing different pathways that lead to these metastable oxoiron(IV) species. Our current understanding of the structures, spectroscopic properties, and reactivities of these novel compounds will be presented. Session Lectures Journal of Inorganic Biochemistry 96 (2003) 13

Recognition of antimony and bismuth by peptides and proteins: insight into the mechanism of action

Hongzhe Sun, Department of Chemistry and Laboratory of Chemical Biology, The University of Hong Kong, Hong Kong Siucheong Yan, Department of Chemistry and Laboratory of Chemical Biology, The University of Hong Kong, Hong Kong Li Zhang, Department of Chemistry and Laboratory of Chemical Biology, The University of Hong Kong, Hong Kong W ei Li, Department of Chemistry and Laboratory of Chemical Biology, The University of Hong Kong, Hong Kong

In spite of extensive use of antimony (SbV) for the treatment of Leishmaniasis and bismuth (BiIII) for ulcers for decades,1 the mechanism of action including their side-effects still remains unclear. One of the targets of the antimony drugs is thought to be trypanothione, which is a major low molecular mass thiol inside the parasite, and overproduction of trypanothione in cells is related to resistance of antimony drugs. Pentavalent antimony (SbV) can be rapidly reduced to its trivalent state by trypanothione, and followed by binding of SbIII to trypanothione to form an SbIII-trypanothione complex.2 Surprisingly, addition of low molecular monothiol ligands such as glutathione to the SbIII-trypanothione complex leads to the formation of a novel ternary complex. The discovery of the ternary complex is important as it may play a role in the transport of antimony and the antileishmaniasis properties of the drugs are probably via the formation of a complex between of SbIII-trypanothione and enzymes. Clinically used bismuth citrate antiulcer drug is a unique polymer. Binding of BiIII to the iron transport protein transferrin and lactoferrin (80 kDa) was unexpected strong along with carbonate as the synergistic anion.1,3 Both NMR and FPLC in combination with ICP-MS have demonstrated that transferrin is the major target for bismuth in blood plasma.4 Binding of BiIII to other proteins will also be discussed and the possible mechanism of action will be proposed. 1 (a) Guo Z, Sadler PJ, Adv. Inorg. Chem. 49, 183-306 (2000). (b) Sun H, ‘Metallodrug’ in ‘Encyclopedia of Nuclear Magnetic Resonance: Advances in NMR’ (Ed: Grant DM & Harris RK.), John Wiley & Sons Ltd, 2002, pp413-427. 2 (a) Yan S-C, Ding K, Zhang L, Sun H, Angew. Chem. Int. Ed. 39, 4260-4262 (2000)(b) Yan S-C, Wong ILK, Chow LMC, Sun H, Chem. Commun. 266-267 (2003). 3 Zhang L, Szeto KY, Loh TT, Sadler PJ, Sun H, Biochemistry, 44, 13281-13287 (2001). 4 Sun H, Szeto KY, J. Inorg. Biochem. 94, 114-120 (2003). We thank the RGC of Hong Kong, NSFC/RGC, Livzon Pharmaceutical Ltd, Area of Excellence Scheme and the University of Hong Kong for support.

A unique bioinorganic mechanism of action of antimalarial aminoquinolines

Timothy J Egan, Department of Chemistry, University of Cape Town, South Africa Jill M Combrinck, Division of Pharmacology, Department of Medicine, University of Cape Town, South Africa Catherine H Kaschula, Department of Chemistry, University of Cape Town, South Africa Roger Hunter, Department of Chemistry, University of Cape Town, South Africa Peter J Smith, Division of Pharmacology, Department of Medicine, University of Cape Town, South Africa Giovanni Hearne, School of Physics, University of the Witwatersrand, South Africa Helder M Marques, School of Chemistry, University of the Witwatersrand, South Africa Donatella Taramelli, Institute of Microbiology, University of Milan, Italy

Considerable evidence now suggests that chloroquine and related antimalarials target haematin, released during the digestion of human haemoglobin in the food vacuole of the malaria parasite. This occurs during the part of its life cycle that takes place inside the red blood cell of the human host. We have shown by chemical analysis of total iron content, Mössbauer spectroscopy of whole malaria parasites and electron spectroscopic imaging of these parasites that at least 95% of the haematin is normally incorporated into haemozoin or malaria pigment. This material, which is a cyclic dimer of iron(III)protoporphyrin IX appears to form in a biomineralisation process in the parasite. Its formation seems to be a detoxifi cation mechanism in the parasite, as haematin is known to be toxic to other microorganisms. Aminoquinoline antimalarials have a unique and specifi c ability to inhibit this process and we have shown that there is a direct correlation between antimalarial activity against cultured malaria parasites and in vitro inhibition of synthetic haemozoin formation. This suggests that antimalarials of this type may form a unique class of bio-crystallisation inhibitors. 14 Journal of Inorganic Biochemistry 96 (2003)

Biolocalization and in vivo Coordination Chemistry of Vanadium Pharmaceuticals

Chris Orvig, University of British Columbia, Canada Katherine H Thompson, University of British Columbia, Canada Barry D Liboiron, University of British Columbia, Canada John H McNeill, University of British Columbia, Canada Violet G Yuen, University of British Columbia, Canada

Vanadyl maltol complexes, e.g. bis(maltolato)oxovanadium(IV) (BMOV) and its ethyl maltol analogue, are among the most extensively investigated vanadium compounds for the treatment of diabetes mellitus. Numerous over-the-counter ‘pharmaceutical’ preparations now contain BMOV. This paper will discuss our latest model of vanadium metabolism, and our investigations of the relationship of structure to glucose-lowering ability in a closely-related series of V compounds. We have used many physical and biological techniques to describe the uptake, biodistribution and speciation of exogeneously-added vanadium. The resulting metabolic model is being used as a working hypothesis to provide insights into the mechanism of vanadium’s insulin-enhancing effects. Various measures of tissue uptake, distribution and accumulation of exogenously added vanadium have been compared for their utility in predicting relative potency of these compounds as anti-diabetic therapeutic agents. Both carrier-added 48V-BMOV and [ethyl-1-14C]BEOV biodistribution studies indicated that bone is the preferred tissue target for vanadium, with a half-life of several months. The emerging picture of the metabolic fate of vanadium maltol complexes is one of rapid de-complexation following administration. Determination of an index of glucose- lowering ability for an extended set of V compounds, based on percentage of glucose-lowering in test compounds compared to untreated STZ-diabetic rats, shows a range of effi cacies, possibly due to different absorption, distribution and excretion profi les.

The antidiabetic potential of vanadium compounds

Dieter Rehder, Institute of Inorganic and Applied Chemistry, University of Hamburg, Germany Jessica Gaetjens, Institute of Inorganic and Applied Chemistry, University of Hamburg, Germany Tamas Kiss, Department of Inorganic and Analytical Chemistry, University of Szeged, Hungary

Vanadium(IV and V) coordination compounds with various ligand systems have been shown to exhibit insulin-mimetic effects [1, 2], i.e. they stimulate glucose degradation and inhibit lipolysis in vitro, in model animals (streptozotozin induced diabetic rats), in naturally diabetic cats, and in humans patients (phase II clinical tests). The compounds can be applied orally, and they are effective in both types of diabetes (insulin defi ciency and insulin tolerance). Picolinatovanadium complexes are particularly interesting in this respect [3].We have developed this specifi c family of vanadium coordination compounds towards the optimisation of the following factors: stability under stomach, intestinal and blood stream conditions; effective intestinal absorption; effective transport and trans-membrane transport; tolerable cytotoxicity; low inhibitory effect towards ATPase; and high insulin-mimetic effi cacy. These optimisations have been carried out by employing derivatives of picolinic acid, HA, with an ester function in the 5 position; see Fig. 1.

Speciation analyses of the complexes VOA2 have revealed that the complexes remain partially intact [in the form of VOL(OH2/ OH)] over the pH range 2 to 9, Fig. 2. They are comparable in effi cacy to insulin both with respect to the inhibition of lipolysis (stimulation of lipogenesis) and glucose intake into cells. The complexes further are not cytotoxic at concentrations below 0.1 mM, and – in contrast to vanadate – only negligibly inhibit of K,Na-ATPase. 1 K.H. Thompson, J.H. McNeill, C. Orvig, Chem. Rev. 99 (1999) 2561; H. Sakurai, Y. Kojima, Y. Yoshikawa, K. Kawabe, H. Yasui, Coord. Chem. Rev. 226 (2002) 187. 2 D. Rehder, J. Costa Pessoa, C.F.G.C. Geraldes, M.M.C.A. Castro, T. Kabanos, T. Kiss, B. Meier, G. Micera, L. Pettersson, M. Ranger, A. Salifoglou, I. Turel, D. Wang, J. Biol. Inorg. Chem. 7 (2002) 384. 3 D. C. Crans, J. Inorg. Biochem. 80 (2000) 123. Journal of Inorganic Biochemistry 96 (2003) 15

Li+/Mg2+ competition for Mg2+-binding sites in G-proteins: implications for bipolar disorder

Duarte Mota de Freitas, Loyola University Chicago, United States Nicole Williams, Loyola University Chicago, United States Carlos Geraldes, University of Coimbra, Portugal

Li+ is the primary drug to treat bipolar disorder, but its mechanism of action is unknown. Guanine nucleotide-binding (G) proteins, which are Mg2+-dependent, are involved in signal transduction and therefore, much effort has been given to elucidating their role in bipolar disorder. We explored the Li+/Mg2+ competition hypothesis with respect to metal ion binding and activation with a model G-protein, isoform 1 of the α subunit from recombinant, inhibitory G-protein (rGiα1), and two of its mutants, T181D and T181V (where Thr-181, a Mg2+ ligand, was mutagenized to Asp or Val). Using intrinsic fl uorescence spectroscopy, we found that for protein samples (2.5 µM) containing excess Mg2+ (1.25 mM), the overall percent activation for the T181V mutant (22.9 ± 0.9 %) was signifi cantly less (p < 0.001) than that seen for wt rGiα1 (37.0 ± 2.0 %) 2+ + 2+ and rGiα1-T181D (32.4 ± 3.0 %). With a Mg fl uorophore (furaptra), we quantifi ed the extent of Li /Mg competition in + 2+ all three proteins, and found that, when [Li ] exceeded 40 mM with T181V or 60 mM with T181D, the [Mg ]f values were + 7 signifi cantly larger (p < 0.05) than with wt rGiα1. To quantitate Li binding, Li NMR T1 relaxation rates were used for the + 2+ app 2+ determination of the apparent and actual Li and Mg binding constants. By plotting the KLi values vs. [Mg ], we found actual evidence for the existence of two distinct high- and low-affi nity metal binding sites in all three proteins, and the KLi and app KMg values for the low-affi nity sites in T181D and T181V were smaller than those for wt rGiα1. These fi ndings indicate that 2+ 2+ mutations at T181 regulate the Mg affi nity for the low-affi nity Mg -binding sites in rGiα1. The authors acknowledge fi nancial support from the NIH, grant # MH-45926.

Signal transduction by PAS-domain containing heme-based sensors: an overview

Marie-Alda Gilles-Gonzalez, Department of Biochemistry, UT Southwestern Medical Center at Dallas, United States Elhadji M Dioum, Department of Biochemistry, UT Southwestern Medical Center at Dallas, United States Jared R Rutter, Department of Biochemistry, UT Southwestern Medical Center at Dallas, United States Gonzalo Gonzalez, Department of Biochemistry, UT Southwestern Medical Center at Dallas, United States Steven L McKnight, Department of Biochemistry, UT Southwestern Medical Center at Dallas, United States Jason R Tuckerman, Department of Biochemistry, The Ohio State University, United States

PAS, or Per-Arnt-Sim, domains occur in more than 1100 proteins throughout all three kingdoms of life. A heme-binding subgroup of PAS domains is specifi cally dedicated to sensing of heme ligands such as molecular oxygen, carbon monoxide, and nitric oxide. This group of proteins includes BjFixL, RmFixL, AxPDEA1, EcDos, and more recently, MmNPAS2 (1-5). They have evolved to employ a variety of signal-transduction strategies, including phosphoryl transfer, hydrolysis of cyclic- nucleotide second messengers, and binding of DNA. These signal-transduction schemes require coupling the heme status to a transmitter within the same protein. We will review those systems and discuss the factors governing their distinctive responses to ligands. References 1. M. A. Gilles-Gonzalez (2001). Oxygen signal transduction. IUBMB Life 51, 165-173. 2. T. Tomita, G. Gonzalez, A. L. Chang, M. Ikeda-Saito, and M. A. Gilles-Gonzalez (2002) A comparative resonance Raman analysis of heme-binding PAS domains: heme-iron coordination structures of the BjFixL, AxPDEA1, EcDos, and MtDos proteins. Biochemistry 41, 4819-4826. 3. J. R. Tuckerman, G. Gonzalez, E. M. Dioum, and M. A. Gilles-Gonzalez (2002) Ligand and oxidation-state specifi c regulation of the heme-based oxygen sensor FixL from Sinorhizobium meliloti. Biochemistry 41, 6170-6177. 4. G. Gonzalez, E. M. Dioum, C. M. Bertolucci, T. Tomita, M. Ikeda-Saito, M. R. Cheesman, N. J. Watmough, and M. A. Gilles-Gonzalez (2002) Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli. Biochemistry 41, 8414-8421. 5. E. M. Dioum, J. Rutter, J. R. Tuckerman, G. Gonzalez, M. A. Gilles-Gonzalez, and S. L. McKnight (2002) NPAS2: a gas-responsive transcription factor. Science 298, 2385-2387. 16 Journal of Inorganic Biochemistry 96 (2003)

Oxygen-sensing and signal transduction by the heme-based sensor protein HemAT-Bs

Shigetoshi Aono, Center for Integrative Bioscience, Okazaki National Research Institutes, Japan Takuya Iwabuchi, Center for Integrative Bioscience, Okazaki National Research Institutes, Japan Hiroshi Nakajima, Center for Integrative Bioscience, Okazaki National Research Institutes, Japan Takehiro Ohta, Center for Integrative Bioscience, Okazaki National Research Institutes, Japan Teizo Kitagawa, Okazaki National Research Institutes, Japan

HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis, where the heme acts as an oxygen sensor. We have characterized the recombinant HemAT-Bs to elucidate the mechanisms of oxygen-sensing and signal transduction by HemAT-Bs. HemAT-Bs shows similar uv/vis spectra to those of myoglobin (Mb). Site-directed mutagenesis reveals that His123 is the proximal ligand of the heme in HemAT-Bs. -1 -1 -1 Oxygen binding and dissociation rate constants are determined to be kon = 32 µM s and koff = 23 s , respectively, revealing that HemAT-Bs has a moderate oxygen affi nity similar to that of sperm whale Mb. The rate constant for autoxidation at 37°C is 0.06 h-1, which is also close to that of Mb. Although the electronic absorption spectra of HemAT-Bs are similar to those of Mb, HemAT-Bs shows some unique characteristics in its resonance Raman spectra. Oxygen-bound -1 HemAT-Bs gives the ν(Fe-O2) band at a noticeably low frequency (560 cm ), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule. Deoxy HemAT-Bs gives the ν(Fe- His) band at a higher frequency (225 cm-1) than those of ordinary His-coordinated deoxy heme proteins. HemAT-Bs consists of two domains, a N-terminal sensor domain and a C-terminal signaling domain. We have also prepared a truncated mutant consisting of the only N-terminal sensor domain. The heme environmental structure is perturbed by truncating the C-terminal domain. The resonance Raman spectroscopy reveals that a hydrogen bonding pattern toward the heme-bound oxygen is different from each other between wild-type and sensor domain mutant. The rate constant for autoxidation is 0.6 h-1 for the sensor domain mutant. The oxygen binding kinetics are also changed for the sensor domain -1 -1 -1 mutant to be kon = 69 µM s and koff = 1.2 s , indicating the binding affi nity of oxygen increases in this mutant compared with wild-type HemAT-Bs.

Structural studies of heme and non-heme iron proteins that sense molecular oxygen

Michael K Chan, The Ohio State University, United States

While aerobic bacteria require O2 for life, too much O2 can be toxic. Hence it is not surprising that nature has developed a variety of O2 sensing proteins that regulate specifi c activities such as the transcriptional activation of genes, or bacterial chemotaxis. Interestingly, a common feature in many of these O2 sensing proteins is the presence of either a heme and non- heme iron center that serves as the site of O2 interaction. To elucidate the mechanisms by which this interaction can lead to signal transduction, we have been working to determine the structures of a variety of oxygen sensing proteins. Our current progress on these systems will be presented. Journal of Inorganic Biochemistry 96 (2003) 17

The role of Fe-S clusters in the stability of the transcription factor FNR

Patricia J Kiley, University of Wisconsin, United States Victoria R Sutton, University of Wisconsin, United States Erin L Mettert, University of Wisconsin, United States Helmut Beinert, University of Wisconsin, United States

The [4Fe-4S] cluster of the transcription factor FNR from Escherichia coli is key to its function as an oxygen sensor and 2+ transcriptional regulator. While it is established that the inactivation of FNR by O2 is mediated by conversion of the [4Fe-4S] cluster to a [2Fe-2S]2+ cluster, the fate of the [2Fe-2S]2+ cluster form of FNR under aerobic growth conditions was previously unknown. To address the fate of the [2Fe-2S]2+ cluster form of FNR, we examined the effect of various redox reagents on the stability of the [2Fe-2S]2+ cluster. The [2Fe-2S]2+ cluster of FNR was stable to oxygen in vitro but was rapidly destabilized in the presence of superoxide, a byproduct of aerobic metabolism. Consistent with this observation, we have found that the predominant form of FNR in aerobically grown cells is apoFNR. However, this apoFNR is not completely stable, since our data indicate that the ClpXP protease increases the turnover rate of the FNR polypeptide specifi cally under aerobic conditions. From pulse-chase radiolabeling experiments of both aerobically and anaerobically grown E. coli containing either WT FNR or various FNR mutants, the increase in proteolysis rate under aerobic growth conditions could be attributed to the absence of an Fe-S cluster. Therefore, we propose that loss of the Fe-S cluster transforms FNR into a ClpXP substrate. We are currently defi ning the region of FNR that serves as a ClpXP target. Taken together, these data indicate the remarkable versatility of Fe-S clusters and show that they can affect regulation of protein function at several steps.

Water Splitting: Nature’s Innovation, Mankind’s Aspiration

G Charles Dismukes, Princeton University, United States Wolfgang F Ruettinger, Princeton University, United States Emilie Bourles, Princeton University, United States Thomas G Carrell, Princeton University, United States Marcel Maneiro, Princeton University, United States

President Bush’s State of the Union Address in January 2003 featured a chemistry tutorial on fuel cells powered by H2 and O2. While the immediate future sources of H2 and O2 will be derived from fossil fuels and the atmosphere, the long term goal is to obtain these by splitting water. The only biological process that can perform water splitting is photosynthesis. This process employs a universal enzyme containing a complex inorganic core, Mn4OxCa1Cly. Bioinorganic modelling studies of the enzyme have focused on a n+ class of manganese-oxo clusters, one of which, contains the reactive [Mn4O4] ‘cubane’ core. The cubane core can be trapped by chelation with six organophosphinate ligands that bridge each face: Mn4O4(Ph2PO2)6. Removal of one phosphinate ligand produces O2 n+ + in high yield (50-100%) with rearrangment to form a reduced [Mn4O2] ‘butterfl y’ core complex: [Mn4O2(Ph2PO2)5] . By use of diarylphosphinate ligands the butterfl y cannot fully open its wings and thus is able to bind water molecules at the vacant bridging-oxo sites. It is thus poised for reoxidation chemistry that is currently under investigation. Our current understanding the chemistry of the Mn-oxo cubanes and their potential as water splitting catalysts will be presented. 18 Journal of Inorganic Biochemistry 96 (2003)

Toward a Better Understanding of Photosystem II Using Synthetic Complexes

William H Armstrong, Boston College, United States Sumitra Mukhopadhyay, Boston College, United States Henry J Mok, Boston College, United States Bhavesh Gandhi, Boston College, United States

A tetranuclear manganese complex resides at the active site of the Photosystem II (PSII) water oxidase. While a crystal structure of PSII has been reported, the structure of the manganese cluster is poorly defi ned. Synthetic compounds of relevance to the enzyme active site complex are being sought. With model complexes, we wish to test various hypotheses regarding the 4+ structure and function of the Sn states. In this presentation, we examine the interesting behavior of [(Mn2O2)2(tphpn)2] upon redox changes. In the 4+ solid state complex, the Mn atoms are arranged in a rectangle, while in the 5+ complex there is a 4+ dramatic rearrangement to a tetrahedral shape. Reduction of [(Mn2O2)2(tphpn)2] by one electron provides a species whose

EPR spectrum closely resembles that of the PSII S0 state. Spectral and magnetic properties of the reduced form (3+) strongly suggest a structural transformation has occurred, as was the case for the oxidized form. We will also present results from recent attempts to prepare novel manganese aggregates relevant to PSII. Included here is the fi rst species with a [Mn4O5] central core and compounds obtained by employing sterically bulky aryl caboxylate ligands. Finally, a role for the PSII water oxidase cofactor chloride will be discussed.

Nitrogenase mechanism: Modulation of energy transduction and electron transfer within the Fe-protein-MoFe protein Complex

Roger N F Thorneley, Department Biological Chemistry, John Innes Centre, Norwich, United Kingdom Haley C Angove, Department Biological Chemistry, John Innes Centre, Norwich, United Kingdom Marcus C Durrant, Department Biological Chemistry, John Innes Centre, Norwich, United Kingdom Shirley A Fairhurst, Department Biological Chemistry, John Innes Centre, Norwich, United Kingdom Simon J George, Department Biological Chemistry, John Innes Centre, Norwich, United Kingdom Andrew Sinclair, Department Biological Chemistry, John Innes Centre, Norwich, United Kingdom John D Tolland, Department Biological Chemistry, John Innes Centre, Norwich, United Kingdom Patrick C Hallenbeck, Department de Micro.et Immunol, Université de Montreal, Canada

The kinetic mechanism of nitrogenase is still described by the original concepts, experimentally determined rate constants and simulations fi rst reported at a nitrogen fi xation symposium held in Canberra, Australia 23 years ago (Thorneley and Lowe 1981). The model was subsequently detailed in a series of fi ve papers (see Lowe-Thorneley 1984 and references therein)and has recently been re-formatted (Wilson et al 2001). The Fe protein cycle comprises the minimum number of partial reactions necessary to effect the transfer on an electron from the Fe protein to the MoFe protein coupled to the hydrolysis of two molecules of MgATP. A major challenge is to understand at atomic resolution the energy transducing reactions, (electron transfer, hydrolysis of MgATP and associated conformation changes) that occur within the transient complex formed between the Fe- and MoFe proteins. We have recently probed the structural determinants of the kinetic and spectroscopic profi les of reactions occurring in the Fe-protein cycle in three ways: 1 Use of the ATP analogue 2’-deoxy ATP 2 SDM of Fe protein residues that interact with MgATP 3 Replacement of the 4Fe4S cluster by a 4Fe4Se cluster in the Fe protein. In addition, stopped-fl ow Fourier transform Infrared spectroscopy has been used to monitor azide reduction and carbon monoxide inhibition in both the pre-steady state and steady state. These data are beginning to contribute to our understanding of the chemistry that occurs at the FeMo cofactor. Lowe D.J.& Thorneley R.N.F.(1984) Biochem. J. 224, 877-886 Thorneley, R.N.F. & Lowe, D.J.(1981)In Proceedings of the 4th International Symposium on Nitrogen Fixation, Canberra 1980 (Eds A.H. Gibson, W.E. Newton). Australian Accademy of Science p.360 Wilson, P.E., Nyborg, A.C. & Watt, G.D.(2001)Biophysical Chemistry 91,281-304 Journal of Inorganic Biochemistry 96 (2003) 19

Density Functional Theory Calculations on the Nitrogenase Cofactor and synthetic analogs

Dimitri Coucouvanis, University of Michigan, United States Jaehong Han, University of Michigan, United States Reinhart Ahlrichs, University of Karlsruhe, Germany Paola Nava, University of Karlsruhe, Germany Uwe Huniar, University of Karlsruhe, Germany

Density functional theory (DFT) calculations were carried out on the FeMo cofactor of nitrogenase including the recently detected central atom X. These calculations were conducted for X= N, O and void. The results were compared to similar calculations on the cuboidal clusters with the MoFe3S4 cores with various numbers of valence electrons. The results of the DFT calculations show that the central atom is very likely N, and its presence does not signifi cantly affect the Fe-Fe distances in the central hexairon unit. These distances refl ect weak Fe-Fe bonding interactions. Energetically closely spaced states with highest occupied molecular orbitals of antibonding Fe-Fe character also are present. Such states when populated are expected to drastically distort the structure of the cofactor and play an important role in the activation and reduction of dinitrogen. A proposed mechanism for the reduction of dinitrogen to ammonia will be presented that is based on the reversible rupture of Fe-Fe bonds as a means of storing or retrieving electrons with concomitant structural changes istrumental for nitrogen binding and activation.

Ferritin: Gated pores and Fe transport

Elizabeth C Theil, CHORI (Children’s Hospital Oakland Research Institute), United States Xiaofeng Liu, CHORI (Children’s Hospital Oakland Research Institute), United States

Fe ions are transported into and out of ferritin through 8 pores in the spherical protein with a large central cavity for forming hyraded ferric oxide. Concentrating Fe to 10-4 M needed by living cells is the main role of the ferritin. Multiple metal sites include: a) ferro/oxygen oxidoreductase sites-subunit center, b) mineralization surfaces-cavity, c) entry/exit-pores. Fe dissolved from the mineral after reduction, exits ferritin through gated protein pores at the 3-fold axes. Pores gates, revealed by unfolding in crystals of mutant protein with rapid chelation, are controlled by pairs of conserved hydrophobic (L/L) and ionic (D-COOH and R-NH) residues, which can be selectively opened by 1mM urea and 54 deg C. The main protein structure is stable to 6 M urea and > 85 deg C (Figure 1) (1). Natural molecules likely occur to control gating and Fe reduction/chelation in vivo. Fe entry for mineralization initiation is independent of pore gating, mainly controlled by catalytic site binding (2). Mossbauer and EXAFS showed a diferric, peroxo intermediate, which decays to diferric mineral precursors and H2O2 (2). Fe ligands, defi ned by amino acid substitutions and crystal structures complexed with Ca, Mg as A: E 23, E 57, 58, H61 and B: E58, E103, Q137 , were confi rmed by stepwise site restoration of catalysis in a degenerate subunit, naturally mixed with active subunits in animal tissues. A-Site residues confer low activity with Q 137 in site B conferring the largest rate increase (~10,000 X). D140, a serine in mitochondrial ferritins, played a key role in the kinetics of the diferric peroxo intermediate, supporting the idea that the ferritin site is derived from sites in di-iron oxidases. Part Support NIH- DK 20251. (1) Liu, X., et al. (2003) Proc. Natíl Acad. Sci. USA (in press). (2) Jamison, G.N. et al.. (2002) Biochemistry 41:13435 Figure 1: Ferritin, (left) pores closed and (right) open. One pore is red. See reference 1 . 20 Journal of Inorganic Biochemistry 96 (2003)

Dioxygen Activation by Copper Complexes Supported by 2-(2-Pyridyl)ethylamine Ligands. Mechanistic Insights into Copper Monooxygenases and Copper Oxidases

Shinobu Itoh, Osaka City University, Japan

Reactions of copper(I) complexes with molecular oxygen have been examined using a series of N-alkyl-bis[2-(2- pyridyl)ethyl]amine tridentate ligands (R´Py2R) and N,N-dialkyl-2-(2-pyridyl)ethylamine didentate ligands (R´Py1R1,R2) at low temperature (Chart 1). The tridentate ligands predominantly provide side-on type peroxo dicopper(II) complex (A), while the didentate ligands enhance O-O bond homolysis of the peroxo species to produce bis(µ-oxo)dicopper(III) complex (B) (Figure 1). With the µ-peroxo dicopper(II) complex (A) supported by the tridentate ligand, effi cient oxygenation of phenolates to the corresponding catechols has been accomplished to provide a good model reaction of tyrosinase. The bis(µ-oxo)dicopper(III) complex (B), on the other hand, undergo aliphatic ligand hydroxylation as well as oxygen atom transfer to sulfi des to give the corresponding sulfoxides. In the reaction of bis(µ-oxo)dicopper(III) complex (B) with 10-methyl-9,10-dihydroacridine

(AcrH2) and 1,4-cyclohexadiene (CHD), a new active oxygen intermediate such as a (µ-oxo)(µ-oxyl radical)dicopper(III) or a tetranuclear copper-oxygen complex has been suggested to be involved as the real active oxygen species for the C-H bond activation of the external substrates. A mixed valence bis(µ3-oxo) trinuclear copper(II,II,III) complex (C) has also been assessed using the didentate ligand with the smallest N- alkyl substituent (methyl). Mechanistic details of the above reactions as well as ligand effects on the copper(I)-dioxygen reactivity are discussed systematically.

Understanding the Mechanism of Superoxide Reduction by the Non-Heme Iron Enzyme Superoxide Reductase (SOR) using a Synthetic Analogue Approach

Julie A Kovacs, University of Washington, United States Sarah Fitch, University of Washington, United States Roslyn Theisen, University of Washington, United States Jason Shearer, University of Washington, United States Terry Kitagawa, University of Washington, United States Robert Scarrow, Haverford College, United States

Superoxide reductases (SORs) belong to a new class of metalloenzymes that degrade superoxide by reducing it to hydrogen peroxide. These enzymes contain a cysteinate-ligated non-heme iron site that cycles between the FeII and FeIII states during catalysis. A key step in the reduction of superoxide has been suggested to involve superoxide binding to FeII, followed by inner-sphere electron transfer to afford an FeIII-OO(H) intermediate. Oxygen does not appear to react readily with the FeII form of SOR. Cyanide inhibits superoxide reduction. Synthetic models prepared in our group have been shown to reduce superoxide at rates similar to the enzyme, and form an III -1 S= 1/2 Fe -OOH intermediate that displays a vO-O stretch at 784 cm in the vibrational -1 18 - spectrum. This vO-O stretch shifts to 753 cm in the O2 labeled compound. Upon binding cyanide, the redox potential of our FeIII model shifts signifi cantly, suggesting that cyanide inhibits SOR by preventing the ferrous form from being regenerated, and thus preventing the enzyme from turning over. Although oxygen reacts with our SOR model to afford a µ-oxo dimer, slight modifi cation of our ligand results in a complex that is unreactive towards oxygen. Journal of Inorganic Biochemistry 96 (2003) 21

Binding and activation of oxygen species by artifi cially designed metalloenzyme models with intramolecular non-covalent interaction groups

Hideki Masuda, Department of Applied Chemistry, Nagoya Institute of Technology, Japan

Binding and activation of dioxygen or oxygen species in biological respiratory and metabolic systems are key processes that are carried out by many heme- and non-heme metalloenzymes. These processes and regulations are performed by metal active sites, such as iron, copper, and manganese ions. Detailed examinations by use of biomimetic model systems are required for understanding in the natural processes, such as dioxygen transport, oxidation, oxygenation and dehydrogenation. These active sites are surrounded by hydrophobic environment in many cases, and their functions are controlled by a combination of non-covalent interactions, such as hydrogen bonding, hydrophobic, and electrostatic interactions. These weak non-covalent interactions are quite important in the recognition, activation and regulation of substrates. On the basis of the active site structures in these biological systems, we designed and synthesized some new metal complexes as the structural and/or functional models, which are consisted of polypyridine ligands having non-covalent interaction sites such as hydrophobic tert-butyl and hydrogen bonding NH groups as well as coordination bonding sites of pyridine donors. Using these ligands, some iron and copper complexes were synthesized, and their complexes were spectroscopically and structurally characterized and examined on the reactivities with oxygen species such as dioxygen, superoxide, hydrogen peroxide, etc. Here we will describe the ligand design concept and especially report the Cu(II) complex with hydroperoxide as a fi rst structural model of reaction intermediates in dopamine β-hydroxylating monooxygenase (DβH), peptidylglycine α-amidating monooxygenase (PHM), galactose oxidase, etc. and the Fe(III) complexes with hydroperoxide or alkylperoxide as the reaction intermediates in lipoxygenase, catechol dioxygenase, bleomycin, etc. Unique diiron complexes that can reversibly bind with dioxygen will also be described as a functional model of hemerythrin, oxygen carrier.

Corrolazines: Novel Porphyrinoid Compounds Capable of Oxygen Binding, Stabilization of High-Valent Metal-Oxo Species, and More

David P Goldberg, Johns Hopkins University, United States Bobby Ramdhanie, Johns Hopkins University, United States Beaven S Mandimutsira, Johns Hopkins University, United States Halin Wang, Johns Hopkins University, United States Joseph P Fox, Johns Hopkins University, United States

The synthesis of novel porphyrinoid ligands has played a key role in providing mechanistic insights into the chemistry of heme proteins, and in yielding novel synthetic catalysts for a variety of important transformations. We have recently reported the facile synthesis of a new class of porphyrinoid molecules called corrolazines (Cz), which are best viewed as hybrids between tetraazaporphyrins (porphyrazines) and corroles.1 There has been a recent surge of interest in the chemistry of corroles, which differ from porphyrins in that they have a direct pyrrole-pyrrole linkage, a formal trinegative charge when deprotonated, and most importantly, show a remarkable ability to stabilize high-valent states. The synthesis, structure, and reactivity of a number of metallocorrolazines will be discussed. In particular, a series of cobalt corrolazines will be presented, for which a combination of spectroscopic and structural analysis has shown that the corrolazine cavity is smaller than a conventional corrole, and that the meso-N atoms have a signifi cant electron-withdrawing effect on the aromatic system as well as the metal center.2 A reduced cobalt(II) complex has been generated and demonstrated to bind dioxygen resulting in a cobalt(III)-superoxo complex, as evidenced by EPR spectroscopy. In addition, the synthesis and characterization (UV-vis, NMR, mass spec., resonance Raman, 18O labeling) of a remarkably stable high-valent manganese(V)-oxo complex3 will be presented together with its reactivity in oxygen-atom-transfer reactions (e.g. PhSMe converted to PhS(O)Me). 1 Ramdhanie, B., Stern, C. L., Goldberg, D. P. J. Am. Chem. Soc, 9447-9448 (2001). 2 Ramdhanie, B., Zakharov, L. N., Rheingold, A. L., Goldberg, D. P. Inorg. Chem., 4105-4107 (2002). 3 Mandimutsira, B. S., Ramdhanie, B., Todd, R. C., Wang, H., Zareba, A. A., Czernuszewicz, R. S., Goldberg, D. P. J. Am. Chem. Soc., 15170-15171 (2002). The generous support of the National Science Foundation (USA) is acknowledged. 22 Journal of Inorganic Biochemistry 96 (2003)

Modeling the Acetyl Coenzyme A Synthase Active Site

Charles G Riordan, University of Delaware, United States Rangan Krishnan, University of Delaware, United States Yoshinobu Ishikawa, University of Delaware, United States

Recent protein crystallographic investigations of acetyl coenzyme A synthase (ACS) revealed a surprising active site composition not anticipated based on prior biophysical and enzymological studies. Specifi cally, copper was identifi ed as a constituent of the A cluster along with the anticipated Ni and 4Fe4S, Figure 1. While copper was identifi ed unambiguously by multiwavelength anomalous dispersion experiments, the composition of the active protein remains in question. Using small molecule synthetic and mechanistic chemistry we are developing an understanding of the activity of ACS. Herein we present the synthesis and characterization of relevant NiCu and NiNi binuclear complexes, e. g. Fig. 2, designed to establish the fundamental reactivity of these small molecules relevant to ACS catalysis.

Gadolinium complexes containing polyamino polycarboxylate ligands for Magnetic Resonance Imaging (MRI) contrast agents

Wing-Tak Wong, The University of Hong Kong, China

Magnetic Resonance Imaging (MRI) is an important non-invasive method for studying the internal structures of the human body. There is a need in developing dose-effective contrast agents with higher specifi city and relaxivity. New Gd(III) complexes endowed with different functionalities were synthesized and studied in this work.

A series of mononuclear Gd(III) polyaminocarboxylates, [Gd(18dtpaxam)(H2O)] C1, [Gd(16dtpapam)(H2O)] C2 and

[Gd(20dtpasul)(H2O)] C3, were synthesized. Hydrophobic moieties, such as aromatic group and phenyl sulphone group, are introduced into these complexes so as to achieve target-specifi city. Moreover, a known compound [Gd(16dtpapnOH)(H2O)] C4 was also investigated for comparison. The syntheses, the solid- and solution-state structural characterization using single crystal X-ray analyses, electrospray ionization-mass spectrometry and luminescence lifetime measurements; thermodynamic stability studies by potentiometry; and relaxometric investigations of the compounds by Nuclear Magnetic Resonance Dispersion (NMRD) profi les, variable-temperature 17O NMR transverse relaxation, pH dependence and temperature dependence relaxivity are discussed. The four Gd(III) chelates, C1-C4, all possess one coordinated water molecule (q = 1) which ensures the inner-sphere contribution to relaxivity. The relaxivities are in the descending order of C1 (4.95 mM-1s-1) > C2 (4.51 mM-1s-1) > C3 (3.92 mM-1s-1) > C4 (3.70 mM-1s-1), measured at 20 MHz and 25°C. The overall formation constants of the four complexes fall in the range of 19-23, which are high and comparable to the clinically used contrast agents. Comparing [Gd(18dtpaxam)(H2O)] C1 with the two commercially available contrast agents 2- - [Gd(DTPA)(H2O)] and [Gd(DOTA)(H2O)] , it is concluded that if the water exchange rate in C1 could be optimized, it would give the highest relaxivity among the three. Journal of Inorganic Biochemistry 96 (2003) 23

Expanded Porphyrins as Potential Therapeutics

Jonathan L Sessler, The University of Texas, United States Darren J Magda, Pharmacyclics, Inc., United States

Expanded porphyrins are emerging as important synthetic targets with a variety of potential uses, including in the area of drug development.[1] In this latter context, considerable effort both at The University of Texas and at Pharmacyclics, Inc. has been devoted to the development of lanthanide(III) texaphyrins as possible therapeutics and two such systems, namely the Gd(III) and Lu(III) complexes of a water solubilized texaphyrin core (motexafi n gadolinium and motexafi n lutetium, respectively), are now in advanced clinical testing as adjuvants for the treatment of inter alia cancer and cardiovascular disease.[2] The fi rst of these systems is ‘MRI active’ whereas the second is highly fl uorescent and emits in the far-red portion of the UV- vis spectrum. These features make these systems of interest for diagnosis in the context of treatment. Separate from this work, effort has also been devoted to the synthesis and study of texaphyrins containing other coordinated cations, including transition metals, as well as to other expanded porphyrins that provide different cavity geometries, structural topographies, or pyrrole-to-pyrrole connectivities. Here, goals have been to develop novel catalysts of potential biomedical utility and to produce anion receptors that could fi nd application as in vivo anion sensors and carriers.[3,4] A summary of this work, with a focus on synthesis, mechanism of action, and potential clinical utility, will form the basis for this lecture.

Support for this work was provided in part by the National Institutes of Health (CA 68682). [1] J.L. Sessler, S.J. Weghorn, Expanded Contracted and Isomeric Porphyrins, Elsevier: Oxford; 1997 (520 pp). [2] T. D. Mody, L. Fu, J.L. Sessler, Progr. Inorg. Chem. 2001, 49, 551-598. [3] W. E. Allen, J.L. Sessler, ChemTech 1999, 29, 16-24. [4] J.L. Sessler, J.M. Davis, Acc. Chem. Res. 2001, 34, 989-997.

Formation of Unphotodissociable CO-Heme Adduct in Soluble Guanylate Cyclase

Teizo Kitagawa, Okazaki National Research Institutes, Japan Biswajit Pal, Center for Integrative Bioscience, Okazaki National Research Institutes, Japan Takehiro Ohta, Center for Integrative Bioscience, Okazaki National Research Institutes, Japan Zhengqiang Li, Key Laboratory for Molecular Enzymology and Engineering, China Shigeo Takenaka, Department of Veterinary Science, Osaka Prefecture University, Japan Shingo Tsuyama, Department of Veterinary Science, Osaka Prefecture University, Japan

Photodissociation of CO from CO adducts of heme proteins is considered as an intrinsic property of heme-CO complexes. Unexpectedly, however, the heme-CO of soluble guanylate cyclase (sGC) is not photodissociated under certain conditions. sGC with an αβ dimer structure contains a single heme in the N-terminal region of β subunit and catalyzes the conversion of GTP to cGMP in the C-terminal regions of both subunits. The catalytic activity increases up to ~200 times when NO binds to its heme but only ~4 times when CO binds to it. Binding of NO and CO to sGC provided the fi ve coordinate (5c) heme-NO and six coordinate (6c) His-heme-CO adducts, respectively, in the ordinary conditions. We report here that the heme-CO adduct is not photodissociable when GTP and YC-1 [3-(5í-hydroxymethyl-2í-furyl)-1-benzanidazole] are present, and that the enzymatic activity becomes to the level of the NO-bound sGC.

The sGC isolated from bovine lung yielded the main bands of the Fe-CO (νFe-CO) and C-O (νCO) stretching modes at 473 and 1984 cm-1, respectively, but minor bands at 488 and 1970 cm-1. Relative intensities of the two sets were appreciably altered by addition of YC-1 or GTP, and the former pair disappeared completely and additional CO-isotope sensitive band appeared at 521 cm-1 when both are present. The original spectral pattern was restored upon extensive dialysis of the mixed -1 solution, demonstrating that binding of YC-1 and GTP is reversible. When the νFe-CO band is present at 473 cm , there are -1 two ν4 bands at 1370 and 1356 cm , the latter of which became stronger with higher laser power. However, when the 473 -1 -1 -1 cm band was replaced by the 488 cm band, a single ν4 band was observed at 1372 cm even with a higher laser power. This means no photodissociation of the CO-heme. A model compound study with iron(II)-protoporphyrin IX in micelles of cetyl-trimethyl ammonium bromide demonstrated that the 521 cm-1 band arises from the 5c CO adduct. DFT calculations on heme-CO adducts indicated that the 6c heme with His-Fe(II)-CO is photolabile but the 5c heme with Fe(II)-CO is not. Consequently, we conclude that YC-1 and GTP synergetically make the heme-CO adduct 5c and raise the activity similar to that of NO adduct. 24 Journal of Inorganic Biochemistry 96 (2003)

A Kinetic Trap Mechanism for Release of NO from Cytochrome c’: Implications for Inactivation of the NO Sensor, Soluble Guanylate Cyclase

Kenton R Rodgers, North Dakota State University, Department of Chemistry, United States Colin R Andrew, Eastern Oregon University, Department of Chemistry, United States Robert R Eady, John Innes Centre, Norwich, United Kingdom

Recent stopped-fl ow kinetic studies of ferrous Alcaligenes xylosoxidans cytochrome c’ (AXCP) have revealed that its thermodynamic NO adduct forms via two NO-dependent steps, reminiscent of the kinetic behavior of soluble guanylate cyclase (sGC) with NO. Moreover, x-ray crystallography has shown the thermodynamic ferrous AXCP-NO complex to be fi ve-coordinate with the NO ligand bound to the proximal side of the heme. Similarity in the kinetics of NO binding to AXCP and sGC have spawned the hypothesis that sGC-NO may also contain a proximal NO ligand. In this study, we have shown by NO recombination experiments that the proximal NO structure facilitates NO release by way of an intermediate in which the endogenous histidine ligand has trapped the proximal coordination site. After photolysis of AXCP-NO, ~5% of the photolyzed hemes are converted to a fi ve-coordinate ferrous state, implying that reattachment of the endogenous His ligand competes kinetically with NO rebinding from the proximal heme face. The NO-binding rates of the conformationally relaxed fi ve-coordinate photoproduct are very similar to those previously determined in the aforementioned stopped-fl ow study. In contrast with the stopped-fl ow results, these recombination results reveal a fraction of unrelaxed ferrous heme that contains a labile Fe-His bond, allowing NO to bind directly on the proximal heme face to give the fi ve-coordinate proximal AXCP-NO complex. The discovery of rapid proximal His binding to NO-dissociated heme reveals a novel ‘kinetic trap’ mechanism for lowering the fi ve-coordinate heme nitrosyl population in response to decreased ambient NO concentrations. Thus, NO dissociation from the fi ve-coordinate heme nitrosyl, whether thermal or photochemical, is followed by rapid, and only slowly reversible His reattachment which kinetically traps the heme in its fi ve-coordinate ferrous state. Since return to the fi ve-coordinate heme nitrosyl requires two NO-dependent steps, the protein uses a kind of kinetic amplifi cation of the thermodynamic dissociation that occurs in response to decreased NO concentrations. Implications of this ‘kinetic-trap’ mechanism for the deactivation of sGC-NO will be discussed. Journal of Inorganic Biochemistry 96 (2003) 25

NMR investigations of the four nitrophorins from the blood-sucking insect rhodnius prolixus

F Ann Walker, University of Arizona, United States Tatjana Kh Shokhireva, University of Arizona, United States Nikolai V Shokhirev, University of Arizona, United States

The nitrophorins are NO-storing, -carrying and -releasing proteins from the saliva of blood-sucking insects. They are stable as ferriheme proteins that have Kd values of ~1 µM and 20 nM for NO and histamine, respectively, which makes them excellent at maintaining the stability of NO for long periods of time, until the insect wants to use it to aid in obtaining a suffi cient blood meal by dilating the capillaries of the victimin the region of the bite. Histamine binding to the nitrophorins, upon release of NO, provides a second means of assuring the insect of having enough time to complete the meal without being detected. We are investigating the four nitrophorins of Rhodnius prolixus, NP1-NP4, by 1H and 13C NMR spectroscopy to determine the similarities and differences in the heme binding pockets of these beta-barrel heme proteins. Multidimensional NMR techniques have been used to assign the heme resonances of all four nitrophorins in both the high-spin (S = 5/2) and low-spin (S = 1/2) Fe(III) states. As ligands to produce the low-spin state, we have used histamine, several imidazoles and cyanide. To simplify the 1H NMR spectra, a symmetrical hemin, 2,4-dimethyldeuterohemin, has been utilized, because among the protohemin-containing proteins only NP2 has mainly one heme orientation. Saturation transfer techniques have allowed assignment of many of the heme resonances of the high-spin proteins. We fi nd that NP1 and NP4 have very similar ligand binding pockets, but NP2 and NP3, previously thought to be fairly similar in structure, have rather different ligand binding pockets. With the information gained by investigation of the symmetrical hemin-containing proteins, we have now been able to assign the two sets of resonances for the two heme orientations of the natural protohemin-containing proteins. NMR investigations of NP2 mutants, of amino acids having bulky side chains that protrude into the distal ligand binding pocket, have provided important insights into how the design of this distal pocket controls ligand binding and orientation.

New insights into the role of metal-thiolate clusters in neuronal growth inhibitory factor (metallothionein-3)

Milan Vasak, University of Zurich, Switzerland

The understanding of neuronal growth is of considerable current interest. Apart from the involvement of growth factors in these processes, it has been recognized that growth inhibitory factors play an equally important role. Human neuronal growth inhibitory factor, metallothionein-3 (MT-3), possesses two mutually interacting domains each encompassing a homometallic I II metal-thiolate cluster. The biological activity has been found for Cu 4,Zn 3-MT-3 isolated from brain and also for recombinant

Zn7-MT-3. Mutational analysis established that the conserved T(5)-C-P-C-P(9) motif in the N-terminal β-domain is II 1-2 I responsible for both biological activity and unprecedented dynamics of its M 3(CysS)9 cluster. Independently, Cu and II Zn containing MT-3 was generated in vitro. The characterization of the Cu4,Zn4-MT-3 form by optical and immunological 3-4 techniques revealed a specifi c formation of a Cu4(CysS)9 cluster in the β-domain and a Zn4(CysS)11 cluster in the α-domain. I In contrast to the Cu 4 cluster, which was found to be stable in air, oxidation of thiolate ligands in the α-domain resulted in a II release of one Zn and formation of a Zn3-thiolate cluster, i.e., the native-like Cu4,Zn3-MT-3 form. Since the oxidation process could be reversed under reducing conditions, a regulatory role for the α-domain of MT-3 is proposed. The implications of these studies for the biological activity of MT-3 will be discussed. 1 Hasler D.W., Jensen L.T., Zerbe O., Winge D.W. and Vasak M., Biochemistry, 39, 14567-14575 (2000) 2 Romero-Isart, N., Jensen L.T., Zerbe O., Winge D.W. and Vasak M, J. Biol. Chem. 277, 37023-37028 (2002) 3 Roschitzki, B. and Vasak M., JBIC, 7, 611-616 (2002) 4 Roschitzki, B. and Vasak M., submitted 26 Journal of Inorganic Biochemistry 96 (2003)

Characterization of unusual multicopper nitrite reductase containing blue and green type 1 Cu ions

Shinnichiro Suzuki, Osaka University, Japan Mayuko Kobayashi, Osaka University, Japan Koushi Itoh, Osaka University, Japan Atsushi Fukui, Osaka University, Japan Kazuya Yamaguchi, Osaka University, Japan Kunishige Kataoka, Kanazawa University, Japan

Dissimilatory nitrite reductase (NIR) located in the periplasm catalyzes one-electron reduction of nitrite to NO. The molecular structures of the Cu-containing NIRs from Achromobacter cycloclastes IAM 1013 (green AcNIR) and Alcaligenes xylosoxidans (blue AxNIR) are a trimer, in which a monomer (ca. 37 kDa) contains a type 1 Cu ion and a type 2 Cu ion. These protein structures are quite similar to each other. The type 1 Cu is involved in electron-transfer processes and the type 2 Cu is the catalytic reduction site of the substrate. Several years ago, we found that blue-green Hyphomicrobium denitrifi cans NIR (HdNIR, ca. 50 kDa) shows unique spectroscopic features of the type 1 Cu, compared with those of AcNIR and AxNIR: the visible absorption spectrum implies that the enzyme has two kinds of the type 1 Cu. The genetic analysis of HdNIR indicated that the polypeptide is consist of blue copper protein-like domain (ca. 14 kDa) containing one type 1 Cu ligand motif (type 1 CuN: His77, Cys114, His119, and

Met124) and NIR-like domain (ca. 35 kDa) containing one type 1 Cu ligand motif (type 1 CuC: His219, Cys260, His268, and Met273) and one type 2 Cu ligand motif (His224, His259, and His416). The preparation and characterization of the C114A and C260A mutants disclosed that the blue type 1 CuN and the green type 1 CuC shows an axial EPR signal like the type 1 Cu of AxNIR and a rhombic signal like that of AcNIR, respectively. The catalytic activity of C114A HdNIR is larger than that of the recombinant enzyme by a factor of about 1.7, but the

C260A mutant shows hardly enzyme activity. The cognate cytochrome c550 functions as an electron donor to the recombinant and C114A HdNIRs not to the C260A mutant. Therefore, the electron acceptor site of HdNIR is the type 1 CuC coupled with 5 -1 -1 the type 2 Cu, and not the type 1 CuN. An electron-transfer rate constant of 4.1 X 10 M s from cyt c550 to the C114A mutant at pH 5.5 is almost similar to that of the recombinant enzyme (4.4 X 105 M-1s-1). Journal of Inorganic Biochemistry 96 (2003) 27

IV 12- Solution Chemistry and Spin Frustration of [(V O)3(SbW9O33)2] Exhibiting a Potent Anti-RNA Virus Activity

Toshihiro Yamase, Chemical Resources Laboratory, Tokyo Institute of Technology, Japan Shiroh Shigeta, Department of Microbiology, Fukushima Medical College, Japan

Polyoxometalates have been proven to inhibit the replication of several enveloped DNA and RNA viruses. We have recently IV 12- described potent antiviral activities of the tris(vanadyl)-18-tungsto-2-antimonate(III) anions, [(V O)3(SbW9O33)2] and V IV 11- [(V O)(V O)2(SbW9O33)2] against a wide variety of the enveloped viruses which infect high-risk individuals, such as infants born with prematurity, cardiovascular failure, pulmonary dysplasia, and human immunodefi ciency virus (HIV) [1]. IV 12- V IV 11- [(V O)3(SbW9O33)2] and [(V O)(V O)2(SbW9O33)2] have been prepared and x-ray crystallographically characterized IV V IV as potassium salts K11[(V O)3(SbW9O33)2]•27H2O (1) and K10Na[(V O)(V O)2(SbW9O33)2]•26H2O (2), respectively (Fig. 1). The cyclic voltammograms of (1) in aqueous solutions at 3cp: centers for (1), which leads to the two total spin quantum number (S)=1/2 states as a result of spin frustration, as supported by measuring accurate heat capacity of (1) polycrystallines (Fig. 2). (2) strongly inhibited the binding of anti-gp-120 antibody to HIV-1 infected MT-4 cells, whereas it did not inhibit the binding of anti-X4 receptor antibodies (anti-CD4 and anti-CXCR4) to Molt-4 cells (Tab. 1). This result indicates that (2) binds viral gp-120 and interferes the interaction with its receptors (CD4 and CXCR4) without any direct interaction with the receptors. The binding of 1 and 2 with gp-120 is likely to occur through the three equatorial K+ ions of the anion center which is associated with IV V the V /V -redox property of the three VO5 square-pyramids (Fig. 1). [1] S. Shigeta, S. Mori, E. Kodama, J. Kodama, K. Takahashi, and T. Yamase, Antiviral Res., (2003) in press. Table 1. Inhibitory effect of (2) and DS5000 on bindings of anitibodies against CD4, CXCR4 and gp-120.

Compound Antibody %inhibition3

DS5000(10µM) Anti-CD41 <5 Anti-CDCR41 <5 Anti-gp-1202 27.0 (2) Anti-CD4 <5 Anti-CDCR4 <5 Anti-gp-120 97.0

A representative resuls is shown. 1 For the binding assay of anti-CD4 and anti-CXCR4 antibodies MT-4 cells were used. 2 For the binding assay of anti-gp-120 antibody, Molt-4/IIIb cells were used. 3 Percent of the antibody-bound cells were determined by FACScan analysis. 28 Journal of Inorganic Biochemistry 96 (2003)

Single-Crystal Voltammetry and In Situ Scanning Tunnelling Microscopy of Redox Metalloproteins: Bioelectrochemistry towards the Single-Molecule Level

Jens Ulstrup, Department of Chemistry, Technical University of Denmark, Denmark Jingdong Zhang, Department of Chemistry, Technical University of Denmark, Denmark Allan G Hansen, Department of Chemistry, Technical University of Denmark, Denmark Hainer Wackerbarth, Department of Chemistry, Technical University of Denmark, Denmark Hans EM Christensen, Department of Chemistry, Technical University of Denmark, Denmark

Diffusion controlled and fi lm voltammetry are established techniques to address thermodynamic and interfacial electron transfer of both small redox metalloproteins and multi-centre redox metalloenzymes. Introduction of single-crystal, atomically planar metal electrode surfaces and in situ scanning tunnelling microscopy (STM) directly in aqueous buffer under electrochemical potential control, has, however, added new perspectives to protein bioelectrochemistry. Single-crystal electrodes enhance signifi cantly voltammetric and interfacial capacitance sensitivity, and in situ STM addresses even single biomolecules in electron transporting action. Other state-of-the-art surface science tools such as microcantilever sensor techniques and X-ray photoelectron spectroscopy have supported this. We overview studies in our group based on single-crystal voltammetry, interfacial capacitance, X-ray photoelectron spectroscopy, microcantilever sensor techniques, and in situ STM of several redox proteins on functionally modifi ed Au(111)- electrode surfaces. The proteins are: Pseudomonas aeruginosa azurin, Saccharomyces cerevisiae cytochrome c, Pyrococcus furiosis [3Fe-4S] ferredoxin, and the trimeric blue copper oxidase Achromobacter xylosoxidans Cu-nitrite reductase, as shown in Figure 1. The data point to new ways of mapping and controlling redox metalloprotein orientation and function at well-characterised electrochemical surfaces. This is important in future electrochemical biosensor design towards the single-molecule level. Zhang, J.; Chi, Q.; Kuznetsov, A.M.; Hansen, A.G.; Wackerbarth, H.; Christensen, H.E.M.; Andersen, J.E.T.; Ulstrup, J. J. Phys. Chem. B 2002, 106, 1131-1152. Fig.1 Molecular structures. In the left column the Cu-proteins azurin (top) and the enzyme nitrite reductase (bottom). In the right column the iron containing (yeast) cytochrome c (top) and ferredoxin (bottom) Journal of Inorganic Biochemistry 96 (2003) 29

Time-Resolved Fluorometry Using Lanthanide Labels for Diagnostic and Biotechnology

Kazuko Matsumoto, Waseda University, Japan

Certain lanthanide (Eu3+, Tb3+, Sm3+, and Dy3+) chelates have specifi c fl uorescence properties that conventional organic dyes do not have: (i) The lifetime of the lanthanide chelates are very long ranging from 50 µs to 2 ms; (ii) the chelates are excited by UV light (310-350 nm) and emit fl uorescence in the visible region (615, 545, 643, 574 nm); (iii) the emission profi les are sharp, having the half-widths of only ~10 nm. By taking advantage of these properties, these chelates have been used as fl uorescent labels for biomolecules, and highly sensitive time-resolved fl uorometry has been developed for immunoassay and DNA detection. The beta-diketonate type chelate (BHHCT-Eu3+, Fig. 1) has been found to give innovatively high sensitivity, improving the detection limit of conventional immunoassay by two to four orders of magnitude. For instance, the detection limit of AFP in serum is 0.0041 pg/ml, which corresponds to 4 orders of magnitude improvement. By using the system, serum SDF-1 in normal and HIV-1 infected people was for the fi rst time measured. The concentration ranges 0.3 to 2 ng/ml in the normal people and 1 to 10.5 ng/ml in the infected people. Poly amine carboxylate chelate (BPTA-Tb3+, Fig. 1) is a nanodentate label and is more stable than BHHCT-Eu3+ to use for DNA labeling. BPTA-Tb3+ was bound to 5´end amino-linker, which was found to go through the PCR thermal cycle, and the homogeneous DNA hybridization assay using FRET (fl uorescence resonance energy transfer) between BPTA-Tb3+ and Cy3 has been developed. The label was also applied to the Invader method for SNP (single nucleotide polymorphism) analysis. The characteristics and applications in biotechnology of other labels in Fig. 1 will also be discussed. 30 Journal of Inorganic Biochemistry 96 (2003)

New Fluorescent and Colorimetric DNAzyme Biosensors for Metal Ions

Yi Lu, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Juewen Liu, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Peter J Bruesehoff, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Andrea K Brown, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Kevin E Nelson, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Hee-Kyung Kim, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Daryl P Wernette, Department of Chemistry, University of Illinois at Urbana-Champaign, United States

While remarkable progress has been made in developing sensors for metal ions such as Ca2+, designing and synthesizing sensitive and selective metal ion sensors remains a signifi cant challenge. Perhaps the biggest challenge is the design and synthesis of a sensor capable of specifi c and strong metal-binding. Since our knowledge about the construction of metal- binding sites is limited, searching for sensors in a combinatorial way is of signifi cant value. Therefore, we have been able to use a combinatorial method called in vitro selection to obtain DNAzymes (DNA with enzymatic activities, also called catalytic DNA) that can bind a metal ion of choice strongly and specifi cally.1, 2 By labeling the resulting DNAzymes with a fl uorophore/quencher pair (Figure), we have made a new class of metal ion fl uorescent sensors.3, 4 Recently we have converted the DNAzymes into colorimetric sensors by labeling the DNA with gold nanoparticles and using DNA for directed assembly of the gold nanoparticles. A novel approach of using an inactive variant of DNAzymes to tune the detection range of the sensors is also demonstrated. The DNA sensors combine the high selectivity of DNAzymes with the high sensitivity of fl uorescence or colorimetric detection, and can be applied to the quantitative detection of metal ions over a concentration range of three orders of magnitude. DNA is stable, cost-effective, environmentally benign, and easily adaptable to optical fi ber and microarray technology for device manufacture. Thus, the DNA sensors shown here hold great promise for on-site and real-time monitoring of metal ions in the fi elds of environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial process monitoring. 1 Li, J. et al., Nucleic Acids Res. 28, 481 (2000); 2. Brown, A. K. et al., Biochemistry, in press. 3. Li, J. and Lu, Y., J. Am. Chem. Soc., 122, 10466 (2000). 4. Lu, Y. et al. Biosensors and Bioelectronics, in press.

Probing Metallopeptide Folding Mechanisms Using de Novo Design Peptides

Vincent L Pecoraro, University of Michigan, United States Brian Farrer, University of Michigan, United States Manolis Matzepetakis, University of Michigan, United States Debdip Ghosh, University of Michigan, United States

The series of amphiphillic peptides called the TRI series has been used to understand the mechanism of Hg(II) binding to proteins. The peptide BabyL9C (sequence GLKALEEKCKALEEKLKALEEKG) is unstructured at micromolar concentrations at pH 8.5. Addition of Hg(II) nucleates the peptide fi rst into a two stranded coiled coil that contains a two coordinated Hg(II). Subsequently, a third peptide joins the aggregate to form a three stranded coiled coil. Depending on the pH, the Hg(II) can be found as either a linear, two coordinate species or a three coordinate, trigonal complex. This system demonstrates that information to adopt a three stranded coiled coil is embedded in the primary sequence of BabyL9C and only requires a metal as a nucleating agent to achieve the desired fold. Unlike BabyL9C, TRI L9C (sequence GLKALEEK CKALEEKLKALEEKLKALEEKG) is folded into a three stranded coiled coil prior to metal insertion. Hence, one expects a different process for the formation of the trigonal Hg(II) environment. Experiments will be presented that address the processes of metal insertion into these distinct metal-helical bundle aggregates. Journal of Inorganic Biochemistry 96 (2003) 31

NMR studies on partially folded and unfolded states of metalloproteins

Paola Turano, CERM – University of Florence, Italy

How does the presence of a metal infl uence the structure and dynamics of a metalloprotein? The NMR characterization of some metalloproteins in non-native states has been performed with the aim to shed light on the thermodynamics, structure and dynamics of the folding/unfolding process with particular emphasis on the role that metal ions play in modulating the conformation and fl exibility of the protein scaffold. The structural changes detected on passing from the native to the alkaline form of mitochondrial cytochrome c constitute the key to rationalize the redox dependent stability of this electron transfer protein and the different behavior of type-1 cytochromes towards unfolding agents. An intimate relationship between structure stability and dynamical properties has also emerged from these studies. The importance of metal ions with respect to the unfolding process has been analyzed also in the case of copper,zinc- superoxide dismutase.

Mapping Protein Folding Energy Landscapes

Harry B Gray, California Institute of Technology, United States Jay R Winkler, California Institute of Technology, United States Jennifer C Lee, California Institute of Technology, United States

The production of functional proteins requires that polypeptides fi nd a unique conformation in a vast space of incorrect folds. The consequences of failure are severe; misfolded proteins are implicated in a rapidly growing list of debilitating illnesses that includes type II diabetes as well as Alzheimer’s and Parkinson’s diseases. Partially folded polypeptides are key intermediates in both the proper assembly of proteins, and in the formation of harmful misfolded structures. We have developed new methods to probe the conformations of polypeptides as they evolve to their native structures in order to acquire a more detailed understanding of this complex self-assembly process. These investigations into the structures, energetics, and dynamics of partially folded polypeptides are being employed to develop a keener understanding of their benign and malignant pathways. 32 Journal of Inorganic Biochemistry 96 (2003)

Cytochrome c and the Suicide Hotline

George L McLendon, Princeton University, United States

Regulation of programmed cell death (apoptosis) is critical both for normal development, and for ongoing maintenance to remove damaged cells, e.g. cancer cells. We will briefl y review current understanding of this cycle paying particular attention to the signal role of cytochrome c and other mitochondrial proteins.

Structural Change of Plastocyanin. Molecular Interaction and Protein Folding

Shun Hirota, Kyoto Pharmaceutical University, Japan Noriaki Funasaki, Kyoto Pharmaceutical University, Japan Yukari Fujimoto, Nagoya University, Japan Hisano Okumura, Nagoya University, Japan Noriko Katagiri, Nagoya University, Japan Yoshihito Watanabe, Nagoya University, Japan Jungkwon Choi, Kyoto University, Japan Masahide Terazima, Kyoto University, Japan Sachiko Arie, The University of Tokyo, Japan Kentaro Tanaka, The University of Tokyo, Japan Mitsuhiko Shionoya, The University of Tokyo, Japan Toshihide Okajima, Osaka University, Japan Teruhiro Takabe, Meijo University, Japan

Plastocyanin (PC) is a mobile electron transfer protein existing in the thylakoid lumen of photosynthetic organisms. It is negatively charged at neutral pH for higher plant and green algae PCs. These PCs contain consecutive acidic residues located at the solvent-accessible site near the tyrosine residue remote from the Cu center [1]. We have been studying the molecular recognition character of proteins and their interaction-induced structural changes with the use of charged peptides, since they do not have any visible absorption [2]. The active site Cu of PC exhibits a longer Cu-S(Cys) bond length and a higher redox potential on binding of lysine peptides, suggesting that lysine peptides induce a structural change in PC to make the copper site adapted for facile electron transfer. We synthesized various peptides with four lysines and three glycines and compared the effect of charge distribution in the peptide on the interaction. We have previously shown that folding of cytochrome c (cyt c) could be studied by modifying Met65 with a photocleavable o-nitrobenzyl derivative and irradiating UV light on the partially unfolded modifi ed cyt c [3]. We applied this method to apoplasotocyanin (apoPC), and the side chain of the cysteine residue of apoPC was site-specifi cally modifi ed with a photocleavable 4,5-dimethoxy-2-nitrobenzyl derivative. An absorption band at 355 nm of the modifi ed protein was attributed to the photocleavable derivative, and this band disappeared by irradiation of 355-nm pulse light on the modifi ed protein. The modifi ed protein exhibited a denatured structure, whereas the native β-sheet structure was recovered by irradiation of UV light. The change in the volume of apoPC during folding was also observed. 1 J. M. Guss, P. R. Harrowell, M. Murata, V. A. Norris, H. C. Freeman, J. Mol. Biol., 192, 361-387 (1986). 2 S. Hirota, O. Yamauchi, Eur. J. Inorg. Chem. (review), 17-25, (2002). 3 T. Okuno, S. Hirota, O. Yamauchi, Biochemistry, 39, 7538-7545 (2000). Journal of Inorganic Biochemistry 96 (2003) 33

Effect of camphor on the stability of heme in Cytochrome P450cam: Urea mediated unfolding studies

Shyamalava Mazumdar, Tata Institute of Fundamental Research, India R Murugan, Tata Institute of Fundamental Research, India

The effect of camphor on the conformational stability of the heme active site of cytochrome P450cam has been investigated by urea mediated unfolding studies of the enzyme. The absorption spectra of the heme moiety showed presence of at least two intermediates in the unfolding path, and the corresponding thermodynamic parameters were obtained by global fi tting of the experimental data to a generalized sequential unfolding model at different wavelengths. Circular dichroism spectra of the enzyme in the visible and far-UV region were used to identify the critical range of denaturant concentration at which the tertiary structure around the heme center was affected with almost no change in the secondary structure of the enzyme. This critical range of urea concentration was 0-2.8 M urea in presence of camphor and 0-1.2 M urea in absence of camphor. The spectral assignments of the intermediate species of the heme active site with the intact secondary structure of the enzyme were made based on the Soret absorption maxima and the results were analyzed to determine stabilization of the heme active site by the substrate in cytochrome P450cam.

Oxygen Activation by Cytochrome P450 and Nitric Oxide Synthase. Evidence for a Second Active Oxidant From Studies Using P450-CAM as a Model System

John H Dawson, Department of Chemistry and Biochemistry, University of South Carolina, United States Shengxi Jin, Department of Chemistry and Biochemistry, University of South Carolina, United States Mary C Lamczyk, Department of Chemistry and Biochemistry, University of South Carolina, United States Heather L Voegtle, Department of Chemistry and Biochemistry, University of South Carolina, United States Thomas A Bryson, Department of Chemistry and Biochemistry, University of South Carolina, United States

Cytochrome P450 is a versatile heme-containing oxygenase that transfers oxygen atoms from dioxygen to a wide range of organic substrates. Nitric oxide synthase is also an oxygenase; it fi rst hydroxylates arginine to an N-hydroxyimine intermediate and then, in a second O-atom transfer step, forms citrulline and NO. Hydroxylation involves substrate binding to the ferric enzyme followed by reduction and oxygen binding to give the oxyferrous state. Addition of a second electron yields a peroxo-ferric intermediate, protonation of which generates a hydroperoxoferric state; lose of water then forms an oxo-ferryl porphyrin radical (Compound I). Although the latter is thought to be the ultimate oxidant, the peroxo- and hydroperoxoferric species have been proposed as secondary oxidants. The T252A mutant of P450-CAM was fi rst prepared by Sligar and Ishimura. Because this mutant does not hydroxylate camphor, it must not form P450 Compound I. However, it still accepts electrons from NADH to give hydrogen peroxide, presumably via the peroxo- and hydroperoxoferric intermediates. Thus, T252A P450-CAM is the ideal mutant to test whether one and/or the other of these two species are capable of O-atom transfer. We have prepared a series of camphor analogues that contain reactive functional groups that are the site of O-atom transfer in order to investigate the mechanism of olefi n epoxidation, ether N-dealkylation, amine N-dealkylation, thioether sulfoxidation and N-hydroxyimine denitrosation (structures below). With 5-methylenylcamphor, for example, we fi nd that the T252A mutant is enzymatically active in olefi n epoxidation, producing epoxide at 20% of the wild type rate. This clearly demonstrates that a second active oxidant is formed in the P450 reaction cycle. Results of our studies of oxygen activation by P450 with these model substrates, with emphasis on the involvement of a second oxidant in O-atom transfer, will be presented. 34 Journal of Inorganic Biochemistry 96 (2003)

Heme oxygen reactive intermediates: catalysis across a rich functional landscape

Stephen G Sligar, University of Illinois, United States Ilia Denisov, University of Illinois, United States Tom Makris, University of Illinois, United States

Phenomenal advances in genome science have provided an evolutionary linkage between organisms in the ‘tree of life’. This rich landscape also provides unprecedented opportunities to understand the coupling of multiple reactivity profi les to the dynamical control of heme protein catalytic intermediates. Pharmacologists, toxicologists and biochemists have long been interested in the precise chemical mechanisms of substrate processing by the cytochrome P450 monoxygenase systems and the associated process of ‘oxygen activation’. Recently, using enzymes from diverse sources and through advances in cryoenzymology, bioorganic chemistry and rapid reaction methodologies, it has been possible to infer three distinct iron- oxygen reactive states: (1) Nucleophilic peroxo [Fe-OO]; (2) Electrophilic hydroperoxo [Fe-OOH]; and (3) ‘Compound I’ [FeO]. (See for instance: Davydov, R., Makris, T. M., Kofman, V., Werst, D. E., Sligar, S. G., and Hoffman, B. M. (2001) J. Am. Chem. Soc., 123, 1403-1415; Denisov, I., G., Makris, T. M., and Sligar, S. G. (2001) J. Biol. Chem., 276, 11648-11652; Denisov, I. G., Makris T. M., and Sligar, S. G. (2002) J. Biol. Chem., 277, 42706-42710 and references therein). The peroxo and hydroperoxo states are clearly observed by resonance and optical spectroscopy, and the ‘Compound I’ intermediate clearly defi ned for the fi rst time. Using various native and mutant meso/thermophilic cytochromes P450 we have been able to stabilize the higher valent ‘Compound I’ state, and measure its formation and breakdown kinetics. Thermal annealing of cryo-trapped intermediates in many heme protein systems, including cytochrome P450, peroxidase and heme oxygenase, individual iron-oxygen states have been characterized and probed for chemical reactivity. Supported by a Merit Award from the National Institutes of Health GM31756.

Model Studies of Cytochrome P450: Reactive Intermediates, O-O Activation, and Catalytic Oxygenation

Wonwoo Nam, Ewha Womans University, Korea

Heme-containing enzymes such as cytochromes P-450, peroxidases, and catalases utilize dioxygen and its partially reduced forms in a variety of enzymatic reactions such as the incorporation of oxygen atoms into organic substrates (cytochrome P-450) and the oxidation of substrates (peroxidase and catalase). As biomimetic models for the heme-containing enzymes, the reactions of iron(III) porphyrin complexes with various oxidants have been extensively studied, with the intention of elucidating the structure of reactive intermediates and the mechanisms of O-O bond activation. It has been generally believed that high-valent iron(IV)-oxo porphyrin cation radicals are reactive intermediates responsible for the oxygenation of hydrocarbons. However, recent studies provided strong evidence that oxidantñiron porphyrin adducts are also able to transfer their oxygen to hydrocarbons prior to the formation of high-valent iron-oxo intermediates. Regarding the O-O bond activation by iron porphyrin models, the O-O bond of peroxyacids is cleaved heterolytically by the iron porphyrin complexes, resulting in the formation of high-valent iron(IV)-oxo porphyrin cation radical intermediates. However, the situation is less clear in the cases where biologically important oxidants such as hydrogen peroxide and alkyl hydroperoxides are used. We reported recently that the O-O bond of the hydroperoxides is cleaved both heterolytically and homolytically, depending on reaction conditions such as solvent systems, electronic nature and anionic ligand of iron porphyrin complexes, and pH of reaction solution. These results will be discussed with our recent observation that new iodosylbenzne-iron(III) porphyrin intermediates are generated in the reactions of high-valent iron-oxo porphyrin cation radicals and iodobenzenes and that the formation of the new intermediates depends on the electronic nature of the iron porphyrin complexes and iodobenzene derivatives. Journal of Inorganic Biochemistry 96 (2003) 35

Direct Electrochemistry of Cytochrome P450cin: new insights into P450 mechanism

Paul V Bernhardt, University of Queensland, Australia Kondo-Francois Aguey-Zinsou, University of Queensland, Australia James J De Voss, University of Queensland, Australia Kate E Slessor, University of Queensland, Australia

The cytochromes P450 (P450s) comprise a superfamily of heme-thiolate proteins, ubiquitous in Nature [1]. They play essential roles in a variety of biosynthetic and biodegradative pathways and catalyse an impressive array of oxidative

transformations. These reactions utilise O2 as the ultimate electron acceptor, and include the energetically demanding insertion of an O-atom into an unactivated C-H bond (equation 1). P450 mechanism continues to be a major research focus [2].

+ - RH + O2 + 2H + 2e → ROH + H2O (1) Crystallography has revealed a ferric heme b active site in the resting state of all P450s thus far characterized structurally and an axially coordinated cysteinate residue [3]. The ‘distal’ sixth coordination site may be occupied by a variety of ligands during catalysis (aqua, hydroxo, oxo or dioxygen) or it may be vacant. Reduction to ferrous P450 in the absence of substrate, but in presence of dioxygen, has the potential to consume electrons (and energy) and release toxic reactive oxygen species such as superoxide and hydrogen peroxide, yet it is believed to be the initial step in P450 catalysis. A defi nitive explanation of selective substrate

oxidation in preference to futile O2 reduction in P450 catalysis has remained elusive. Here we present some of our recent work [4] comprising the electrochemical characterization of recently isolated

bacterial P450cin (CYP176A) that raises important questions in P450 electron transfer and catalysis. As we will demonstrate, its electrochemical properties are unique among P450s. References [1] P. R. Ortiz de Montellano, ‘Cytochrome P450: Structure, Mechanism, and Biochemistry,’ 2nd Ed., 1995. [2] P. R. Ortiz de Montellano and J. J. De Voss, Nat. Prod. Rep., 2002, 19, 477. [3] T. L. Poulos, Curr. Opin. Struct. Biol., 1995, 5, 767. [4] K.-F. Aguey-Zinsou, P. V. Bernhardt, J. J. De Voss and K. E. Slessor, Chem. Commun., 2003, 418.

The Role of Spin in Oxo-Transfer Chemistry of Reactions Mediated by

Cytochrome P450 and Methane Monooxygenases Sunney I Chan, Institute of Chemistry, Academia Sinica, Taiwan, Province of China

In this lecture, I shall review the role of spin conservation in the oxo-transfer chemistry of reactions mediated by cytochrome P450 and the methane monooxygenases. It is argued that the hydroxylation of small alkanes mediated by both the soluble methane monooxygenase (sMMO) and the particulate methane monooxygenase (pMMO) proceeds via a direct concerted ‘singlet oxene’ insertion across the C-H bond. The transition state involving the ‘oxene’ released by the metal cluster and the ‘C-H’ bond of the alkane should be primarily singlet in character. This feature of the transition state promotes rapid ‘C--O’ bond closure, and the resultant process that ensues should be a ‘concerted’ one. The hydroxylation of small alkanes mediated by the pMMO occurs with total retention of confi guration at the carbon center attacked, consistent with this mechanism of oxo- insertion. On the other hand, spin crossover into the triplet manifold could occur. Since the transition-state is short- lived, no more than 200 femoseconds, the amount of spin-crossover could not be large. This ‘spin-crossover’ could be the origin of the short-lived radical formed in the so-called ‘concerted yet non-synchronous’ radical mechanism advocated for the hydroxylation chemistry mediated by the sMMO. The chemistry mediated by cytochrome P450 is more complex, as the ‘O’ species could be delivered as either the singlet or triplet oxene. If the oxene species is delivered in its triplet state, there would be no ambiguity in the outcome, as the C-center and OH-center radicals must necessarily be long-lived. Since the radicals are formed in the ground state, the level of radicals formed is also relatively higher. Within this scenario, it would be more appropriate to invoke the classical hydrogen-abstraction radical-rebound mechanism to describe the ‘oxo-transfer’ chemistry. 36 Journal of Inorganic Biochemistry 96 (2003)

Preparation of peroxo-bridged heme-copper complex and its crystal structure

Yoshinori Naruta, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan Takefumi Chishiro, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan Yuichi Shimazaki, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan Fumito Tani, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan Jin-gang Liu, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan Yoshimitsu Tachi, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan

Cytochrome c oxidase (CcO) and ubiquinole oxidase, which catalyze four electron reduction of O2 to water, have a unique heme (heme a3)/non-heme copper (CuB) binuclear core in their active sites. Though the role of the Cu ion in the enzymes is interesting, the proposed reaction mechanisms remain controversial. We fi rst reported tetraarylporphyrin-FeII-linked tris(2-pyridylmethyl)amine (TPA)-CuI binuclear complex forms peroxo-bridged binuclear II III species Cu -(O2)-Fe in acetonitrile with a moderate stability at room temperature. Peroxo- II III bridged binuclear species Cu -(O2)-Fe formed and was characterized only by spectroscopic methods in detail, due to the lack of enough thermal stability. We found the introduction of methyl groups on the heme and the ligand for a Cu ion increase the stability of the corresponding peroxy complexes, in spite of the decrease of the O-O bond order. In order to II I maximize the thermal stability, we prepared sterically protected [(TMP)Fe -(5MeTPA)Cu ]BPh4 (1). The oxy form (2) showed characteristic features in UV-vis, ESI-MS, and resonance Raman spectra as a peroxy complex. The obtained peroxy complex showed extremely high thermal stability as expected and its half-life is ca. 20 days at room temperature. This allows us to prepare crystals at -30 °C. We obtained dark-purple crystals, which showed the exactly same spectroscopic features as those in the solution. The magnetic susceptibility and Mössbauer spectrum of 2 supported the oxidation states of the iron and copper are high-spin FeIII and CuII, and the two metal ions are strongly coupled in an antiferromagnetic fashion. Its single crystal is obtained and the detailed 3D structure of the peroxy complex will be discussed in detail (Fig. 1).

Dynamics and structure of Mn(II) binding sites in protein single crystals determined by high fi eld EPR and ENDOR

Daniella Goldfarb, Weizmann Institute of Science, Israel

EPR and electron-nuclear double resonance (ENDOR) experiments carried out at high fi elds offer new opportunities in the investigation of structural and dynamic properties of metal binding sites in proteins and enzymes. Among the many advantages is the high sensitivity for size limited samples, which permits studies of small single crystals. In terms of structure, this is particularly important because it allows the exact determination of protons’ positions in the site, which are usually not detected by X-ray crystallography. This will be fi rst demonstrated on the the Mn(II) site of the saccharide binding protein concanavaline A. In this protein the Mn(II) has a structural role and it is essential for the saccharide binding. Using 1H and 2H W-band (95 GHz) pulsed ENDOR experiments on single crystals the coordinates of the protons of the two water ligands and their involvement in hydrogen bonds were determined. In addition, preliminary ENDOR measurements on single crystals of enolase , aimed at the elucidation of the interaction between the catalytic Mn(II) and an enzyme inhibitor will be presented. The high resolution of the high fi eld Mn(II) EPR spectrum allows to detect and characterize dynamics processes at the metal site. Analysis of the 95 GHz EPR rotation patterns of a single crystal of concanavalin A revealed that while at room temperature there is one type of Mn(II) in the protein, at low temperature two types of Mn(II) are clearly distinguished. Temperature dependence measurements showed the presence of a two state exchange dynamic process which averages the two types at room temperature. This process is attributed to a conformational equilibrium within the Mn(II) binding site which freezes into two conformations at low temperatures. Similar measurements on the enzyme enolase did not detect any dynamics. The differences are interpreted in terms of the fl exibility of the protein structure in the region of the metal binding site. Journal of Inorganic Biochemistry 96 (2003) 37

Mechanistic Implications for the Formation of the Diiron Cluster in Ribonucleotide Reductase from Quantitative EPR Spectroscopy

Mike Hendrich, Carnegie Mellon University, United States Brad Pierce, Carnegie Mellon University, United States Tim Elgren, Hamilton College, United States

The small subunit of E. coli ribonucleotide reductase (R2) is a homodimeric (β2) protein in which each β-peptide contains a diiron cluster. In this work, the paramagnetic Mn(II) ion is used as a spectroscopic probe to characterize the assembly of the R2 site with EPR spectroscopy. Upon titration of Mn(II) into samples of apoR2, we have been able to quantitatively follow three species (aquaMn(II), mononuclear Mn(II)R2, and dinuclear Mn2(II)R2) and fi t each to a sequential two binding site model. The binding affi nities for the fi rst and second Mn are: K1 = 5.5x105 M-1, K2 = 3.9x104 M-1, which are assigned to the B and A sites, respectively. In multiple titrations, only one dinuclear Mn2(II)R2 site was created per homodimer of R2, indicating that only one of the two β-peptides of R2 is capable of binding Mn(II) following addition of Mn(II) to apoR2. Under anaerobic conditions, addition of only 2 equivalents of Fe(II) to R2 completely prevented the formation of any bound

MnR2 species. Upon reaction of this sample with O2 in the presence of Mn(II), both radical Y122 and Mn2(II)R2 were produced in equal amounts. Based on these observations, we propose a model for R2 metal incorporation that invokes an allosteric interaction between the two β-peptides of R2. Upon binding the fi rst equivalent of metal to a β-peptide (βI), the aforementioned protein conformational change prevents metal binding in the adjacent β-peptide (βII) approximately

25 A away. Furthermore, we show that metal incorporation into βII occurs only during the O2 activation chemistry of the βI-peptide. This is the fi rst direct evidence of an allosteric interaction between the two β-peptides of R2. This model for R2 metallation can explain the generally observed low Fe occupancy of R2.

Structural Insights in Bioinorganic Chemistry from X-ray absorption Spectroscopy.

Graham N George, Stanford Synchrotron Radiation Laboratory, United States Ingrid J Pickering, Stanford Synchrotron Radiation Laboratory, United States Hugh H Harris, Stanford Synchrotron Radiation Laboratory, United States Eileen Y Yu, Stanford Synchrotron Radiation Laboratory, United States

One of the fundamental goals of bioinorganic chemistry is an understanding of the structure-function relationship of metal sites in biological systems. X-ray absorption spectroscopy is a technique that is capable of providing accurate structural details under a variety of conditions. The strengths and limitations of the technique will be discussed, and illustrated with recent examples from the authorís laboratory. Comparisons of results from protein crystallography and X-ray absorption spectroscopy will be made, and the utility of combining computational chemistry and X-ray absorption spectroscopy will be discussed. 38 Journal of Inorganic Biochemistry 96 (2003)

Quantum Chemical Modelling of the Spectroscopic Properties of Metalloprotein Active Sites

Frank Neese, Max Planck Institute for Radiation Chemistry, Germany

Transition metal ions play a key role in the active sites of metalloproteins and effi ciently catalyse a wide variety of chemically complicated reactions which often involves the activation of inert small molecules. Most importantly, the geometric and electronic structures of the active sites and their reactivities are being probed by a wide variety of spectroscopic methods. Since many of the transition metal ions occurring in proteins are paramagnetic they are amenable to study by high-level EPR techniques as well as other magnetic techniques such as Moessbauer and MCD spectroscopy. Our research has been focused on developing quantum chemical methods to predict these spectra from fi rst principles. Recently, methods were developed for the predictions of all parameters occurring in EPR spectroscopy as well as several quantities that are accessible by optical spectroscopy. This lecture will deal with the theoretical techniques themselves and how they are combined with experimental methods in order to obtain insight into the structures and reactivities of metalloprotein active sites.

Studies of Electron Transfer between Metal Complexes and DNA by the DFT method and Photochemistry

Liangnian Ji, School of Chemistry and Chemical Engineering, Zhangshan University, Guangzhou, China Kangcheng Zheng, School of Chemistry and Chemical Engineering, Zhangshan University, Guangzhou, China

n+ In order to probe trends in electron transfer between metal complexes binding to DNA, a series of complexes [M(phen)3] n+ n+ (M = Os(II), Ru(II), Co(III), Zn(II))), [M(phen)2L] and [M(bpy)2L] (M = Ru(II), Co(III); L = ip, pdphen, tatp, 2R-tatp, pip, hpipz) were designed, synthesised and studied by spectroscopy, photochemistry and the DFT method. 2+ 3+ The energy difference between the HOMO of [Ru(phen)3] and the LUMO of [Co(phen)3] may be regarded as a rough microcosmic driving force for electron transfer between both metal complexes binding to DNA. Similar trends were found for the other complexes. A series of luminescence quenching experiments among these complexes in DNA media were designed and performed. These confi rmed the notion of a microcosmic driving force for electron transfer between two complexes bound to DNA. The relation between the electron transfer trends of complexes in DNA and the binding mode was also studied in detail. 2+ Trends in DNA-binding and some spectral properties of the complexes [M(phen)2] can be explained theoretically using the DFT method. Acknowledgements. The fi nancial support of National Natural Science Foundation of China and the Natural Science Foundation of Guangdong Province are gratefully acknowledged. Journal of Inorganic Biochemistry 96 (2003) 39

Quantum refi nement – a method to determine protonation and oxidation states of metal sites in protein crystal structures

Ulf Ryde, Lund University, Sweden Kristina Nilsson, Lund University, Sweden

We have developed a method to combine crystallographic refi nement of protein structures with quantum chemical calculations [1]. In essence, we replace the molecular mechanics potential used in protein refi nement by density functional calculations for a small part of the protein, e.g. the active site. For a metal site, this can lead to an improvement of the structure, because no accurate force fi elds exist for metals. We have shown that we bring the geometry of the haem group in a medium-resolution structure of cytochrome b553 closer to that of a atomic-resolution structure of the same protein [2]. Thus, we may locally improve low- and medium-resolution structures. Moreover, we can also interpret crystal structures. In particular, we can deduce the oxidation states of metals and the protonation state of their ligands. This is done by refi ning the structure using various protonation or oxidation states in the quantum chemical calculation. The best interpretation is found by comparing how well the various refi ned structures fi t the electron density in terms of the Rfree factor or electron-density maps and how much the quantum system is distorted in the crystal [2,3]. We have shown that we can reproduce the experimental protonation state of the zinc-bound solvent molecule in alcohol dehydrogenase [3]. We have also applied the method to determine the protonation state of the iron-bound water in superoxide dismutase and to determine the protonation and oxidation states of compound II in myoglobin and of several states in Ni-Fe hydrogenase [3 and work in progress]. 1 U. Ryde, L. Olsen & K. Nilsson (2002), J. Comp. Chem., 23, 1058-1070. 2 U. Ryde & K. Nilsson (2003) J. Mol. Struct., in press. 3 K. Nilsson & U. Ryde (2003), Nature, Struct. Biol., submitted.

New Designs for Metallodrugs : Targeting DNA and Proteins

Peter J Sadler, University of Edinburgh, United Kingdom

We have designed Pt(IV) complexes which are unreactive towards G bases on DNA until photoactivated by visible light [1], a potential basis for photoactivated chemotherapy. Certain ruthenium(II) arene complexes show promising anticancer activity [2] and through variations in the arene and the other ligands it is possible to tune both the kinetics and thermodynamics of targeting of DNA bases [3,4]. Zinc recruitment can be used to enhance the activity of organic drugs. The major transport protein for Zn(II) in blood is albumin [5]. Carboxylates induce time-dependent changes in the structure of the Zn(II) complex of the potent antiHIV drug xylylbicyclam and specifi c confi gurations of Zn2-xylylbicyclam can be recognised by the CXCR4 co-receptor for HIV [6]. Our interest in the design of novel metalloantibiotics which might block iron uptake by bacterial ferric-ion binding proteins (bacterial transferrins) has led to the characterization of transferrins with metal clusters bound in the interdomain cleft [7]. These fi ndings provide new insights into protein-mineral interfaces and the mechanisms of the metal uptake by this transport protein. References [1] P. Müller, et al. Angew. Chem. Int. Ed. 2003, 42, 335-339. [2] R.E. Aird, et al. Br. J. Cancer 2002, 86, 1652-1657. [3] H. Chen, et al. J. Am. Chem. Soc. 2002, 124, 3064-3082. [4] H. Chen, et al. Proc. Natl. Acad. Sci. USA, 2003, in press. [6] X. Liang, et al. J. Am. Chem. Soc. 2002, 124, 9105-9112. [7] D. Alexeev, et al. Nat. Struct. Biol. 2003, 10, 297-302. We thank The Wellcome Trust, EC, ETF, EPSRC, BBSRC, Royal Society, Wolfson Foundation and Delta Biotechnology for support. 40 Journal of Inorganic Biochemistry 96 (2003)

Metal Carbonyls – A New Class of Pharmaceuticals?

Brian E Mann, University of Sheffi eld, United Kingdom Tony R Johnson, University of Sheffi eld, United Kingdom James E Clark, Northwick Park Institute of Medical Research, United Kingdom Roberta Foresti, Northwick Park Institute of Medical Research, United Kingdom Colin Green, Northwick Park Institute of Medical Research, United Kingdom Roberto Motterlini, Northwick Park Institute of Medical Research, United Kingdom

Carbon monoxide, a notoriously dangerous gas, is produced naturally by the human body and has recently been shown to possess important biological functions. In a similar fashion to nitric oxide, CO has been identifi ed as a signalling molecule in mammals. Furthermore, CO gas has been shown to be biologically benefi cial as it elicits vasodilatation, suppresses organ graft rejection and reduces the damaging effects caused by ischemia. Metal carbonyl compounds, a long established area of chemistry, could be used as CO carriers and potentially be developed as pharmaceuticals for the therapeutic delivery of CO in humans. This presentation largely focuses on the chemistry of compounds belonging to the class of [Ru(CO)3Cl(amino acidate)], organometallic complexes of low toxicity. The amino acids used are enantiomerically pure; however, the fac- arrangement of the ligands results in metal chirality. Preliminary results show the glycinate complex to be a highly effective CO carrier functioning as a solid form of CO that can be rapidly released in biological systems. Mechanistic studies have therefore concentrated on this species. Compounds of this type have proved to exhibit rich aqueous chemistry. Ligands are easily replaced resulting in CO loss, whilst the CO ligands are readily attacked and undergo Water-Gas Shift reactions (see below). NMR studies have identifi ed a number of compounds that are formed, depending on pH and temperature. Tony R. Johnson, Brian E. Mann, James E. Clark, Roberta Foresti, Colin Green and Roberto Motterlini, Angew. Chem., Int. Ed. Engl., in press.

Interaction of the Antitumour Metallocenes with Cellular Components

Margaret M Harding, School of Chemistry, University of Sydney, Australia Jenny B Waern, School of Chemistry, University of Sydney, Australia Carolyn T Dillon, School of Chemistry, University of Sydney, Australia

The antitumour activity of titanocene dichloride has attracted signifi cant interest as the fi rst non-platinum metal complex to enter clinical trials [1]. The poor solubility and stability of the drug at physiological pH are drawbacks in the development of a suitable formulation for administration and in the design of titanocene based drugs with improved activity. Current evidence suggests that titanocene dichloride forms a Ti(IV) active species that is stabilized and/or transported into cells under physiological conditions and that interaction with DNA is directly related to anticancer activity.

In contrast to titanocene dichloride, the structurally related metallocenes (Cp2MCl2 M = V, Mo, Nb, Re) have been much less studied. The vastly different chemical stabilities of these complexes at physiological pH and different coordination chemistries of each complex point to signifi cantly different mechanisms of antitumour action for each drug [1]. There are no structure-activity data on these compounds, and with the exception of Cp2VCl2, the cellular distribution of the compounds is unknown. Our recent studies have focused on establishing the mechanism of action of the less-well studied metallocenes. Systematic studies of the interaction(s) with cellular components including human serum albumin, glutathione [2], amino acids and blood plasma have been performed. These studies are a precursor to using micro-SRIXE (Synchroton Radiation Induced X-Ray Emission) to visualize the intracellular locations of the metals upon treatment of mammalian cells [3], and hence to increase the understanding of the antitumour mechanism of action of these compounds. [1] Harding, M. M.; Mokdsi, G. Current Medicinal Chemistry, 2000, 7, 1289-1303. [2] Mokdsi, G; Harding, M. M. J. Inorg. Biochem. 2001, 86, 611-616. [3] Dillon, C. T.; Lay, P. A.; Kennedy, B. J.; Stampfl , A. P. J.; Cai, Z.; Ilinski, P.; Rodrigues, W.; Legnini, D. G.; Lai, B.; Maser, J. J. Biol. Inorg. Chem. 2002, 7, 640-645. Journal of Inorganic Biochemistry 96 (2003) 41

The chemical biology of NO. Insights into Potential Therapeutic Strategies of NO.

David A Wink, National Insitutes of Health, United States Micheal G Espey, National Insitutes of Health, United States Douglas D Thomas, National Insitutes of Health, United States Katrina M Miranda, University of Arizona, United States Nazareno Paolocci, Johns Hopkins Medical Institutes, United States

Since the discovery of NO as production by biological system, the relevant chemistry under a variety of homeostatic as well as pathophysiological mechanisms has been discussed. The chemistry of NO is the most important determinant of its effect on biological systems and could be important in the treatment of different conditions and diseases. The chemical biology of NO is divided into to two distinct categories, direct and indirect. The direct reaction are those where NO binds to the biological target, which is primarily heme complexes. Conversely, indirect effects are those that are derived from chemistry of NO - with molecules such as oxygen and superoxide generating reactive nitrogen oxide species such as N2O3, HNO and ONOO . These species can be further subdivided into oxidative and nitrosative stress. Nitrosative stress is mediated by reaction of - N2O3 while oxidative stress is mediated ONOO and HNO. This paper will discuss different potential reactions in context of there formation from NOS as well as potential use of NO donor and other redox complexes and what we could learn about some in vivo conditions and therapeutic strategies. By far and away the most predominant reaction in vivo is NO with metal complexes in particular heme proteins. However, the interplay of reactive nitrogen oxide and reactive oxygen species can have important consequences. The talk will concentrate on three basic aspects of NO, NO interaction with reactive oxygen species; nitrosative stress and fi nally chemical and biological HNO. These chemistry will be individually discussed in context of not only the chemical reaction but in terms of a ischemia reperfusion models as well as effects on, on treatment of heart disease and cancer.

Novel cytotoxic trans-diaminedichloro platinum(II) complexes with nonplanar heterocyclic ligands, preparation, cytotoxicity and DNA Binding properties

Dan Gibson, The Hebrew University, Jerusalem, Israel Yousef Najajreh, The Hebrew University, Jerusalem, Israel Jana Kasparkova, Institute of Biophysics, Academy of Sciences of the Czech Republic, Czech Republic Viktor Brabec, Institute of Biophysics, Academy of Sciences of the Czech Republic, Czech Republic Jose-Manuel Perez, Departamento de Química Inorgánica. Facultad de Ciencia, Spain Carmen Navarro-Ranniger, Departamento de Química Inorgánica. Facultad de Ciencia, Spain

Positively-charged, water soluble cis/trans-[PtCl2(piperazine)(Am1)] (where Am1 = NH3, n-butylamine, isopropylamine, 4- picoline, piperidine, and piperazine) and their neutral piperidine analogs, have signifi cant cytotoxic activity against cisplatin resistant ovarian cancer cells. The charged complexes are taken up by cancer cells much more rapidly than cisplatin and bind to cellular DNA and to Calf Thymus DNA much faster than cisplatin or transplatin. The results show that the replacement of the NH3 group in cisplatin by the heterocyclic ligands does not considerably affect DNA binding mode of this compound. The work correlates DNA binding mode in a cell-free medium of the bifunctional analogues of cisplatin and transplatin containing one piperidine, piperazine or 4 picoline ligand with their activity in several tumor cell lines. The results offer a strong experimental support for the view that one strategy to activate trans geometry in bifunctional platinum(II) compounds and to circumvent resistance to cisplatin consists in a chemical modifi cation of the ‘classical’ transplatin which would result in an increased stability of the intrastrand CLs of these trans-platinum compounds in double-helical DNA and/or in their increased effi ciency to form interstrand CLs. The present work also suggests that such a modifi cation may also consist in the replacement of one ammine group by non-planar heterocyclic ligand, such as piperidine and piperazine or aromatic planar heterocyclic ligand, such as 4-picoline. The platinum-piperazine complexes bind proteins (Ubiquitin and Myoglobin) very slowly compared to cisplatin and to their neutral piperidine analogs.

Altogether, the results reported here suggest that combination of positively charged ligands with a trans-Pt(II)Cl2 center may lead to the discovery of platinum complexes that are able to circumvent cisplatin resistance. 42 Journal of Inorganic Biochemistry 96 (2003)

Mechanism of Reaction of Platinum-Based Anticancer Drugs with Sulfur- Containing Biomolecules and Novel Drug Design

Zijian Guo, Nanjing University, China

Sulfur-containing biomolecules such as methionine and glutathione are believed to play important roles in the metabolism and mechanism of action of platinum based anticancer drugs. As one of the key metabolites, [Pt(Met-N,S)2] has been isolated from the urine of patients treated with cisplatin, cis-[Pt(NH3)2Cl2]. We have shown recently by ESMS and NMR that the major products for the reaction of cisplatin and carboplatin with selenomethionine and methionine are [Pt(Se-Met-Se,N)2] and [Pt(Met-N,S)2] [1,2]. Although these metabolites has been proved to be inactive towards biological targets such as DNA due to the inertness and stability of the S,N-chelated L-methionine, we show that glutathione can readily replace the L-MetH from [Pt(L-Met-S,N)2], and forms the S-bridged polynuclear (upto pentanuclear) adducts. The cis-[Pt(L-Met-S,N)2] isomer is more reactive than the trans isomer [3]. Most of the structure-activity rules emerged from the initial studies by Rosenberg et al has recently been questioned. Many Pt (II) complexes which are trans in geometry, or charged, or having only one leaving group have been found active. Here we report the design of several new types of platinum-based anticancer complexes, which will include: a) chelate and ring-opening Pt(II) antitumor complexes [4]; b) highly cytotoxic monofunctional Pt(II) complexes; c) polynuclear Pt(II) complexes with robust structural features. [1] Q. Liu, J.Y. Zhang, X.K. Ke, Y.H. Mei, L.G. Zhu and Z.J. Guo*, J. Chem. Soc., Dalton Trans., 2001, 911. [2] Q. Liu, J. Lin, P.J. Jiang, J.Y. Zhang, L.G. Zhu, Z.J. Guo*, Eur. J. Inorg. Chem., 2002, 2170. [3] Q. Liu, J. Lin, P.J. Jiang, H.Y. Wei, L.G. Zhu, Z.J. Guo*, Submitted. [4] J.Y. Zhang, Q. Liu, C.Y. Duan, Y. Shao, J. Ding, Z.H. Miao, X.-Z. You and Z.J. Guo*, J. Chem. Soc., Dalton Trans., 2002, 591.

X-ray Absorption Studies of Copper Chaperones

Ninian J Blackburn, Oregon Health & Sciences University, United States Martina Ralle, Oregon Health & Sciences University, United States Svetlana Lutsenko, Oregon Health & Sciences University, United States Jack Kaplan, Oregon Health & Sciences University, United States John Eisses, Oregon Health & Sciences University, United States Jay Stasser, Oregon Health & Sciences University, United States

The copper chaperone HAH1 transports Cu to Menkes (MNK) and Wilson (WND), believed to provide Cu for important enzymes such as Cp, PAM and DBM. Although structural data exists for HAH1 and its homologues, the Cu coordination remain elusive, and suggestive of the presence of two protein cysteine ligands and a third exogenous thiol ligand. Here we report the reconstitution of HAH1 with Cu(I) using a protocol which minimizes the use of thiol reagents believed to be the source of the third ligand. We show by XAS that this protocol generates a Cu(I) site with linear bis cysteinate coordination, as evidenced by (a) an intense edge absorption at 8982.5 eV, and (b) an EXAFS spectrum which fi ts to 2 Cu-S at 2.16 angstroms, typical of digonal Cu(I). When exogenous ligands were titrated into Cu(I)-HAH1, all formed adducts, but with differing affi nities. Thus GSH and DTT showed KD values of 10 – 25 mM, while TCEP showed stronger affi nity with KD 5 mM. When the N-terminal domain of WND was reconstituted with HAH1, a 2-coordinate Cu(I) species was obtained but with a much lower intensity of the 8983 eV peak suggestive of distorted digonal geometry. On the other hand individual domains 2 and 3 of WND gave spectra identical to HAH1 with linear 2-coordinate geometry. Hence, structural elements of the folded WND distort the Cu(I) binding geometry and expose a potential open position on Cu(I). These data support the mechanism in which chaperone docks with its target to form a transient 3-coordinate intermediate involving shared cys residues from each protein. CCS is the chaperone for the Cu-Zn SOD1. Early XAS studies from our laboratory had described the Cu(I) binding as a bis-cysteinate-bridged dinuclear cluster formed between domains I and III of the same monomer. Recent mutagenesis experiments have now shown that the cluster must form between two domain III CXC motifs within a protein dimer and suggest that the current heterodimer model for CCS-SOD interaction may be oversimplifi ed. Journal of Inorganic Biochemistry 96 (2003) 43

Beta-amyloid cuproprotein: a therapeutic target for alzheimer’s disease

Ashley I Bush, University of Melbourne, Australia, and Massachusetts General Hospital, United States

A-Beta is the principal component of the plaque pathology which is the hallmark of Alzheimer’s disease (AD). It is a ubiquitous metalloprotein, with selective high-affi nity binding sites for zinc (Ka = 100 nM) and copper (Ka = 10 attoM), which resembles those of SOD1, and a lower affi nity Fe binding site. Abnormal A-Beta metabolism is related to the cause of AD, and our model proposes that marked elevations of brain Cu and Fe, that occur as an inevitable consequence of aging, are responsible for corrupting this protein. The plaque pathology of Alzheimer’s disease is formed by the reaction of A-Beta with Zn, Cu and Fe. The A-Beta:Cu complex is highly redox active, with a strong reducing potential (+550 mV vs

Ag/AgCl), similar to those of the blue copper proteins like laccase. A-Beta:Cu complexes form H2O2 catalytically from O2, with biological reducing agents such as dopamine and cholesterol as electron donors (Km = 5 µM, Vmax = 30 nM/min). This is the source of the toxicity of the peptide, which is fostered by Cu and these reducing agents. The toxicity of A-Beta species is proportional to the peptide’s ability to reduce Cu and generate H2O2 (A-Beta42> A-Beta40 >rat A-Beta). Chelators of copper both disaggreagate A-Beta deposits from post-mortem brain tissue and also shut down H2O2 production. One such chelator, clioquinol, markedly inhibits brain amyloid pathology in the APP2576 mouse model for AD. This compound is a retired antibiotic and has been tested in a Phase 2 clinical trial of moderately demented patients, where its therapeutic effi cacy was accompanied by a decrease in plasma A-Beta42.

Misfolded Superoxide Dismutase and ALS

P John Hart, University of Texas Health Science Center San Antonio, United States Jennifer Stine Elam, University of Texas Health Science Center San Antonio, United States Alexander B Taylor, University of Texas Health Science Center San Antonio, United States Richard Strange, CLRC Daresbury, United Kingdom S Samar Hasnain, CLRC Daresbury, United Kingdom Peter A Doucette, University of California, Los Angeles, United States Joan S Valentine, University of California, Los Angeles, United States Lawrence J Hayward, University of Massachusetts Medical School, United States

Mutations in copper-zinc superoxide dismutase (SOD1) cause the autosomal dominant, neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). In spinal cord neurons of human patients and in transgenic mice expressing these proteins, high molecular weight, insoluble protein complexes (IPCs) containing FALS SOD1 are observed. Although the molecular basis for FALS has remained obscure, accumulation of SOD1 IPCs is believed to interfere with axonal transport, protein degradation, and anti-apoptotic functions of the neuronal cellular machinery. Here, we show that metal-defi cient, pathogenic SOD1 mutants S134N and H46R crystallize in three different crystal forms, all of which are characterized by higher order assemblies of aligned β-sheets. Linear, amyloid-like fi laments and helical, water-fi lled nanotubes arise through extensive interactions between loop and β-barrel elements of neighboring SOD1 molecules. In all cases, non-native conformational changes permit the gain-of-interaction between dimers that leads to formation of higher order arrays. Normal β-sheet-containing proteins avoid such self-association by preventing their edge strands from making intermolecular interactions, often by covering them with loop elements. We suggest that loss of this protection through conformational rearrangement in the metal-defi cient enzyme is a toxic property that may be common to mutants of SOD1 linked to FALS. 44 Journal of Inorganic Biochemistry 96 (2003)

Metallochaperone Protein, CCS, Has Sulfhydryl Oxidase Activity: Post-translational Modifi cation of Superoxide Dismutase

Yoshiaki Furukawa, Department of Chemistry, Northwestern University, United States Nina M Brown, Department of Chemistry, Northwestern University, United States Thomas V O’Halloran, Department of Chemistry, Northwestern University, United States

Copper is an essential cofactor in several biochemical processes such as electron transfer and oxygen-utilization. We have proposed that cells operate on a copper budget that does not involve the intermediacy of a pool of free copper ion in the cytosol (1). Recent calibration of CueR, a copper sensing metalloregulatory protein in E.coli, corroborates this model. One of the better understood metallochaperone proteins is the yeast Atx1 protein, which specifi cally donates a Cu(I) ion to its physiological partner, Ccc2. Between the metal-binding sites of these two proteins, copper ion rapidly partitions via formation and decay of two- and three-coordinate Cu(I)-thiolate intermediates (2). We have recently found that another copper chaperone, CCS, plays a more complex role in the cytoplasm; it acts to modulate the amount of the copper loading into its target, superoxide dismutase (SOD1), in response to changes in oxygen tension. In the absence of O2, there is no transfer of copper from CCS to SOD1. Upon exposure to O2, Cu-CCS exhibits both sulfhydryl oxidase and protein disulfi de isomerase activities with SOD1 as the substrate. We are testing the proposal that oxidation of Cu(I) or its thiol ligand in Cu-CCS triggers release of copper from CCS to SOD1, followed by the formation of the disulfi de in SOD1. In light of these fi ndings, we propose a dual role of CCS in the activation of SOD1: the ‘copper insertion’ and the ‘catalysis of disulfi de formation’. In clinical point of view, furthermore, point mutations in SOD1 can cause the amyotrophic lateral sclerosis (ALS), which is a neurodegenerative disease, but the exact role of SOD1 in ALS has yet to be established. On the basis of the disulfi de formation in SOD1 by CCS, we will also discuss the regulation mechanism of ALS, which might be induced by perturbation of the stability of the SOD1 disulfi de bond. (1) Rae, T.D. et al. Science 1999 284 805 (2) Huffman, D.L. et al. Annu.Rev.Biochem. 2001 70 677

Proteins involved in the Metallation of Copper Centers in Cytochrome c Oxidase

Dennis R Winge, University of Utah, United States Andrew Maxfi eld, University of Utah, United States Heather Carr, University of Utah, United States Paul Cobine, University of Utah, United States Keith McCall, University of Utah, United States

Assembly of cytochrome c oxidase in the inner mitochondrial membrane requires a number of accessory proteins. The delivery and insertion of copper ions in cytochrome c oxidase appear to require at least four proteins, Cox17, Cox19, Sco1 and Cox11. Cox17 is a Cu(I) binding putative metallochaperone for delivery of copper ions to the mitochondrion. Cox19, like Cox17, is a Cu(I) binding protein found in both the cytosol and the mitochondrial intermembrane space (IMS). Cox17 is functional if predominantly localized within the IMS either as a soluble protein or tethered to the inner membrane. The Cu(I) binding function of Cox17 occurs within a central Cys-rich portion of the molecule, whereas the C-terminal segment of Cox17 is essential for mitochondrial uptake and a secondary function within the IMS. Insertion of Cu ions within the two mitochondrially encoded subunits Cox1 and Cox2 appears to involve co-metallochaperones Sco1 and Cox11 for metallation of CuA and CuB sites, respectively. Sco1 and Cox11 are inner membrane proteins tethered by a single transmembrane (TM) helix. Both proteins bind a single Cu(I) ion per subunit. The function of both proteins is dependent on its own TM segment and their Cu(I) binding residues. Mutation of the Cu(I) ligating residues abrogates function in all three proteins. This correlation is consistent with Cox17, Sco1 and Cox11 functioning in the Cu ion metallation of cytochrome c oxidase. Two cysteinyl residues present as a CxxxC motif in Sco1 and a distant His form a Cu(I) binding site, but this complex can convert to a Cu(II) complex with optical transitions similar to the red nitrosocyanin complex. In contrast, the dimeric Cox11 forms a stable Cu(I)-thiolate site with the Cu(I) ion in each monomer bridged in a binuclear cluster. Journal of Inorganic Biochemistry 96 (2003) 45

Nickel traffi cking and urease: recent developments

Stefano Ciurli, University of Bologna, Italy

Urease catalyzes the last step of nitrogen mineralization, the hydrolysis of urea to ammonia and carbamate. The active site of urease contains two Ni(II) ions. The recent developments of our studies of the catalytic mechanism based on crystal structures of Bacillus pasteurii urease complexed with inhibitors, transition state analogues and substrate analogues will be illustrated (1). The in vivo assembly of B. pasteurii urease active site requires four accessory proteins, UreE, UreF, UreG, and UreD. Our recent discoveries on the structural biochemistry of these metallochaperones (2) will be described and discussed. (1) (a) Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Ciurli, S.; Mangani, S. JBIC., 1998, 3, 268-273. (b) Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Miletti, S.; Ciurli, S.; Mangani, S. Structure. Fold Des., 1999, 7, 205-216. (c) Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Miletti, S.; Ciurli, S.; Mangani, S. JBIC., 2000, 5, 110-118. (d) Musiani, F.; Arnofi , E..; Casadio, R.; Ciurli JBIC., 2001, 6, 300-314. (e) Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Ciurli, S.; Mangani, S. JBIC., 2001, 6, 778-790. (f) Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Ciurli, S.; Mangani, S. Submitted. (2) (a) Remaut, H.; Safarov, N.; Ciurli, S.; Van Beeumen J. J. Biol. Chem. 2001, 276, 49365-49370. (b) Ciurli, S.; Safarov, N.; Miletti, S.; Dikiy, A.; Christensen, S. K.; Kornetzky, K.; Bryant, D. A.; Vandenberghe, I.; Devreese, B.; Samyn, B.; Remaut, H.; Van Beeumen, J. JBIC 2002, 7, 623-631. (c) Musiani, F.; Zambelli, B.; Stola, M.; Ciurli, S. Submitted; (d) Musiani, F.; Mangani, S.; Safarov, N.; Ciurli, S. Submitted; (e) Musiani, F.; Ciurli, S. Submitted.

[NiFe] hydrogenases under aerobic conditions

Bäerbel Friedrich, Humboldt-Universitaet zu Berlin, Institute of Biology, Germany Oliver Lenz, Humboldt-Universitaet zu Berlin, Institute of Biology, Germany Tanja Burgdorf, Humboldt-Universitaet zu Berlin, Institute of Biology, Germany Thorsten Buhrke, Humboldt-Universitaet zu Berlin, Institute of Biology, Germany

[NiFe] hydrogenases, enzymes capable of hydrogen oxidation, contain an unusual bimetallic active site consisting of a nickel and an iron atom. One CO and two CN ligands are bound to the iron. This metal site is generally reversibly inactivated under aerobic conditions and is energetically expensive to synthesize, requiring the aid of at least six accessory (Hyp) proteins [1,2]. The beta proteobacterium Ralstonia eutropha utilizes three [NiFe] hydrogenases to grow chemolithoautotrophically using CO2 as carbon source and H2 as the energy source in the presence of oxygen. An H2-sensing hydrogenase (RH) is used to control expression of the hydrogenase genes [3]. Signal transduction is believed to involve a redox active non-metal cofactor located in the H2 sensor and gene transcription is fi nally achieved by a two-component system. The RH is not directly involved in energy generation. However, R. eutropha synthesizes two additional [NiFe] hydrogenases for energy generation: a membrane-bound heterodimer (MBH) which is exposed to the periplasm and a bidirectional soluble hydrogenase (SH) which is a heterotetrameric NAD-reducing enzyme consisting of a hydrogenase and an NADH oxidoreductase module [4,5]. Spectroscopic analysis has shown that the SH contains a modifi ed form of the standard [NiFe] active site, namely two additional cyanide ligands, one at the iron and one at the nickel. This modifi ed active site architecture may account for the oxygen tolerance of the SH. Furthermore, in addition to the six Hyp proteins required for the standard [NiFe] active site synthesis, another Hyp protein, HypX, is required for MBH and SH function under aerobic conditions [6]. Evidence is accruing that HypX is involved in the addition of the extra cyanide to the Ni of the SH active site. [1] R. Cammack, R. Robson, and M. Frey (ed.). 2001. Hydrogen as a fuel: Learning from nature, Taylor & Francis, London, U. K. [2] Blokesch et al. 2002. Biochem. Soc Trans. 30:674-80. [3] Lenz et al. 2002. J. Mol. Microbiol. Biotechnol. 4:255-62. [4] Bernhard et al. 1996. J. Bacteriol. 178:4522-9. [5] Happe et al. 2000. FEBS Lett. 466:259-63. [6] Buhrke and Friedrich. 1998. Arch. Microbiol. 170:460-3. 46 Journal of Inorganic Biochemistry 96 (2003)

NiSOD: A New Mechanism for Removing Superoxide, or a Strep. Tease?

Michael J Maroney, University of Massachusetts, United States Peter A Bryngelson, University of Massachusetts, United States Jennifer L Pinkham, University of Massachusetts, United States Patrick R DeCourcy, University of Massachusetts, United States Sumonu E Arobo, University of Massachusetts, United States

Ni-containing superoxide dismutases (NiSODs) are found in Streptomyces species and represent a novel approach to the elimination of superoxide in biological systems. SODs catalyze the conversion of the superoxide to hydrogen peroxide and dioxygen, and are involved in the protection of cells from oxidative damage caused by superoxide or reactive oxygen species generated therefrom. Several SODs have been characterized from a variety of sources, and may be classifi ed by their metal content as CuZnSOD, MnSOD, or FeSOD. Whereas aqueous solutions of Cu2+, Mn2+, and Fe2+ ions are capable of catalyzing the dismutation of superoxide, solutions of Ni2+ are not. Nonetheless, kinetic studies using pulse-radiolytic generation of superoxide show that NiSOD catalyzes the reaction at a rate near the diffusion limit. We report the successful expression in E. coli and reconstitution to full activity of S. coelicolor NiSOD. NiSOD is expressed in E. coli as an apo-monomeric protein that requires precise processing of the N-terminus before Ni incorporation leads to active NiSOD. A fusion protein containing a protease cleavage site was used to generate NiSOD with the correctly processed N-terminus. Following reduction of a disulfi de formed between two Cys residues that are critical for Ni binding, incorporation of Ni leads to the formation of hexameric enzyme that is fully active and spontaneously produces the rhombic EPR signal typical of resting, oxidized NiSOD. XAS studies have established that the Ni sites in the hexamer feature 3 S-donor ligands and 1-2 O- or N-donor ligands. Only three S-donors are present (Cys2, Cys6, and Met28) in the monomer. Mutation of Met28 to Leu produces an enzyme with full activity that exhibits the WT EPR spectrum. Thus, Met28 is not a Ni ligand. This result, plus the role of Ni in assembling a hexameric enzyme point to a dinuclear Ni site formed at subunit interfaces. The role of additional residues in forming the active site will be discussed using results from site-directed mutagenesis.

Unique Nickel Sites in Proteins

Catherine L Drennan, Massachusetts Institute of Technology, United States Eric Schreiter, Massachusetts Institute of Technology, United States Tzanko I Doukov, Massachusetts Institute of Technology, United States Peter T Chivers, Washington University, United States Javier Seravalli, University of Nebraska, United States Stephen W Ragsdale, University of Nebraska, United States

Nickel is essential in anaerobic metabolism. For example, it is required for the activity of carbon monoxide dehydrogenases/ acetyl-CoA synthases (CODH/ACSs) in many microbes. These enzymes allow organisms to live on carbon monoxide and carbon dioxide as sources of carbon and energy. X-ray analyses of CODH from Rhodospirillum rubrum and of CODH/ACS from Moorella thermoaceticum have revealed the architecture of Ni sites in the complex metallocenters referred to as the C- and A-clusters. Here we will describe our current understanding of the structure and function of these Ni-containing metalloclusters. We will also describe our crystallographic studies of the Ni regulatory protein NikR. While Ni is essential for enzymes such as CODH/ACS, excess Ni can be toxic to the organism. Crystallographic studies of NikR allow us to examine the structural mechanism by which Ni regulates its own uptake into the cell. Journal of Inorganic Biochemistry 96 (2003) 47

Modelling the Active Site of [NiFe] Hydrogenase: The Electronic Structure of a Charge-Delocalised Radical

Martin Schroder, University of Nottingham, United Kingdom Wang Quiang, University of Nottingham, United Kingdom Andrew C Marr, University of Nottingham, United Kingdom Jonathan McMaster, University of Nottingham, United Kingdom Alexander J Blake, University of Nottingham, United Kingdom Claire Wilson, University of Nottingham, United Kingdom Stephen Davies, University of Nottingham, United Kingdom Eric JL McInnes, University of Manchester, United Kingdom

The publication in 1995 of the crystal structure determination of [NiFe] hydrogenase isolated from Desulfovibrio gigas has generated much interest in the synthesis of low molecular weight thiolate-bridged heteronuclear Ni-Fe complexes that may model the structural, redox and catalytic features of the parent enzyme. We describe the synthesis, structures and redox properties of a series of Ni-Fe complexes that show, for the fi rst time, Ni-Fe distances of ca 2.5 angstroms, very similar to that proposed for the active and catalytic form of the enzyme.

Of particular interest is the complex [(L)Ni(µ-S,S´)Fe2(CO)6] (1) (see Figure) which shows a fully reversible one-electron reduction at E1/2 = -0.77 V vs. SCE. Controlled potential electrolysis demonstrates the reversibility of this redox process and this was confi rmed further by wide-ranging spectroelectrochemical studies using uv/vis, ir and epr spectroscopy. Epr spectroscopic analysis on natural and 61Ni labelled samples shows high electron delocalization within the cluster, with 24% unpaired electron based on the Ni centre and the nickel contribution to the SOMO being overwhelmingly dominated by the dxz orbital contribution. This observation is also consistent with ir spectroscopy which shows a shift to lower frequency in v(CO) for the reduced species (1-). Density functional calculations provide a framework for the interpretation of the spectroscopic properties of (1-) and confi rm a Ni contribution to the SOMO of 24% with 20.3% from Ni dxz orbital and

3.7% from the Ni dyz orbital as revealed by epr spectroscopy. The calculations also suggest that the SOMO is delocalised over the whole NiS4Fe2(CO)6 core of the cluster and that the 1/1- redox process is not localized soly on the NiS4 fragment. The similarity and relevance of the structural, electronic and redox properties of 1 to the active site in [NiFe] hydrogenase will be discussed. 48 Journal of Inorganic Biochemistry 96 (2003)

A biochemical rationale for the orthogonal behavior of nitroxyl (HNO) and nitric oxide (NO) in the cardiovascular system

Katrina M Miranda, University of Arizona, United States Nazareno Paolocci, The Johns Hopkins Medical Institutions, United States Tatsuo Katori, The Johns Hopkins Medical Institutions, United States Douglas D Thomas, National Institutes of Health, United States David A Wink, National Institutes of Health, United States

The redox siblings nitroxyl (HNO) and nitric oxide (NO) have often been assumed to undergo casual redox reactions in biological systems. However, several recent studies have demonstrated distinct pharmacological effects for donors of these two species. Infusion of the HNO donor Angeli’s salt (AS) into normal dogs resulted in elevated plasma levels of calcitonin gene related peptide (CGRP) whereas neither the NO donor diethylamine/NONOate (DEA/NO) nor the nitrovasodilator nitroglycerin (NTG) had an appreciable effect on basal levels. Conversely, plasma cGMP was increased by infusion of DEA/ NO or NTG but was unaffected by AS. These results suggest the existence of two mutually exclusive response pathways that involve stimulated release of disparate signaling agents from HNO and NO. In light of both the observed dichotomy of HNO - and NO and the recent determination that, in contrast to the O2/O2 couple, HNO is a weak reductant, the relative reactivity of HNO with common biomolecules was determined. This analysis suggests that under biological conditions the lifetime of HNO with respect to oxidation to NO, dimerization or reaction with O2 is much longer than previously assumed. Rather, HNO is predicted to undergo addition reactions with thiols and ferric proteins. CGRP release is suggested to occur via altered calcium channel function through binding of HNO to a ferric or thiol site. The orthogonality of HNO and NO may be due to differentially reactivity toward metals and thiols, and in the cardiovascular system, may ultimately be driven by respective alteration of cAMP and cGMP levels.

Metal Ion Binding to Human Hemopexin

A Grant Mauk, University of British Columbia, Canada Marcia R Mauk, University of British Columbia, Canada Bao Lige, University of British Columbia, Canada Geoffrey R Moore, University of East Anglia, United Kingdom

Although purifi cation of human hemopexin (Hx) by metal affi nity chelate chromatography was fi rst reported in 1983 (1), the metal ion binding properties of Hx have apparently not been characterized. We have now initiated a variety of thermodynamic and spectroscopic studies to address this issue. As part of this effort, we have developed an effi cient method for purifying human Hx from cryosupernatant that employs anion exchange chromatography followed by Ni(II) and Zn(II) affi nity chromatography on iminodiacetate-based metal affi nity chelate resin and enables isolation of 30-60 mg of purifi ed Hx from 200 mL of cryosupernatant. Initial potentiometric titrations of apo-Hx and holo-Hx with Ni(II) establish that Ni(II) binds to high-and low-affi nity sites on both forms of the protein, so the binding of metal ions and heme are apparently not mutually exclusive. The 1D 1H-NMR spectrum of the heme-Hx complex exhibits the broad paramagnetically-shifted resonances that are characteristic of Hx from most species (2). As one of the His ligands identifi ed by the crystallographically-determined structure (3) occurs in the fl exible hinge region linking the N- and C-terminal domains of Hx, the fi eld-strength dependence of the line width of these resonances was investigated and found to vary with fi eld strength in a manner consistent with a mobile paramagnetic center. In addition, 1D 1H-NMR spectra of apo-Hx following addition of 1-3 eq of Ni(II) indicated the presence of at least three paramagnetically-shifted resonances not observed in the spectrum of the apo-protein and were consistent with the involvement of three nitrogen-based ligands in coordination of the metal ion. Supported by the Canadian Institutes of Health Research (Grant MOP-53131 (AGM) and a Canadian Blood Services Postdoctoral Fellowship (BL)). (1) Porath & Olin, Biochemistry 22, 1621-30 (1983). (2) Deeb, Muller-Eberhard, & Peyton, Biochim Biophys Acta 1200, 161-6 (1994). (3) Paoli, Anderson, Baker, Morgan, Smith, A. & Baker, Nat. Struct. Biol. 6, 926-31 (1999). Journal of Inorganic Biochemistry 96 (2003) 49

Role of Heme-Propionate Side Chains in Myoglobin Function

Takashi Hayashi, Kyushu University, PRESTO in JST, Japan Takashi Matsuo, Kyushu University, PRESTO in JST, Japan Katsuyoshi Harada, Kyushu University, Japan Yoshio Hisaeda, Kyushu University, Japan Shun Hirota, Kyoto Pharmaceutical University, Japan Noriaki Funasaki, Kyoto Pharmaceutical University, Japan

Myoglobin, an oxygen storage protein, has a protoheme IX as a prosthetic group in the protein matrix where there are unique interactions between the heme and the protein. To discuss why the myoglobin can strongly bind dioxygen, many mutations at the distal and proximal sites have been performed and the obtained mutants demonstrate which amino acid residues are important to stabilize the bound dioxygen. In contrast, less attention is being denoted to the role of two heme-propionate side chains in the stabilization of the bound dioxygen. However, according to the three dimensional structure of myoglobin, each heme propionate rigidly interacts with polar residues. Thus, we started to evaluate the contribution of two heme-propionates to the myoglobin function using ‘chemical mutation’ of the prosthetic group as shown in the following scheme. To understand the role of the propionates, we have prepared two myoglobins rMb(1) and rMb(2) reconstituted with monopropionate-hemin, 1 or 2, where either the heme-6-propionate or heme-7-propionate is replaced by a methyl group, respectively. From the kinetic studies on ligand binding, the following aspects are clarifi ed. The dissociation of the dioxygen for oxy-rMb(1) is clearly accelerated compared to oxy-rMb(2) and native oxymyoglobin. In addition, the autoxidation from oxymyoglobin to metmyoglobin for rMb(1) was 6-fold faster, indicating that oxy-rMb(1) is relatively unstable. These results indicate that the 6-propionate plays an important role on the stabilization of dioxygen. In contrast, Fe-His stretching bands determined by RR spectra for these myoglobins indicate that the band of rMb(2) shifts up by 2 cm -1 compared with that of native myoglobin and rMb(1). This fi nding suggests that the 7-propionate might regulate the strength of Fe-His coordination. In this presentation, we wish to discuss the structure and function of the reconstituted myoglobins to understand the physiological property of myoglobin. 50 Journal of Inorganic Biochemistry 96 (2003)

Nuclear Resonance Vibrational Spectroscopy (NRVS): An Exciting New Technique for the Study of Iron Porphyrinates and Heme Proteins.

W Robert Scheidt, University of Notre Dame, United States Graeme RA Wyllie, University of Notre Dame, United States Mary K Ellison, University of Notre Dame, United States J Timothy Sage, Northeastern University, United States Brakesh K Rai, Purdue University, United States Stephen M Durbin, Purdue University, United States Wolfgang Sturhahn, Argonne National Laboratory, United States E Ercan Alp, Argonne National Laboratory, United States

Nuclear Resonance Vibrational Spectroscopy (NRVS) is a new spectroscopic technique that provides information on all vibrational modes with an iron contribution in an iron-containing molecule. The method is based on features arising from a combination of the recoilless Mössbauer line and vibrational quanta. This allows observation of a range of low-frequency modes hitherto unobserved. Data collection is carried out at the Advanced Photon Source at Argonne National Laboratory. We briefl y describe both the basic theory and experimental details of NRVS. We have collected and analyzed NRVS for a number of model iron porphyrinates with coordination numbers ranging from four to six and incorporating a wide variety of axial ligands. Selected single crystal measurements have confi rmed the identifi cation of bands as arising from in-plane or out-of-plane motion of the iron. Peripheral substituents are found to signifi cantly contribute to many very low-frequency modes. The assignments show that these modes are very mixed. We are examining the effects of peripheral substitution with a series of fi ve-coordinate (nitrosyl)iron(II) porphyrinates. Complete assignments of the modes for deoxy- and carbon monoxy-myoglobin model complexes have also been carried out. The assignment of the FeñIm stretching frequency has been obtained for both compounds; this is the fi rst time this mode has been observed for a six-coordinate heme. We thank the NIH, NSF and DOE for funding this research.

Molecular Mechanism of the Catalase Reaction Studied by Myoglobin Mutants

Yoshihito Watanabe, Nagoya University, Japan Takafumi Ueno, Nagoya University, Japan Shunichi Fukuzumi, Osaka University, Japan Shigeru Kato, The Graduate University for Advanced Studies, Japan

Catalase is a heme enzyme, which catalyzes the disproportionation of hydrogen peroxide to H2O and O2. In the fi rst step, IV +• H2O2 serves as a two-electron oxidant to generate a ferryl porphyrin cation radical (O=Fe Por ) called compound I and

H2O. In the second step, compound I serves as a two-electron oxidant of H2O2 affording O2 (catalatic reaction) accompanied by regeneration of the ferric form of catalase. However, the detailed mechanism of the oxidation of H2O2 by compound I has yet to be clarifi ed. Diffi culty in preparation of catalase compound I by a stoichiometric amount of H2O2 or peracetic acid has precluded direct observation of the reaction of compound I with H2O2. Recently, we have prepared a series of myoglobin (Mb) mutants that afford compound I as observable species. More importantly, the successful observation of Mb-I has allowed us to follow directly the oxidation step of H2O2. Thus, the catalase reaction has been studied in detail by using Mb mutants. Upon the addition of H2O2, Mb-I is reduced back to the ferric state without forming any intermediates. This reveals that Mb-I is capable of performing two-electron oxidation of H2O2 18 16 (catalytic reaction). GC-MS analysis of the evolved O2 from a 50:50 mixture of H2 O2/H2 O2 solution shows two peaks for 18 16 16 18 O2 (m/e = 36) and O2 (m/e = 32) but no indication of O O (m/e = 34) formation. Deuterium isotope effects on rates of the catalytic reaction of Mb mutants suggest that the catalytic reactions of F43H/H64L Mb proceed via an ionic mechanism, since the distal histidine is located at a proper position acting as a general acid-base catalyst in the ionic reaction to give a small isotope effect of less than 2.1. In contrast, other Mb mutants such as H64X (X: A, S, D) and L29H/H64L Mb oxidize

H2O2 via a radical mechanism in which hydrogen is abstracted by the ferryl species with very large isotope effects in a range of 10 to 29, due to the lack of the general acid-base catalyst. Journal of Inorganic Biochemistry 96 (2003) 51

The Reaction Mechanism of Xanthine Oxidase

Russ Hille, The Ohio State University, United States

The molybdenum-containing hydroxylases represent a unique solution to the problem of C-H bond cleavage and substrate hydroxylation as they utilize water rather than O2 as the source of the oxygen atom incorporated into product. Xanthine oxidase has long been the paradigm for this important group of enzymes, and the reaction mechanism of this enzyme has been investigated using a variety of methods. Isotope labeling experiments demonstrate that an equatorial hydroxyl group in the molybdenum coordination sphere is the proximal oxygen atom donor to product, being regenerated by hydroxide from solvent at the completion of each catalytic sequence. The pH dependence of the enzyme kinetics with xanthine as substrate exhibits a bell-shaped curve, consistent with enzyme acting on neutral substrate (pKa 7.4) via a mechanism involving nucleophilic attack assisted by an active site base (pKa 6.6) tentatively assigned as Glu 1261. Computational studies indicate that neutral substrate tautomerizes in the course of the reaction, resulting in a 24 kcal/mol reduction in the energy of activation. This tautomerization is possibly facilitated by Glu 802. Further computational work identifi es a transition state with signifi cant negative charge accumulation on the proton being transferred from substrate to the molybdenum center in the course of the reaction, indicating hydride rather than hydrogen atom transfer. Kinetic studies using a homologous series of substrates also indicate that a mechanism based on sequential one-electron steps rather than a single two-electron step is unlikely. These studies lead to a comprehensive chemical mechanism by which xanthine oxidase and related molybdenum hydroxylases carry out substrate hydroxylation.

Molybdenum Hydroxylases: Relevant Thio-Mo Chemistry

Charles G Young, University of Melbourne, Australia

The Mo hydroxylases feature a unique oxo-thio-Mo(VI) active site responsible for the hydroxylation of various purine and aldehyde substrates. Synthetic models for these enzymes are limited due to the redox interplay of sulfi de and Mo(VI) and the enhanced reactivity of the thio ligand. Here we describe the synthesis and full characterization of oxo-thio-Mo(VI) and -Mo(V) complexes of hydrotris(3-isoprop ylpyrazolyl)borate (TpPr) as structural, spectroscopic and chemical models for these enzymes. Mononuclearity in the solid and solution states and at the Mo(VI) and Pr VI s Mo(V) levels is achieved in a system based on Tp Mo OS(OC6H4X-2) (X = Bu, t Pr IV Bu, Ph etc) prepared in the reactions of Tp Mo O(OC6H4X-2)(OPEt3) with sulfur atom donors. Structural and spectroscopic studies confi rm the presence of the terminal thio ligand and provide insights into the electronic control of enzyme action. Pr VI t The X-ray crystal structure of Tp Mo OS(OC6H4 Bu-2) has been determined (Figure 1). Other less sterically encumbered derivatives undergo reactions involving the terminal thio ligand. These include internal redox leading the formation of disulfi do-bridged dimers, hydrolysis forming disulfi do-oxo-bridged species, and novel aromatic C-H ring activation reactions. The latter produce molybdenyl species (see, e.g., Figure 2) and are consistent with the extreme electrophilicity of the thio ligand. The outcomes of these reactions and the factors inducing them have been defi ned by a systematic study of available derivatives, including dioxo-Mo(VI) and phosphine oxide precursors. 52 Journal of Inorganic Biochemistry 96 (2003)

Variable Frequency Pulsed EPR Studies of Molybdenum Enzymes

John H Enemark, Department of Chemistry, University of Arizona, United States Andrei V Astashkin, Department of Chemistry, University of Arizona, United States Arnold M Raitsimring, Department of Chemistry, University of Arizona, United States Changjian Feng, Department of Chemistry, University of Arizona, United States Heather L Wilson, Department of Biochemistry, Duke University Medical Center, United States K V Rajagopalan, Department of Biochemistry, Duke University Medical Center, United States

Variable frequency pulsed EPR spectroscopy provides detailed structural information about the Mo(V) active sites that is not accessible by other structural techniques. Chicken liver and recombinant human sulfi te oxidase (SO) have been investigated by electron spin echo envelope modulation (ESEEM) and pulsed ENDOR spectroscopy. The chicken and human spectra are identical and show a single exchangeable proton for the low pH (lpH) form and two nearby exchangeable protons in the high pH (hpH) form. These spectra also reveal nearby non-exchangeable protons. The closest proton is the α-proton of the coordinated cysteinyl residue, and the variation in this signal with pH is ascribed to a small change in torsional angle of the coordinated cysteinyl residue. The proton environments of the SO active site of several mutant variants of human SO will also be described. In the Tyr343Phe mutant, the nearby OH of the conserved Tyr in the active site has been removed. In the Arg160Gln mutant the charge on the active site has been changed. The fi rst direct investigations of the exchangeable Mo-O unit in SO by HYSCORE experiments using 17O-enriched water will also be presented.

2-Hydroxyacyl-CoA Dehydratases, a novel family of molybdenum enzymes

Wolfgang Buckel, Philipps-Universitaet Marburg, Germany

The reversible syn-elimination of water from (R)-2-hydroxyacyl-CoA to (E)-enoyl-CoA is of considerable mechanistic interest, since a proton has to be removed from the non-activated 3-position (pK = 40), whereas a hydroxyl ion is expelled from the 2-position adjacent to the electron withdrawing CoA-ester. The 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans (Clostridiales), which catalyses the formation of glutaconyl-CoA, consists of two protein components, A and D. The extremely oxygen sensitive homodimeric component A (2 x 27 kDa) contains one [4Fe-4S]1+/2+ cluster between the two subunits and acts as an activator of the dehydration. Each subunit of component A comprises an ADP/ATP-binding site related to actin [1]. The heterodimeric component D (55 + 45 kDa), the actual dehydratase, carries 2+ one [4Fe-4S] cluster and exhibits 1.0 FMNH2 and ca. 0.1 molybdenum. Component D from Clostridium symbiosum with an approximately 7-fold higher specifi c activity contains 2.0 [4Fe-4S], 1.0 FMNH2 and 0.7 Mo. Component A is reduced by a 2[4Fe-4S] ferredoxin or by fl avodoxin. In the presence of ATP the electron is further transferred to component D, in which Mo(VI) is reduced to Mo(V). The now oxidised component A hydrolyses ATP and returns to the resting state with bound ADP. It is postulated that the electron is transferred from Mo(V) further to the thiol ester of the substrate to yield a nucleophilic ketyl radical anion, which eliminates the adjacent hydroxyl group. The thereby formed enoxy radical (pK = 14) is deprotonated to the ketyl radical anion of the product glutaconyl-CoA. A further electron transfer back to Mo(VI) affords the product and completes the catalytic cycle [2]. References: [1] Locher, K. P., Hans, M., Yeh, A. P., Buckel, W. & Rees, D. C. (2001) J. Mol. Biol. 307, 297-308. [2] Hans, M., Bill, E., Cirpus, I, Pierik, A. J., Hetzel, M., Alber, D. & Buckel, W. (2002) Biochemistry, 41, 5873-5882. Journal of Inorganic Biochemistry 96 (2003) 53

Site-directed mutagenesis of dimethylsulfoxide reductase from Rhodobacter capsulatus: the critical roles of tyrosine-114 and tryptophan-116

Alastair G McEwan, University of Queensland, Australia Justin P Ridge, University of Queensland, Australia Kondo-Francois Aguey-Zinsou, University of Queensland, Australia Paul V Bernhardt, University of Queensland, Australia Graeme R Hanson, University of Queensland, Australia

The crystal structure of six functionally-distinct enzymes of the DMSO reductase family of molybdenum enzymes has revealed that the tertiary structure of the polypeptide that binds the bis(MGD)Mo cofactor is highly conserved. Differences in the catalytic properties of enzymes of this family are almost certainly dependent upon differences in the structure of the Mo active site. In DMSO reductase from Rhodobacter species tryptophan-116 (W116) hydrogen-bonds to an oxo group coordinated to the Mo ion. In addition a second amino acid side chain from tyrosine-114 (Y114) is in close proximity to the oxo group. We have investigated the role of Y114 and W116 in DMSO reductase using site-directed mutagenesis. The Y114F mutant had an increased kcat and increased Km towards DMSO compared to the native enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfi de (DMS) with phenazine ethosulfate (PES) as electron acceptor but with a lower kcat than that of the native enzyme. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant but the mid-point potential of the Mo(VI)/Mo(V) couple was raised by about 50mV.

The W116F mutant enzyme also showed a greatly reduced kcat towards DMSO but only relatively small changes in the value of Km. The mutant enzyme was unable to oxidize DMS with PES as electron acceptor and did not form a characteristic pink complex with DMS. The UV/visible spectrum of the Mo(VI) form of the W116F mutant revealed the loss of the characteristic transition at 720 nm. Results of electrochemistry experiments were consistent with a single protonation event accompanying the conversion of Mo(VI) to Mo(V) and Mo(V) to Mo(IV) in the W116F mutant enzyme while in the native enzyme protonation only accompanies electron transfer during the conversion of Mo(VI) to Mo(V). The above data are interpreted with reference to existing structural information for DMSO reductase.

Copper Homeostasis in E. hirae

Marc Solioz, University of Berne, Switzerland Reto Portmann, University of Berne, Switzerland Jivko Stoyanov, University of Berne, Switzerland

The E. hirae cop operon is located on the chromosome and is required for copper homeostasis. It consists of four genes in the order: copY, copZ, copA and copB. CopY encodes a copper responsive repressor, copZ encodes a copper chaperone, and copA and copB encode CPx-type copper ATPases. If cytoplasmic copper becomes excessive, it is secreted by CopB.

Tetrathiomolybdate inhibits the purifi ed Enterococcus hirae CopB copper ATPase with an IC50 of 34 nM. Inhibition could be reversed by copper or silver, suggesting inhibition by substrate binding. This allowed an estimate of the high affi nity of CopB for copper and silver. The fate of cytoplasmic copper remains unclear in several regards. A major function in intracellular copper routing is taken by the CopZ copper chaperone, which has been shown to deliver copper to the CopY repressor. In its zinc form, CopY binds to the cop promoter and represses transcription. When CopZ donates copper to CopY, its bound zinc is displaced by copper and the repressor dissociates from the promoter, allowing expression of the downstream genes. The interaction of CopZ with other proteins has been analyzed by surface plasmon resonance analysis. CopZ was shown to interact with CopA, which may be the site of copper loading of CopZ, and with Gls24, a protein of unknown function. The interaction of CopZ with the CopY repressor is currently being analyzed by the same technique. It was observed that CopZ of E. hirae is being degraded under high copper stress. CopZ levels increased only up to 0.5 mM copper and declined at higher copper concentrations. Proteolysis of CopZ could be demonstrated in vitro. When cytosolic extracts were mixed with purifi ed CopZ, it was rapidly degraded. The proteolytic activity was stimulated by Cu(I) or Ag(I). The serine protease inhibitors p-phenylmethylsulfonyl fl uoride and p-aminobenzamidine inhibited the degradation of CopZ. It was concluded that the protease degrading CopZ is a serine type protease. On zymograms, the CopZ degrading activity was tentatively identifi ed as a protein of 58 kDa. 54 Journal of Inorganic Biochemistry 96 (2003)

The CueR regulator and copper tolerance in E. coli.

Nigel L Brown, The University of Birmingham, United Kingdom Christopher J Kershaw, The University of Birmingham, United Kingdom Jivko V Stoyanov, The University of Birmingham, United Kingdom

Aerobic copper tolerance in Escherichia coli appears to be maintained primarily by regulation of expression of the copA and cueO genes, encoding a copper-exporter and a periplasmic copper-dependent phenoloxidase. We have shown that the regulatory protein, CueR, regulates gene expression in response not only to Cu(I) and Ag(I) but also to gold salts (probably Au(I)), although resistance to gold salts is apparently not conferred by CopA/CueO. We also describe experiments examining the expression of other E. coli genes in response to copper salts.

Structure-function and regulation of the Menkes copper-translocating P-type ATPase (MNK ; ATP7A)

James Camakaris, Department of Genetics, University of Melbourne, Australia Ilia Voskoboinik, Department of Genetics, University of Melbourne, Australia Mark Greenough, Department of Genetics, University of Melbourne, Australia Cinnamon Lane, Department of Genetics, University of Melbourne, Australia

The Menkes transmembrane copper-translocating P-type ATPase (MNK ; ATP7A) has a special role in maintaining copper homeostasis and mutations in the MNK gene can lead to the severe and usually fatal copper-defi ciency disorder, Menkes disease. MNK is regulated primarily at the level of sub-cellular localisation ñ its basal localisation is in the trans Golgi network where it delivers Cu to Cu-dependent enzymes in the secretory pathway whilst at elevated Cu levels MNK traffi cks to the plasma membrane where it pumps Cu out of cells to restore normal Cu homeostasis. Traffi cking and catalytic activity appear to be linked, although evidence from mutants we have studied suggests that complete loss of catalytic activity needs to occur for Cu-regulated traffi cking to be signifi cantly compromised. We have identifi ed an important role for 1230Asp within the DxxK motif in coupling ATP binding and acylphosphorylation with copper translocation. MNK also undergoes copper-stimulated protein kinase-dependent phosphorylation which may be associated with regulating its localisation and traffi cking to sub-cellular sites. Unique phosphopeptides have been identifi ed in response to elevated Cu levels and phosphorylation occurs on serine residues. Our current evidence suggests that MNK endocytosis occurs via both clathrin-dependent and clathrin-independent processes and the effects of Cu on these processes are being investigated. Journal of Inorganic Biochemistry 96 (2003) 55

Ni-Thiolate and Fe-Thiolate-Cyanocarbonyl Complexes Modeling the Nickel/Iron Site and Reactivity of [NiFe] Hydrogenases

Weng-Feng Liaw, Department of Chemistry, Tsing Hua University, Taiwan, Province of China Chien-Ming Lee, Department of Chemistry, Tsing Hua University, Taiwan, Province of China I-Jui Hsu, Department of Chemistry, Tsing Hua University, Taiwan, Province of China Shyue-Chu Ke, Department of Chemistry, Tsing Hua University, Taiwan, Province of China Gene-Hsiang Lee, Department of Chemistry, Tsing Hua University, Taiwan, Province of China Yu Wang, Department of Chemistry, Tsing Hua University, Taiwan, Province of China

During catalysis, the active center cycles through different redox states Ni(I, II, III) with the iron site remaining as Fe(II) in [NiFe] hydrogenases.1-4 Recent studies of the reduced active site of [NiFe] hydrogenase isolated from D. vulgaris Miyazaki F implicate that the Ni-C intermediate is a formal Ni(III) oxidation state with a hydride (H-) bridging between the Ni and the

Fe atoms, and both of Ni and Scys atoms participating in the heterolysis of hydrogen molecule is essential for the hydrogen molecule activation by the [NiFe] hydrogenase.1-4 The preparation of Ni-hydride chalcogenolate derivatives to better mimic the features of the paramagnetic Ni-C intermediate of the catalytic cycle of [NiFe] hydrogenases could uncover the active- site structure of Ni-C state and the hydrogen-activation mechanism. Model compound studies on the synthesis and reactivity - - II n- investigation of complexes [Ni(SePh)(P(ο-C6H4S)2(ο-C6H4SH))H] , [Ni(SePh)(P(ο-C6H4S)3)] and [Fe (CO)x(CN)y(SR)z]m (x = 1, 2, 3; y = 3, 2, 1; z = 1, 2, 3; m = 1, 2; n = 1, 2) may yield answers to these questions. 1 De Lacey, A. L.; Hatchikian, E. C.; Volbeda, A.; Frey, M.; Fontecilla-Camps, J. C.; Fernandez, V. M. J. Am. Chem. Soc. 1997, 119, 7181-7189. 2 Niu, S.; Thomson, L. M.; Hall, M. B. J. Am. Chem. Soc. 1999, 121, 4000-4007. 3 Ogata, H.; Mizoguchi, Y.; Mizuno, N.; Miki, K.; Adachi, S.-I.; Yasuoka, N.; Yagi, T.; Yamauchi, O.; Hirota, S.; Higuchi, Y. J. Am. Chem. Soc. 2002, 124, 11628. 4 Foerster, S.; Stein, M.; Brecht, M.; Ogata, H.; Higuchi, Y.; Lubitz, W. J. Am. Chem. Soc. 2003, 125, 83.

Redox Activation of FeMoco and hydrogenase H-cluster model compounds

Stephen P Best, University of Melbourne, Australia Stacey J Borg, University of Melbourne, Australia Christopher J Pickett, John Innes Centre, Norwich, United Kingdom Kylie A Vincent, Oxford University, United Kingdom

The development of novel high pressure spectroelectrochemical techniques [1] have facilitated the study of redox modulation of the chemistry of the isolated FeMoco cofactor of the nitrogenase enzyme and of models of the hydrogenase H-cluster

over a range of CO, H2 and N2 gas pressures. In the case of FeMoco this has revealed the redox state dependence of CO binding and has led to the identifi cation of further reduced states of the cluster that are made accessible following CO coordination. The relationship between the various reduced and CO-bound species deduced from the spectroelectrochemical measurements have recently been reported [2]. The similarity between the behaviour of the NMF-solvated and the protein- bound cofactor in terms of the redox state dependence of CO binding suggests an alternative interpretation of the ENDOR experiments conducted on the enzyme under turnover conditions in the presence of CO [3], this experiment being critical to the assignment of the oxidation states of the metals in the resting state form of protein-bound FeMoco. The H-cluster of the all-iron hydrogenase is a remarkable organometallic species that, superfi cially, is closely related to dithiolate-bridged di-iron carbonyl compounds. A major difference, however, is the presence of a bridging CO group in the oxidised form of the H-cluster. Reduction of the model compounds leads to a complex set of chemical reactions, including the transient formation of species containing bridging CO species. It has recently been shown that the oxidation of related model compounds leads to transient formation of a species that has EPR and IR spectra closely related to that of the enzyme- bound H-cluster [4]. In addition to providing structural and spectroscopic models of the H-cluster, this class of compound also provide functional models (the observation of electrocatalytic proton reduction reactions). [1] Borg, S.J., Best, S.P., J. Electroanal. Chem., 2002, 535, 57-64. [2] Pickett, C.J., et. al., Chem. Eur. J., 2003, 76-87. [3] Lee, H-I., Hales, B.J., Hoffman, B.M., J. Amer. Chem. Soc., 1997, 119, 11395-11400. [4] Razavet, M., et. al., Chem. Commun, 2002, 700. 56 Journal of Inorganic Biochemistry 96 (2003)

Synthesis of Biomimetic Models of Iron-only Hydrogenases Active Site Linked to Ru-Polypyridine Photosensitizers

Licheng Sun, Department of Organic Chemistry, Arrhenius Laboratory, Sweden Sascha Ott, Department of Organic Chemistry, Arrhenius Laboratory, Sweden Szabolcs Salyi, Department of Organic Chemistry, Arrhenius Laboratory, Sweden Henriette Wolpher, Department of Organic Chemistry, Arrhenius Laboratory, Sweden Mikael Kritikos, Department of Structure Chemistry, Arrhenius Laboratory, Sweden Bjoern Akermark, Department of Organic Chemistry, Arrhenius Laboratory, Sweden

Hydrogen as energy carrier becomes more and more important in our society which demands sustainable developments. Sulphur containing binuclear complexes of iron are interesting synthetic targets, because they closely resemble the active site of hydrogenases, a naturally occurring class of enzymes, which regulate hydrogen production and consumption in microorganisms. We became intrigued by the possibility to covalently link biomimetic models of the Fe-only hydrogenase to ruthenium polypyridine complexes as photosensitizers, in an attempt to make hydrogen production by sun light. As schematically illustrated in the fi gure below, the photo-excited ruthenium complex is oxidatively quenched by the

Fe-dimer. The reduced Fe2-species will then drive the reduction of protons, forming hydrogen. The synthesis of novel systems such as 1 where a Fe-dimer is covalently linked to a ruthenium photosensitizer will be presented. All complexes were characterized by common techniques(NMR, IR, UV-Vis, etc.), including X-ray crystal structure analysis for selected Fe-dimers. Reference: S. Salyi, M. Kritikos, B. Akermark, L. Sun, Chem. Eur. J. 2003, 9, 557-560. Journal of Inorganic Biochemistry 96 (2003) 57

67Zn NMR of Carbonic Anhydrase: Distinguishing Water and Hydroxide Bound to Zinc.

Paul D Ellis, Environmental Molecular Sciences Laboratory, United States Andrew S Lipton, Environmental Molecular Scieces Laboratory, United States Robert W Heck, Environmental Molecular Sciences Laboratory, United States

We present, herein, a general method for the direct determination of the state of ionization of bound oxygen to metals in metalloproteins and illustrate this method by examining the water and hydroxide bound to Zn2+ in human carbonic anhydrase II. We have employed low temperature (10K) solid-state NMR utilizing cross polarization from protons to zinc. We have utilized ab initio molecular theory as a means to predict the observed quadrupole coupling constants. The agreement between theory and experiment is encouraging. This methodology enables one to address a key issue as to the functional role the metals may play in the chemistry and associated biology of the protein. Hence, low temperature solid state NMR of metals in metalloproteins represents a complimentary experiment to x-ray crystallography of this class of proteins. Acknowledge: NIH GM26295 and DOE KP11 24931.

Zinc catalyzed alkyl-transfer enzymes

James E Penner-Hahn, The University of Michigan, United States Daniel Tobin, The University of Michigan, United States Rowena G Matthews, The University of Michigan, United States Carol A Fierke, The University of Michigan, United States Katrina Peariso, The University of Michigan, United States David A Grahame, Uniformed Services University of the Health Sciences, United States Timothy A Garrow, The University of Illinois, United States

In the last decade, a new class of catalytic zinc metalloproteins has been characterized. These enzymes contain Zn coordinated to at least one, and up to three cysteine residues, and catalyze transfer of an alkyl group to a nucleophilic thiolate substrate. It has been suggested that coordination of the substrate thiol to the zinc ion lowers the pKa of the thiol, there by increasing the concentration of thiolate at physiological pH. However, the detailed mechanism of the alkyl transfer in the individual members of this class of metalloproteins has yet to be determined. X-ray absorption spectroscopy (EXAFS and XANES) has been used to investigate the local structural and electronic environment of the Zn in methionine synthase, betaine:homocysteine methyl transferase, methylcobamide:coenzyme M methyltransferase, and farnesyl protein transferase. These enzymes, which span the range of Zn ligation from 1 to 3 endogenous cysteines, show both signifi cant structural similarities and some surprising spectroscopic differences. By using selenium-substituted substrates, it has been possible to obtain geometric information about the active site. This, together with EXAFS data for both reactant-bound and product-bound forms of the enzymes provides insight into the mechanism of alkyl transfer. Data for both the enzymes and relevant model compounds will be presented. 58 Journal of Inorganic Biochemistry 96 (2003)

Model Study For The Bio-coordination Sphere Of Active Center In Carbonic Anhydrase And Application Into DNA Cleasvage Study

Zong-Wan Mao, School of Chemistry & Chemical Engineering, Sun Yat-Sen, China Heng Fu, School of Chemistry & Chemical Engineering, Sun Yat-Sen, China Xin-Man Xu, School of Chemistry & Chemical Engineering, Sun Yat-Sen, China Jie Li, School of Chemistry & Chemical Engineering, Sun Yat-Sen, China Si-Dong Liu, School of Chemistry & Chemical Engineering, Sun Yat-Sen, China Shu-Yi Deng, School of Chemistry & Chemical Engineering, Sun Yat-Sen, China Liang-Nian Ji, School of Chemistry & Chemical Engineering, Sun Yat-Sen, China

Recent progress on carbonic anhydrase (CA) revealed that a hydrogen bond network consisting of the Thr-199 hydroxyl group acts as an important role in the catalytic process [1,2]. In model study, we successfully isolated two important bicarbonate intermediate complexes, Lipscomb- and Lindskog- type structures [3], and observed a carbonation process going with hydration and dehydration past a few years [4-5]. In order to mimic the hydrophobic environment and functional Thr-199 of the Zn(II) ion in the CA, we further focused our work on mimicing the active center of the CA and its bio- coordination sphere. One of our strategies was constructing a supramolecular system by the inclusion complex of ß-cyclodextrin containing hydroxyl groups with Zn(II) chain or macrocyclic polyamine complexes containing hydrophobic group as a new model. An inclusion complex of Cu(II) and beta- cyclodextrin has been structurally characterized. The further studies show that in the presence of ß-cyclodextrin carbonate-bridged tetranuclear Zn(II) complex was crystallized from the aqueous solution of Zn(II) complex with 4-benzyldiethylenetriamine in air, but in the absence of beta-cyclodextrin a hydroxyl-bridged binuclear Zn(II) complex was obtained. The potentiometric titration further demonstrated that the presence of ß-cyclodextrin decreases the pKa values of metal complexes in 0.5-1.0 range. The results being presented will indicate that the hydroxyl groups of beta-cyclodextrin can effectively mimic catalytic action of the Thr-199 by weak supramolecular interaction. The corresponding Cu(II) complexes were applied to study of DNA cleavage. All above-mentioned and further studied results will be presented in detail. [1] W. N. Lipscomb, N. Strater, Chem. Rev. 1996, 96, 2375. [2] E. Kimura, Acc. Chem. Res. 2001, 34, 171 [3] Z.-W. Mao, G. Liehr, R. van Eldik, J. Am. Chem. Soc. 2000, 122, 4839. [4] Z.-W. Mao, G. Liehr, R. van Eldik, J. Chem. Soc., Dalton Trans. 2001, 1593. [5] Z.-W. Mao, F W Heinemann, G. Liehr, R. van Eldik, J. Chem. Soc., Dalton Trans. 2001, 3652. We thank the national and Guangdong provincial NSFC and Ministry of Education for fi nancial support. Journal of Inorganic Biochemistry 96 (2003) 59

‘Molybdopterin’ Complexes

C David Garner, University of Nottingham, United Kingdom Tarnjeet N Bhachu, University of Nottingham, United Kingdom Stephen E Davies, University of Nottingham, United Kingdom Christopher J Lowe, University of Nottingham, United Kingdom Jonathan McMaster, University of Nottingham, United Kingdom Josephine M Tunney, University of Nottingham, United Kingdom James P Dicks, University of Nottingham, United Kingdom Ben Bradshaw, Manchester University, United Kingdom David Collison, Manchester University, United Kingdom John A Joule, Manchester University, United Kingdom Rehana Karim, Manchester University, United Kingdom

Molybdenum enzymes, e.g. sulfi te oxidase, xanthine oxidase, DMSO reductase, and the assimilatory and dissimilatory nitrate reductases, involve a single Mo atom bound to one or two molecules of a special dithiolene, ‘molybdopterin’ (MPT). However, as MPT (Figure 1) also binds W, it is preferable to regard this moiety as a Metal-binding Pyranopterin ene-1,2-diThiolate. In Mo-MPT enzymes of prokaryotes, the phosphate may be part of a diphosphate bound to a nitrogenous base such as adenine, guanine, or cytosine. Virtually all of the Mo-MPT and W-MPT enzymes catalyse the transfer of an oxygen atom to, or from, the substrate X (Figure 2). Beyond the dithiolene group of MPT defi ning part of the metal’s coordination sphere and tuning the redox potential of the catalytic site, the pyrazine ring could act in concert with the metal centre to facilitate the two-electron redox change that is concomitant with oxygen atom transfer. Other roles for MPT could include mediation of the proton fl ux and/or the electron- transfer involved in the regeneration of the catalytically active state. We are seeking an understanding of the role(s) of MPT in the catalyses effected by the Mo-MPT and W-MPT enzymes and have synthesised MPT (1) and related systems (2) in protected forms. We have commenced studies directed towards the synthesis and characterisation of complexes of these pro-ligands, complemented by investigations of other metal dithiolene complexes. The results of these researches will be described. 1 B. Bradshaw, A. Dinsmore, W. Ajana, D. Collison, C. D. Garner, and J. A. Joule, J. Chem. Soc., Perkin Trans. 1, 2001, 3239-3244. 2 B. Bradshaw, D. Collison, C. D. Garner, and J. A. Joule, Org. Biomol. Chem., 2003, 1, 129-133. We thank the BBSRC and EPSRC for fi nancial support. 60 Journal of Inorganic Biochemistry 96 (2003)

An Electronic Structure Description of Atom and Electron Transfer in Sulfi te Oxidase

Martin L Kirk, The University of New Mexico, United States Katrina Peariso, The University of New Mexico, United States

The sulfi te oxidase (SO) family of mononuclear molybdenum enzymes functions to catalyze various two-electron redox reactions coupled to formal oxygen atom transfer. In vertebrates SO is found in the mitochondrial intermembrane space, where the physiologically important oxidation of sulfi te represents the terminal step in the oxidative degradation of cysteine and methionine. Individuals who suffer from isolated sulfi te oxidase defi ciency, which derives from specifi c mutations in the SO gene, display a variety of deleterious effects including neurological abnormalities, dislocation of the ocular lens, mental retardation, and even attenuated brain growth.

We have used small molecule analogues of oxidized (SOox) and reduced (SOred) SO in order to provide deeper insight into their mechanism of activity. Deconvoluting the different contributions of thiolate and ene-1,2-dithiolate donors to the underlying electronic structure of the Mo site in SO has proven to be a diffi cult task. We have used detailed bonding calculations in order to probe the effects of these S donor ligands on SOox in the oxygen atom transfer process. Regarding the oxidative half-reaction, these differences in S donor ability may be illuminated by selectively substituting Se for S in model complexes which possess multiple sulfur donor ligand environments. As such, we will discuss the synthesis, structures, and detailed spectroscopy of new oxo-Mo(V) complexes as effective models for the one electron reduced active site of SO. These studies have provided much needed insight into the electronic structure of the reduced SO site, and allowed for increased understanding of the individual roles played by these different thiolate donors in electron transfer regeneration of the enzyme. The results of this work are also being used to ascertain the electronic origin of the enzyme electronic absorption, EPR, ESEEM, and ENDOR spectra, and to develop deeper insight into the electronic structure of the SO site.

Function of NH—S Hydrogen Bond in Dioxo W-oxidase Model Complexes

Norikazu Ueyama, Department of Macromolecular Science, Graduate School of Science, Osaka University, Japan Koji Baba, Japan Taka-aki Okamura, Department of Macromolecular Science, Graduate School of Science, Osaka University, Japan

Tungsten-oxidases have been considered to catalyze oxo-transfer reaction with kinetic advantages at high temperature. A VI 2- model complex, [W O2(bdt)2] (bdt = benzenedithiolate), exhibits relatively high oxo-transfer reactivity, when compared VI 2- with the corresponding [Mo O2(bdt)2] , in a model C-H oxidation reaction using benzoin. The origin of such a reactivity comes from a mutal trans infl uence of thiolate sulfur at the trans position of W=O. The presence of NH—S hydrogen bond between pterin amine NH and dithiolene sulfur is proposed in both oxidized and reduced states based on the crystallographic VI data reported for various Mo- and W-oxidases. The crystal structure of a novel model complex, (NEt4)[W O2{1,2-S2-3,6-

(MeCONH)2C6H2}2], indicates the existence of strong NH—S hydrogen bond between amide NH and dithiolene sulfur (2.46 angstroms for NH—S distance) at trans position of W=O but no NH—S hydrogen bond in dithiolene sulfur at the cis position of W=O, as supported by IR data for amide NHs in the solid state and in CH2Cl2 solution. Two W-S bonds at the cis position of W=O have a strong covalent character like Hg-S bond in a linear Hg(II) thiolate complex and other W-S bonds at the trans position of W=O have a relatively ionic one. The NH—S hydrogen bond does not affect the W-S bond distances at the trans position of W=O while defi nitely decreases oxo-transfer reactivity in a model oxidation reaction using benzoin. In addition, the observation of only a weak W=O Raman signal suggests the decrease of LMCT from sulfur pπ to W(VI)=O antibonding LUMO. Thus, NH—S hydrogen bond toward dithiolene sulfur at the trans position of W=O stabilizes the W(VI) state to decrease oxo-transfer reactivity. Journal of Inorganic Biochemistry 96 (2003) 61

Structure/Function Correlations over Non-heme Iron Enzymes

Edward I Solomon, Stanford University, United States

Non-heme iron active sites are found in a wide range of enzymes which perform different biological functions requiring dioxygen. These reactions often involve dioxygen activation by a ferrous active site which is generally diffi cult to study with most spectroscopic methods. A new spectroscopic methodology has been developed utilizing variable temperature, variable fi eld magnetic circular dichroism (VTVH MCD) which enables one to obtain detailed insight into the geometric and electronic structure of the non-heme ferrous active site and probe its reaction mechanism on a molecular level. This spectroscopic methodology will be presented and applied to a number of key non-heme iron enzymes leading to a general mechanistic strategy for O2 activation.

Theoretical studies of oxygen activation by non-heme iron enzymes

Arianna Bassan, Stockholm Center for Physics, Astronomy and Biotechnology, Sweden Margareta RA Blomberg, Stockholm Center for Physics, Astronomy and Biotechnology, Sweden Per EM Siegbahn, Stockholm Center for Physics, Astronomy and Biotechnology, Sweden

Density functional theory with the B3LYP functional has been employed to shed light on oxygen activation by non-heme iron enzymes, such as tetrahydrobiopterin-dependent amino acid hydroxylases and Rieske dioxygenases. In the catalytic core of both these enzymes iron is coordinated by the 2-His-1-carboxylate motif. Since synthetic catalysts play an important role in the understanding of enzyme catalysis, theoretical studies have also been performed on the catalytic activity of an Fe(TPA) complex (TPA=tris-(2-pyridylmethyl)amine), which is a functional model for non-heme iron oxygenases. Tetrahydrobiopterin-dependent amino acid hydroxylases form a family of oxygen activating iron proteins that catalyze the hydroxylation of aromatic amino acids using the cofactor tetrahydrobiopterin. In the catalytic core of these enzymes, molecular oxygen reacts with the pterin cofactor and is likely to be activated by forming an Fe(IV)=O complex. Calculations indicate that this process passes through the formation of an iron(II)-peroxy-pterin-intermediate. The high-valent iron-oxo species then hydroxylates the aromatic amino acids phenylalanine and tryptophan. Activation of molecular oxygen in Rieske dioxygenases has been investigated by focusing on catalysis of naphthalene 1,2- dioxygenase (NDO), which carries out cis-dihydroxylation of naphthalene. The possibility that a high-valent iron-oxo species is formed along the reaction pathway of the cis-dihydroxylation reaction has been investigated, showing that a too high activation energy is needed for this process to take place. A concerted mechanism, where O-O bond cleavage is combined with C-O bond formation is suggested. It is interesting to compare the reaction mechanism of this enzyme with the one of the Fe(TPA) catalyst, which has also been found capable of cis-dihydroxylation. In this case the theoretical studies corroborate the experimental data, which point towards the involvement of a high-valent iron-oxo intermediate, the Fe(V)=O species. 62 Journal of Inorganic Biochemistry 96 (2003)

Characterization of a High-Spin Fe(IV) Intermediate in the Reaction of Taurine/ Alpha-Ketoglutarate Dioxygenase (TauD)

J Martin Bollinger, Jr, Penn State University, United States John C Price, Penn State University, United States Eric W Barr, Penn State University, United States Bhramara Tirupati, Penn State University, United States Carsten Krebs, Penn State University, United States

The Fe(II)- and alpha-ketoglutarate-dependent dioxygenases activate molecular oxygen to couple oxidative decarboxylation of alpha-ketoglutarate (2-oxo-1,5-pentanedoic acid) with hydroxylation of unactivated alkyl groups on a variety of substrates. Enzymes in this class catalyze reactions of tremendous biomedical and environmental importance. A consensus mechanism, involving a substrate-hydroxylating Fe(IV)=O complex and as many as three additional intermediate states after addition of oxygen, has often been proposed, but none of the postulated intermediates has ever been detected. We have recently succeeded in characterizing an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli, by rapid kinetic and spectroscopic methods. Its formation and decay are fast enough for the complex to be on the taurine-hydroxylation pathway. The Mössbauer and EPR properties of the intermediate and its one-electron cryo-reduced form (obtained by low-temperature gamma-radiolysis) establish that it is a high-spin, formally Fe(IV) complex. To our knowledge, this is the fi rst direct demonstration of a catalytically relevant Fe(IV) intermediate in a mononuclear, non-heme-iron enzyme. Ongoing spectroscopic characterization and kinetics experiments to defi ne the timing of its formation and decay relative to transformation of taurine and alpha-ketoglutarate will permit the structure of the novel complex to be defi ned. Knowledge of its structure and position in the TauD reaction cycle will provide insight of unprecedented detail into the mechanism of this important enzyme family. Journal of Inorganic Biochemistry 96 (2003) 63

Tyrosyl Radicals in Iron Enzymes: Structural Information from High-Field EPR on Frozen Solutions and Single Crystals

Friedhelm Lendzian, Technical University Berlin, Germany Marcus Galander, Technical University Berlin, Germany Martin Högbom, University Stockholm, Sweden Pär Nordlund, University Stockholm, Sweden Christiane Jung, Max-Delbrück-Center Berlin, Germany Volker Schünemann, Medical University Lübeck, Germany Anne-Laure Barra, CNRS Grenoble, France

High-Field EPR is an important tool for identifi cation and investigation of structure and interactions of protein-based radicals [1]. This is demonstrated on two examples: Freeze-quenched intermediate radicals observed during the shunt reaction of substrate-free cytochrome P450cam are identifi ed as hydrogen bonded tyrosyl radicals by 94 GHz and 285 GHz EPR. Their resolved hyperfi ne structure enables an assignment to a specifi c residue (Y96 in the wild type and Y75 in mutant Y96F, respectively) [2]. In class I ribonucleotide reductase of E. coli a functional essential tyrosyl radical is harboured at residue Y122, close to the diferric iron center in subunit R2 of the active enzyme. X-ray structure data are only available for the inactive enzyme, where Y122 is in its reduced non-radical form (met-R2). In our study the active radical Y122* was generated with reasonable yield (10%) in single crystals of the enzyme. High-fi eld EPR experiments on these single crystals reveal a signifi cant reorientation of the radical with respect to the reduced tyrosine molecule, moving the tyrosyl oxygen away from the diiron site by > 0.8 Angstrom (Fig.) [3]. Implications for the proposed catalytic mechanism (coupled electron/proton transfer) are discussed. [1] G. Bleifuss, M. Kolberg, S. Pötsch, W. Hofbauer, R. Bittl, W. Lubitz, A. Gräslund, G. Lassmann, and F. Lendzian (2001) Biochemistry, 40, 1562-1568. [2] V. Schünemann, F. Lendzian, C. Jung, J. Contzen, A.-L. Barra, S.G. Sligar, and A. X. Trautwein(2002) Biochemistry, submitted. [3] M. Högbom, M. Galander, M. Andersson, M. Kolberg, W. Hofbauer, G. Lassmann, P. Nordlund, and F. Lendzian (2002) Proc. Natl. Acad. Sci. USA, in press. 64 Journal of Inorganic Biochemistry 96 (2003)

Oxygenation Kinetics of Iron and Copper Protein Active Site Model Complexes

Matthew E Helton, Johns Hopkins University, United States Eunsuk Kim, Johns Hopkins University, United States Kenneth D Karlin, Johns Hopkins University, United States Marcus Honecker, University of Basel, Switzerland Susan Kaderli, University of Basel, Switzerland Yorck-Michael Neuhold, University of Basel, Switzerland David Lahti, University of Basel, Switzerland Andreas D Zuberbuehler, University of Basel, Switzerland

The reactivity of O2 toward iron and copper is of fundamental interest. Iron-O2 and copper-O2 interactions are intrinsic to a number of important biological functions such as O2-transporting [e.g., in hemoglobin and hemocyanin], oxygenase activity [e.g., in methane monooxygenase and tyrosinase], and oxidase activity [e.g. in heme-copper oxidases such as cytochrome c oxidase (CcO)]. As part of our ongoing efforts to elucidate fundamental aspects of iron-O2 and copper-O2 interactions, with particular interest in heme/Cu/O2 chemistry relevant to the CcO active site, we have studied the oxygenation kinetics II of the following systems: 1) copper (I) complexes with tridentate and tetradentate N-donor ligands; 2) (F8TPP)Fe ïH2O 2 II I 2 [where F8TPP = tetrakis(2,6-difl uorophenyl)porphyrinate]; 3) ( L)Fe /Cu [where L is a heterobinucleating, aryl substituted, porphyrinate]; and 4) various combinations of 1) and 2). These complex oxidations were monitored by stopped-fl ow UV-vis spectroscopy in a number of different solvents, and over a wide range of temperatures. II 2- II Oxygenation of our Cu(I) complexes yields the LCu -(O2 )-Cu L µ-peroxo species (where O2 coordinates in either a trans- 2 2 III 2- III µ-1,2 end-on or µ-η :η side-on fashion), and at times we also observe the LCu -(O )2-Cu L bis-µ-oxo species. Oxygenation III - of our heme complexes, in the absence of copper, reveals rapid formation of a (heme)Fe -(O2 ) superoxo species which is quite stable at low temperatures. Oxidation of our heme complexes in the presence of Cu gives rapid formation of the III - I heme-superoxo complex (heme)Fe -(O2 ) followed by subsequent reactivity with Cu to form the heterobinuclear µ-peroxo complex. This peroxo species transforms thermally to a µ-oxo species [(heme)FeIII-(O)-CuIIL]. Quantitative aspects of the reaction kinetics and possible relevance to enzymes will be discussed. Acknowledgement: This work is supported by the NIH [KDK: GM60353 and MEH: GM20805 (Postdoctoral Fellowship)] and the Swiss National Science Foundation [ADZ]. Journal of Inorganic Biochemistry 96 (2003) 65

The sulfur-oxidizing enzyme system of Paracoccus pantotrophus: proteins and reaction

Cornelius G Friedrich, Technische Mikrobiologie, Fachbereich Chemietechnik, Universitaet Dortmund, Germany Armin Quentmeier, Technische Mikrobiologie, Fachbereich Chemietechnik, Universitaet Dortmund, Germany Frank Bardischewsky, Technische Mikrobiologie, Fachbereich Chemietechnik, Universitaet Dortmund, Germany Dagmar Rother, Technische Mikrobiologie, Fachbereich Chemietechnik, Universitaet Dortmund, Germany Petra Hellwig, Institut für Biophysik, Universität Frankfurt, Germany Wolfgang Lubitz, Max-Planck-Institut für Stahlenchemie, Germany Tresfore Dambe, Institut für Biophysik, Universität des Saarlandes, Hom, Germany Axel Scheidig, Institut für Biophysik, Universität des Saarlandes, Hom, Germany

Oxidation of sulfur to sulfate is a major reaction of the global sulfur cycle and performed by aerobic chemotrophic and anaerobic phototrophic bacteria. The enzyme system (Sox) of the chemotrophic bacterium P. pantotrophus consists of four proteins: SoxYZ, SoxXA, SoxB and SoxCD, and oxidizes hydrogen sulfi de, sulfur, thiosulfate and sulfi te (1). SoxYZ is reversibly redox-active and the SoxY subunit covalently binds the sulfur substrate to an invariant cysteine residue to form SoxY-S-thiocysteine (2). The SoxXA c-type cytochrome complex is proposed to link the the sulfur substrate to the cysteine- thiol of SoxY (1). SoxXA has 3 redox-potentials ranging from 198 to 236 mV. The crystal structure of SoxXA was resolved at 1.7 Angstrom resolution. The molybdoprotein SoxC-c-type cytochrome SoxD complex Sox(CD)2 oxidizes the sulfane sulfur of SoxY-S-thiocysteine to SoxY-cysteine-S-sulfate, and the sulfate moiety is proposed to be hydrolyzed off by the dimanganese SoxB to regenerate SoxY. SoxYZ is inactivated by reduction. The fl avoprotein SoxF (42.832 Da) is not required for thiosulfate oxidation in vitro but reactivates the reconstituted enzyme system inactive from reduced SoxYZ. This activity represents a novel reaction of fl avoproteins. Analysis of genome sequences uncovered homologues of the Sox proteins of P. pantotrophus in other chemo- and phototrophic sulfur-oxidizing bacteria (1). No sox genes exist in acidophilic sulfur-oxidizing archaea and bacteria and these prokaryotes are proposed to form enzyme systems different from that of P. pantotrophus. (1) Friedrich et al. (2001) Appl. Environ. Microbiol. 67:2873-2882. (2) Quentmeier, A. and C. G. Friedrich (2001) FEBS Lett. 503:168-172. Fig. 1. Model of thiosulfate oxidation by the Sox-system of P. pantotrophus. Grey circles represent sulfur atoms, black circles oxygen atoms. 66 Journal of Inorganic Biochemistry 96 (2003)

Biomaterials at the Beach: Metal-Protein Interactions in Mussel and Barnacle Adhesives

Jonathan J Wilker, Purdue University, United States Mary J Sever, Purdue University, United States Jaime T Weisser, Purdue University, United States Jennifer Monahan, Purdue University, United States

Marine organisms such as the common blue mussel (Mytilus edulis) affi x themselves to surfaces by producing a protein-based glue. The soluble precursor protein of this biomaterial contains high levels of 3,4-dihydroxyphenylalanine (DOPA) and cross-links into a hardened matrix for adhesion. Interesting properties of this adhesive include a transition metal (e.g., iron, zinc, copper, manganese) content up to 100,000 times that of the surrounding waters. To better understand the bonding interactions of these marine biomaterials, we are working with live mussels, whole protein extracted from mussels, model peptides, synthetic complexes, and materials engineering techniques. We will present results indicating that metal ions are essential for surface adhesion of marine organisms.

The fi gure right shows a mussel adhering to a sheet of poly(tetrafl uoroethylene) (Tefl on).

Bacterial Iron (III) respiration

David Richardson, University of East Anglia, United Kingdom Francisca Reyes, University of East Anglia, United Kingdom Katy Pitts, University of East Anglia, United Kingdom Andrew Hemmings, University of East Anglia, United Kingdom Harriet Seward, University of East Anglia, United Kingdom Andrew Thomson, University of East Anglia, United Kingdom Paul Dobbin, University of Essex, United Kingdom Gary Sawers, John Innes Centre, Norwich, United Kingdom

Iron respiration is a process in which Fe(III) ions act as a terminal electron acceptor for energy-conserving bacterial respiratory electron transport chains. It is an anaerobic process that provides bacteria with a means of respiration when oxygen is absent. The capacity for Fe(III) respiration is phylogenetically widespread and the ecological impact in micro- oxic and anoxic soils and sediments is considerable. At near-neutral pH Fe(III) speciation is very complex with most iron being present as insoluble polynuclear (hydr)oxo-bridged complexes or as soluble organic chelates, depending on the ligands available in a particular environment. To respire extracellular insoluble substrates, the bacteria must possess a means of moving electrons from the site of carbon metabolism in the cell cytoplasm to the outside of the cell. This talk will discuss the molecular nature of an iron respiration system, both at the level of gene regulation at the level of the operation and assembly of a multi-component electron transport system that spans the inner membrane, the periplasmic compartment and the outer-membrane. The organisms under study are members of the Shewanella genus, which are widespread facultative anaerobes that can express a number of terminal respiratory reductases including nitrate, nitrite, fumarate and trimethylamine N-oxide reductases. This organism an synthesise a number of tetra-heme and deca-heme cytochromes, located in the inner membrane, periplasm and outer membrane, which appear to be involved in iron (III) respiration. The characterisation of these, using a combination of structural and spectro-potentiometric methods will be described. Protocols for expressing these complex metalloproteins in Escherichia coli, such that they are functionally assembled in the correct subcellular compartment or membrane have been established and using this we have begun to piece together the minimal molecular requirements for respiration of soluble and insoluble Fe(III) species. Anaerobic growth with Fe(III) results in a large increases in the number of cytochromes expressed and an Fe(III)-responsive transcriptional regulation system that may be important in this regulatory process will also be presented. Journal of Inorganic Biochemistry 96 (2003) 67

Catecholamide siderophores of Azotobacter vinelandii: a comparison of their iron and molybdenum binding properties

Anne K Duhme-Klair, Department of Chemistry, University of York, United Kingdom

Iron and molybdenum, both components of the conventional nitrogenase, are essential for the optimal growth of the nitrogen- fi xing cells of Azotobacter vinelandii. Depending on the relative availability of iron and molybdenum in the growth medium A. vinelandii produces three different catecholamide siderophores with very characteristic metal-binding properties. All three siderophores are able to solubilise ferric hydroxide and support cell growth. However, the most effective siderophore is the hexadentate ligand protochelin which forms an extremely stable 1:1 complex with Fe(III). In contrast, the tetradentate ligand azotochelin [1] and the bidentate ligand aminochelin [2] do not only bind iron but also molybdenum with high affi nity. Under environmental conditions, molybdenum competes with iron for the siderophore ligands. Iron hydroxide solubilisation studies in the presence of equimolar concentrations of molybdate (fi g 1) revealed that the production of differently structured siderophores help the cells to adapt to variations in metal ions concentrations in their environment. While protochelin retains its iron-sequestering activity even in the presence of high molybdate levels, azotochelin and aminochelin are less active under these conditions. The solubilisation delay is most signifi cant in the case of azotochelin which is almost completely inactivated through complexation with molybdenum. Interestingly, the effect is less pronounced for aminochelin, due to its simple bidentate structure and high hydrophilicity. [1] Duhme, A.-K., Hider, R. C., Naldrett and M. J., Pau, R. N., J. Biol. Inorg. Chem., 3, 520-526 (1998). [2] Khodr, H. H., Hider, R. C. and Duhme-Klair, A.-K., J. Biol. Inorg. Chem., 7, 891-896 (2002). 68 Journal of Inorganic Biochemistry 96 (2003)

Microbial Siderophore Infl uence on Plutonium Biogeochemistry

Mary P Neu, Los Alamos National Laboratory, United States Hakim Boukhalfa, Los Alamos National Laboratory, United States Christy E Ruggiero, Los Alamos National Laboratory, United States Joe G Lack, Los Alamos National Laboratory, United States Larry E Hersman, Los Alamos National Laboratory, United States Sean D Reilly, Los Alamos National Laboratory, United States

To what extent can the environmental behavior of major elements be used to forecast the fate of trace actinide contaminants? For example, in response to low iron availability microorganisms produce and secrete siderophores, which bind Fe(III) and facilitate its cellular uptake. These chelating agents have the potential to similarly affect Pu(IV). The trihydroxamate siderophore, desferrioxamine B (DFB), forms highly stable complexes with both Fe(III) and Pu(IV). Interestingly, DFB affects their solubility likewise, being far less effective at solubilizing Fe and Pu solids in buffered neutral solution than simple chelators. Solubilization of Pu(OH)4 by EDTA was much slower after pre-treating the solid with DFB, suggesting that the siderophore passivates the surface. The Fe(III) reduction potential is shifted by 1.239 V by adding excess DFB; while for Pu(IV) the shift is 1.449 V. DFB can mediate Pu(IV) uptake into the bacterium A. fl avesens JG-9 in a metabolically active process that is in competition with Fe(III)-siderophore uptake. Preliminary results suggest that pyoverdin siderophores also interact similarly with Fe(III) and Pu(IV), supporting the hypotheses that siderophore- mediated uptake of Pu(IV) is a general phenomenon and that the environmental behavior of low-valent Pu can be estimated from that of Fe (III).

How microbes detoxify superoxide, hydrogen peroxide and nitric oxide. The non-heme iron reductive paradigm

Donald M Kurtz, University of Georgia, United States Joseph P Emerson, University of Georgia, United States Radu Silaghi-Dumitrescu, University of Georgia, United States Irene Kung, University of Georgia, United States Amaresh Das, University of Georgia, United States Lars G Ljungdahl, University of Georgia, United States

A novel reductive paradigm has emerged in recent years for detoxifi cation of superoxide, hydrogen peroxide and nitric oxide in air-sensitive bacteria and archaea. Among the enzymes that catalyze these reductions are superoxide reductase (SOR), rubrerythrin (Rbr), functioning as a peroxide reductase, and fl avoprotein A (FprA), functioning as a nitric oxide reductase. All three of these enzymes contain novel non-heme iron active sites. SORs contain a fi ve-coordinate, square-pyramidal

[Fe(His)4(Cys)] site that reacts with superoxide in a diffusion-controlled fashion to form a transient ferric-(hydro)peroxo species. This transient species decays with loss of hydrogen peroxide to a six-coordinate ferric site containing an extra glutamate ligand. The mechanism of this SOR reaction and evidence for the ferric-(hydro)peroxo intermediate will be discussed. Rbr contains a [Fe2(µ-O)(µ-Glu)2(Glu)3(His)] diferic active site that is related to those of diiron O2-activing enzymes, but is distinguished by an extra terminal glutamate ligand and one rather than two histidine ligands. Results will be described showing that Rbr functions as an effi cient peroxide reductase in which the diferrous site reduces hydrogen peroxide to water and that the diferrous/diferric interconversion occurs with interchange of one of the terminal glutamates with a second histidine ligand and a 1.8- angstrom movement of one of the irons. We recently described a fi ve-gene ‘oxidative stress protection’ cluster from the strictly anaerobic, acetogenic bacterium, Moorella thermoacetica. Within this cluster are genes encoding not only Rbr and SOR, but also two co-transcribed genes encoding FprA and Hrb (high-molecular weight rubredoxin). Results will be described showing that: i) FprA is a non-heme diiron fl avoprotein that functions as an effi cient nitric oxide reductase both in vitro and in vivo, and ii) Hrb contains both fl avin and rubredoxin-type sites and functions as an effi cient NADH:FprA oxidoreductase. Journal of Inorganic Biochemistry 96 (2003) 69

Superoxide dismutases from hyperthermophiles: clues to metal-ion specifi city

Geoffrey B Jameson, Massey University, New Zealand Julian J Adams, Massey University, New Zealand Paul D Hempstead, Massey University, New Zealand Bryan F Anderson, Massey University, New Zealand Irene Morgenstern-Badarau, Universite Paris-Sud, France James W Whittaker, Oregon Graduate Institute School of Science and Engineering, United States Edward N Baker, University of Auckland, New Zealand

The structures of the apo, Fe and Mn forms of the manganese-preferring superoxide dismutase from the hyperthermophilic microaerobic archaeon Pyrobaculum aerophilum (Pa-SOD) have been determined at 2.0, 2.2 and 1.9 angstrom, respectively, and the structure of the Fe-specifi c superoxide dismutase from the thermophilic methanogenic anerobic archaeon Methanobacterium thermoautotrophicum (Mt-FeSOD) has been determined at 2.6 angstrom. Corresponding values for R

(Rfree) are, presently, 0.16 (0.20), 0.19 (0.24), 0.17 (0.20), and 0.22 (0.24); the asymmetric unit is a dimer for Pa-SODs and a tetramer and half-tetramer for Mt-FeSOD. The extreme thermal stability of Pa-SOD, which survives the autoclaving necessary to insert metal ions into the E. coli- expressed apo-protein, appears to arise from a combination of previously identifi ed structural determinants for thermal stability. In particular, both the Pa-SODs and the Mt-FeSOD form extremely compact tetramers. In common with all other structurally characterised Mn- and FeSODs, the central metal ions of Pa-FeSOD, Pa-MnSOD and Mt-FeSOD have an identical primary coordination sphere. Three histidines, a monodentate aspartate and a solvent- derived ligand (OH- in oxidised MIII forms) coordinate in a trigonal bipyramidal manner, in which the solvent-derived ligand and a histidine ligand are trans to each other. No differences exist in the second coordination sphere – for both species a histidine C-H moiety hydrogen bonds to the solvent-derived ligand, although in many other SODs this aprotic C-H moiety is substituted by a glutamine NH2 moiety. It is only in the third coordination sphere, involving the hydrogen-bonding network to the amine moiety of the coordinated axial histidine that signifi cant differences can be found not only for these SODs but also for other SODs that have been well-characterised both structurally and functionally. A methionine thioether moiety appears to direct a different hydrogen-bonding network to the axial histidine for Mn-specifi c SODs, in contrast to Fe-specifi c SODs where an aliphatic residue is found at this location, position n on the helix supporting the axial histidine at position n+3.

Electronic/structural properties and enzymatic activity inspired by imidazolate-bridged heterodinuclear model compound as active site of Cu,Zn-superoxide dismutase

Kazuhiko Ichikawa, Graduate School of Environmental Earth Science, Hokkaido University, Japan Noritaka Hayashi, Graduate School of Environmental Earth Science, Hokkaido University, Japan Satoshi Hirakawa, Graduate School of Environmental Earth Science, Hokkaido University, Japan

Superoxide dismutase (SOD) is ubiquitous in living things. The enzyme serves a vital role in defending oxygen-utilizing life forms from oxidative damage due to superoxide ion. For the purpose of biofunctional modeling, the small-weight molecule is synthesized by molecular design mimicking the Cu,Zn-SOD active-site. In this work the model complex of Cu,Zn-SOD active site is prepared by self-assembly among mononuclear Cu(II) and Zn(II) complexes with their biomimetic ligands, and a bridging imidazolate. The ligands were synthesized by the tailor- made technique for the needs of the higher active heterodinuclear complex; the synthesized ligands for Cu(II) and Zn(II) were tris[(N-methyl-2-benzimidazolyl)methyl]amine L1′ and N,N-bis[(N-methyl-2-benzimidazolyl)methyl]glycine L2′, respectively. - The SOD-like IC50 activity of the heterodinuclear model complex L1′Cu(im )ZnL2′ 1 was much higher among the literature data for the Cu,Zn model compounds; the reason why 1 has the highest activity is shown as follows: 1) Its stability in solution was successfully examined by the measurements of the cyclic voltammogram(CV), ESR, and UV-vis spectra. 2)

The anode and cathode peak-potentials (Epa and Epc) showed the cathode shift in the order of Cu(aq) cp: may take place on time. 70 Journal of Inorganic Biochemistry 96 (2003)

Bicarbonate Dependent Peroxidase Activity of Human Copper, Zinc Superoxide Dismutase Induces Covalent Aggregation of Protein – Intermediacy of Tryptophan-Derived Oxidation Products

Balaraman Kalyanaraman, Medical College of Wisconsin, United States Hao Zhang, Medical College of Wisconsin, United States Christopher Andrekopoulos, Medical College of Wisconsin, United States Joy Joseph, Medical College of Wisconsin, United States John P Crow, University of Alabama at Birmingham, United States Hakim Karoui, Université de Provence, Marseille, France

This study addresses the mechanism of covalent aggregation of human Cu, Zn SOD (hSOD1WT) induced by bicarbonate - WT (HCO3 )-mediated peroxidase activity. High molecular weight species (apparent dimer and trimer of) hSOD1 were formed WT - - from incubation mixtures containing hSOD1 , H2O2, and HCO3 . HCO3 -dependent peroxidase activity and covalent WT WT + aggregation of hSOD1 were mimicked by UV photolysis of hSOD1 in the presence of a [Co(NH3)5CO3] complex that •- WT generates the carbonate radical anion (CO3 ). Human SOD1 has but one aromatic residue--a tryptophan residue (Trp-32) on the surface of the protein. Substitution of Trp-32 with phenylalanine produced a mutant (hSOD1W32F) which exhibits - WT HCO3 -dependent peroxidase activity similar to wild-type enzyme. However, unlike hSOD1 , incubations containing W32F - hSOD1 , H2O2, and HCO3 did not result in covalent aggregation of SOD1. These fi ndings indicate that Trp-32 is crucial •- WT for CO3 -induced covalent aggregation of hSOD1 . Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1WT, but not from hSOD1W32F. Spin traps also inhibited the aggregation of hSOD1WT. Fluorescence experiments •- - revealed that Trp-32 was further oxidized by CO3 , forming kynurenine-type products in HCO3 /H2O2-dependent aggregation of hSOD1WT, implicating a role for a Trp-32/oxygen adduct in the covalent aggregation of hSOD1WT. Taken together, these results indicate that TRp-32 is fundamentally involved in the oxidative aggregation of hSOD1.

The Structure of MntR from Bacillus subtilis

Arthur Glasfeld, Reed College, United States Emmanuel Guedon, ENSAIA – INPL, France John D Helmann, Cornell University, United States Richard G Brennan, Oregon Health and Science University, United States

MntR from Bacillus subtilis is a manganese-specifi c repressor protein responsible for regulating manganese uptake. In the presence of elevated Mn2+ concentrations, MntR binds to operators upstream of the mntABCD operon and the mntH gene, both of which encode manganese transporters. The 142-residue MntR shares 26% sequence identity with the 226-residue DtxR from Corynebacterium diphtheriae, a metalloregulatory protein that regulates iron homeostasis and the expression of the tox gene. While DtxR is activated in vivo by iron, MntR is selective for manganese. We have explored the origins of this selectivity by site-directed mutagenesis and x-ray crystallography. Comparison of the MntR and DtxR sequences suggested that mutations to suspected metal-binding residues of MntR might alter the protein’s selectivity. One such mutation, substituting Asp 8 of MntR with methionine, the residue appearing at the homologous position in DtxR, yielded a mutant of MntR (D8M) that is activated by both manganese and iron in vivo. We have solved the manganese-bound structures of wild-type MntR and D8M, to 1.75 and 1.61 angstroms respectively. The MntR homodimer displays signifi cant structural homology to DtxR, but lacks the C-terminal SH3-like domain of DtxR. However, the metal-binding pocket of MntR is substantially different from that of DtxR, as MntR binds two manganese ions that are separated by 3.3 angstroms, in contrast to the 9 angstrom cation-to-cation distance between the two DtxR-bound metal ions. In MntR residue Glu 11, which is not present in DtxR, permits formation of the binuclear Mn2+ cluster. By contrast, the D8M mutant binds only a single Mn2+ per monomer, due to alteration of the metal-binding site. The sole retained metal site shrinks from pseudo-heptacoordinate geometry to pseudo-hexacoordinate geometry. Thus, binding of a single metal ion is suffi cient to activate DNA-binding by the MntR repressors, but formation of a binuclear cluster contributes to the normal selectivity for manganese. Journal of Inorganic Biochemistry 96 (2003) 71

Regulation of Tumor Necrosis Factor Alpha (TNFα) mRNA by NUP-475, a Novel Zinc Binding Protein

Sarah LJ Michel, The Johns Hopkins University School of Medicine, United States Barbara T Amann, The Johns Hopkins University School of Medicine, United States Anthony L Guerrerio, The Johns Hopkins University School of Medicine, United States Jeremy M Berg, The Johns Hopkins University School of Medicine, United States

Zinc-binding domains have been found to mediate a wide range of macromolecular interactions including the regulation of gene expression. The protein NUP-475 (also named Tristetraprolin and Tis11) belongs to a newly discovered family of zinc binding proteins with tandem CCCH repeats. NUP-475 binds to the AU-rich sequence elements (AREs) of certain mRNA molecules, such as tumor necrosis factor alpha (TNF α), and favors the degradation of these mRNAs. As part of an effort to understand the macromolecular implications of sequence and the role of these novel zinc binding domains in RNA regulation, we are studying peptides that correspond to the zinc binding domains of NUP-475. To this end, we have expressed and synthesized a series of peptides that correspond to the zinc binding domains of NUP-475 and have begun to identify the domains required for nucleic acid binding using a combination of fl uorescence anisotropy and NMR spectroscopy. Although earlier studies on NUP-475 using gel shift assays determined that both zinc binding domains are necessary for nucleic acid binding, using our more sensitive fl uorescence anisotropy based assay, we have found that a single CCCH domain is capable of binding single-stranded RNA with considerable affi nity and selectivity. These results, as well as the peptides’ metal binding, folding capabilities and structural studies will be presented.

Metallocenter characterization of AlkB, a mononuclear non-heme Fe(II) DNA repair enzyme

Robert P Hausinger, Michigan State University, United States John McCracken, Michigan State University, United States Timothy F Henshaw, Michigan State University, United States

Alkylation of Escherichia coli DNA produces an array of modifi cations that are reversed by DNA repair enzymes. AlkB, an α-ketoglutarate (αKG)-dependent non-heme Fe oxygenase, directly repairs lesions involving 1-methyl-deoxyadenine and 3-methyl-deoxycytosine by hydroxylation of the methyl group followed by spontaneous loss of formaldehyde. Under anaerobic conditions, the metallocenter of Fe(II)AlkB exhibits no discernable electronic or EPR spectrum. Addition of αKG results in a weak absorption (λ ~500 nm, ε ~200 M-1 cm-1) attributed to an MLCT transition, as observed for other members of the αKG-dependent dioxygenase superfamily. Subsequent exposure to oxygen produces a blue chromophore (λ ~590 nm, ε 640 M-1 cm-1 as a lower limit) resulting from self-hydroxylation of an aromatic side chain and binding to the oxidized metal ion. Mass spectrometric investigation revealed the presence of OH-Trp at position 178, which likely coordinates the Fe(III) to produce the observed chromophore. When exposed to the oxygen analogue NO, anaerobic Fe(II)AlkB develops an S = 3/2 EPR signal. This signal is perturbed by the addition of αKG and is further altered by inclusion of DNA. The precise nature of this perturbation is dependent on the methylation status of the included DNA. Further characterization of the NO-FeAlkB complex with methylated and control DNA will be described. 72 Journal of Inorganic Biochemistry 96 (2003)

The Role of the Zinc Ribbon in Transcription Initiation in Archaea

Robert A Scott, University of Georgia, United States Matthew B Renfrow, University of Georgia, United States

Archaea and Eucarya initiate transcription of protein-coding genes through upstream TATA consensus promoter sequences. Formation of the transcription pre-initiation complex (PIC) begins with promoter recognition by the TATA- binding protein (TBP), then recruitment of the major general transcription factor IIB (TFIIB). The C-terminal two-thirds of TFIIB binds to TBP and to DNA both upstream and downstream of the TATA element, in a sequence- specifi c manner. Crystallographic analyses of the ternary (TFIIB-TBP-DNA) complex did not involve the N-terminal domain of TFIIB, which we have now structurally characterized by solution NMR in both archaeal and human versions, as a ‘zinc ribbon’ structural motif. We have used base-specifi c DNA photocrosslinking studies to localize the site of binding of TBP, TFIIB, and RNA polymerase on promoter DNA, providing a molecular topography of the archaeal PIC. In contrast to similar studies on eucaryal promoters, we fi nd that archaeal TFIIB contacts the DNA at locations within one or two base pairs of the transcription start site, suggesting roles for the N-terminal zinc ribbon in recruitment of RNA polymerase and transcription start-site selection through proper positioning of the RNA polymerase active site on the DNA.

Structure, DNA Binding, and Comparative Properties of Zinc, Cadmium, Lead, and Cobalt Finger-3 Peptides from Xenopus laevis Transcription Factor IIIA

David H Petering, Department of Chemistry, University of Wisconsin-Milwaukee, United States Dmitriy Krepkiy, Department of Chemistry, University of Wisconsin-Milwaukee, United States Holger Forsterling, Department of Chemistry, University of Wisconsin-Milwaukee, United States Charles Walsby, Department of Chemistry, Northwestern University, United States Brian Hoffman, Department of Chemistry, Northwestern University, United States

Zinc Finger proteins such as transcription factor IIIA (Zn9-TFIIIA) comprise the most common eukaryotic transcription factors. They have varying affi nity for Zn2+ and have been suggested as targets for toxic metal ions. Cd2+ or Pb2+ compete for binding to Zn-TFIIIA and destroy its ability to bind to DNA. Finger 3 (F3) of TFIIIA has been used as a model peptide to study these effects. Using competitive metal ion exchange or ligand substitution methods, the -log Kd for Co-, Zn-, Cd-, and Pb-F3 are 5.4, 7.4, 8.3, and 8.7, respectively. Zn-F3 binds to C Block DNA of the binding site for TFIIIA with a -log Kd of 4.2; Cd-F3 has a lower affi nity of 3.8. CD spectral analysis shows that, upon formation of the Zn-F3-C Block adduct, the peptide maintains its conformation but the DNA structure is slightly perturbed. 1D 1H NMR spectra reveals that apo-F3 binds to Zn2+ and Cd2+ but 2D NMR analysis is precluded by substantial line broadening, indicative of highly fl uxional structures. To stabilize the structures by increasing the peptide’s affi nity for metal ions, the inter-ligand amino acids were replaced with those from a consensus peptide to form mF3. Zn-mF3 and Cd-mF3 form kinetically stabilized complexes that yield 3D NMR structures that differ from one another in the angle between the fi nger’s α-helix axis and the plane of the β sheet. The Cd structural perturbation is consistent with the diminution in DNA-binding affi nity observed for Cd-F3 vs. Zn-F3. Pb-mF3 does not form an ordered structure. Notably, mF3 and F3 display similar affi nities for Zn2+ and Cd2+. The Co(II)-F3-C Block structure was examined by EPR and ENDOR. Co(II), 1H, and 14N spectra of the peptide do not change upon DNA binding. A 31P resonance is observed and assigned to the closest DNA backbone phosphorus in the native structure based on its calculated Co-P distance of 5.5 Α(Angstrom). Thus, Co-F3, but not Cd or Pb peptides, appears to retain the specifi c conformation of Zn-F3. Supported by NIH grants ES-04026, ES-04184, and HL-13531. Journal of Inorganic Biochemistry 96 (2003) 73

Controlling Structure and Activity of DNA: Metal Cations

Jerzy Leszczynski, Jackson State University, United States

Among the techniques successfully applied in investigations of features of different species including large biomolecules, computational methods have increasingly gained popularity. Fast development of hardware and constant improvements in computer coding make high-performance computational techniques a promising alternative to experimental studies. The metal ions are of the greatest biological importance because of their involvement in many natural processes. Their interactions with nucleic acids might cause heavy-metal toxicity in our environment, but from the other hand, interactions with metals provide foundation for application of cisplatin and its derivatives as anticancer agents. Metal ions are among the number of factors, which govern the structure and activity of DNA. The metal cations are usually located around the phosphate groups and stabilize the DNA double helix by electrostatic interactions with a negatively charged sugar-phosphate backbone. However, in addition they can also interact with nucleic acid bases (NABs). Such interactions are vital for stabilization of the triple and quadruple helices of DNA and the ribose-base stacking in Z-DNA. Recent computational studies reveal details of the experimentally observed phenomena. Recent comprehensive ab initio studies on structures and properties of DNA bases and their complexes with metal ions are reviewed. The consequences of metal ion base pair interactions could be altered by involvement of a polar solvent (water). The presence of a solvent could be crucial for the ionic systems, and many previously predicted effects might be profoundly exaggerated. The details of the infl uence of polar solvents on the molecular structures and stabilities of isolated and hydrogen-bonded bases are revealed Also the role of metal cations in metal cation-assisted stabilization of triplexes and tetrads of the DNA bases has been explained. In addition, a mechanisms of interaction of cisplatin and its analogues with DNA fragments has been suggested.

Copper-Dioxygen Chemistry with N3 Tripodal Ligands

Kiyoshi Fujisawa, Department of Chemistry, University of Tsukuba, Japan

The synthesis, spectroscopic characterization, and reactivity of copper(II) dioxygen complexes including peroxo, hydroperoxo (or alkylperoxo), and superoxo copper(II) complexes have been received much attention for recent years since such species are suggested to be involved as possible reactive intermediates in the enzymatic oxidations catalyzed by copper containing monooxygenases. Dopamine ß-hydroxylase (DBH) and peptidylglycine α-hydroxylating monooxygenase (PHM) contain a mononuclear copper ion in the reaction center. On the other hand, tyrosinase and catechol oxidase contain binuclear copper ions in their active sites. In an effort to gain insight into the reaction mechanism of their catalytic reactions, model studies have been explored. The highly sterically demanding hydrotris(pyrazolyl)bo - - rate ligands, HB(3,5-iPr2pz)3 (L1), HB(3-tBu-5-iPrpz)3 (L3), and the related tris(pyrazolyl)methane ligands, HC(3,5-iPr2pz)3(L1´) and HC(3-tBu-5-iPrpz)3 (L3´) were utilized as supporting ligands for preparing binuclear peroxo copper(II) complexes and mononuclear superoxo copper(II) in this work. There are some differences between borate ligands and methane ligands in the coordination structures and bonding interaction of copper(II) complexes as well as copper(I) complexes and reactivity toward dioxygen of copper(I) complexes. The details of the structures and properties of these copper(II) and copper(I) complexes will be presented. 74 Journal of Inorganic Biochemistry 96 (2003)

Metal Ion Mediated Activation of Dioxygen: Formation of Non-Heme Complexes with Terminal Oxo and Hydroxo Ligands

Andrew S Borovik, University of Kansas, United States Rajeev Gupta, University of Kansas, United States Matthew K Zart, University of Kansas, United States Peter L Larsen, University of Kansas, United States

Non-heme enzymes are known to activate dioxygen during activity. In several of these metalloproteins, the unique protein-derived microenvironment surrounding the metal ion active site aids in regulating function. To model these desirable effects, we have developed synthetic systems that control the structure and function around metal ions by creating bio-inspired microenvironments. The tripodal ligands tris[(N´-tert-butylureaylato)-N-ethyl)]amine ([1]) and tris(N-alkylcarbamoylmethyl)amine ([2]) stabilize monomeric MO and MOH complexes by creating unique cavities around the coordinated metal ion. We have successfully isolated a series of monomeric metal complexes with terminal oxo and hydroxo ligands, which are derived directly from dioxygen. This talk will describe the preparation, structural properties, and reactivity of these complexes.

Carboxylate shifts in labile Fe and Mn complexes

Christine J McKenzie, University of Southern Denmark, Odense, Denmark Birgit Jensen, University of Southern Denmark, Odense, Denmark Martin Mortensen, University of Southern Denmark, Odense, Denmark

We have used carboxylate-containing tetradentate and pentadentate ligands in the preparation of labile mono-, di- and tetra- nuclear Fe and Mn complexes. These complexes model aspects of the dynamic carboxylate donor coordination indicated in mechanisms of dioxygen activation by non-heme iron enzymes. The carboxylate groups can act both as a terminal and bridging donors in a series of complexes with metal ions ranging from +2 to +4 oxidation states. Shifts between these limiting structural forms coincide with changes in oxidation state and reactivity. The trends in redox potentials compared to analogous complexes of neutral ligand counterparts are those expected. However factors like chelate ring size and relative donor orientation can be signifi cant. In M(III)-OH (M=Fe, Mn) compounds, the orientation of a carboxylate donor is cis to the terminal hydroxide ligand, like for Fe- lipoxygenase. This orientation probably favours a high-spin Fe state. In the M(II) precursors of the mononuclear M(III)-OH compounds, the carboxylate bridges via one oxygen atom between the two metal ions of a dinuclear complex. Journal of Inorganic Biochemistry 96 (2003) 75

IV III 3+ The Mixed-valent {Fe (µ-O)(µ-carboxylato)2Fe } Core: Structure and Reactivity

Karl E Wieghardt, Max-Planck-Institut fuer Strahlenchemie, Germany Leonardo D Slep, Max-Planck-Institut fuer Strahlenchemie, Germany Thomas Weyhermueller, Max-Planck-Institut fuer Strahlenchemie, Germany Eckhard Bill, Max-Planck-Institut fuer Strahlenchemie, Germany Frank Neese, Max-Planck-Institut fuer Strahlenchemie, Germany Eberhard Bothe, Max-Planck-Institut fuer Strahlenchemie, Germany

In 1983 – exactly 20 years ago – two groups independently published the synthesis, structures and spectroscopic features of two non-heme model complexes containing the (µ-oxo)bis(µ-carboxylato)diiron(III) core.1,2 We have now discovered that IV III 3+ the compounds undergo a reversible one-electron oxidation yielding the mixed-valent {Fe (µ-O)(µ-carboxylato)2Fe } core with either an St=3/2 or an St=1/2 ground state, which are attained via intramolecular antiferromagnetic coupling of a low spin (SFe=1) or high spin (SFe=2) Fe(IV) ion with a high spin ferric ion (SFe=5/2), respectively. Spectroscopic features (UV-vis, EPR, Mössbauer) of this class II core are discussed as well as DFT calculations. The reactivity of this core has been examined and hydrogen abstraction reactions from weak C-H bonds occur in solution. 1) W.H. Armstrong, S.J. Lippard J. Am. Chem. Soc. (1983) 105, 4837. 2) K. Wieghardt, K. Pohl, W. Gebert Angew. Chem., Int. Ed. Engl. (1983) 22, 727.

Intracellular Coordination Chemistry of Zinc Receptors in Metal Traffi cking and Zn(II) Sensing

Thomas V O’Halloran, Northwestern University, United States

Zinc is frequently referred to as a trace element in biology; however this designation is misleading: Whether we consider the zinc quota for bacteria such as E.coli, yeast or mammalian cells, total zinc concentration falls in a narrow range centered around 0.1 millimolar. Clearly zinc is abundant but how is its speciation controlled in the cell? How much of this zinc is bound by biopolymers and how much is available in the putative ‘free zinc pool’? In the recent calibration of two zinc-sensing metalloregulatory proteins, factors which control metal homeostasis genes in response to metal concentration changes, it has been proposed that in E.coli, [Zn(II)]free is optimal in femtomolar range (Outten and O’Halloran, Science, 292, 2488, 2001). This is 6 orders of magnitude less than one free zinc ion per cell, consistent with an extraordinary chelation capacity for the intracellular milieu. We propose that availability of free Zn(II) ions in the cytoplasm is signifi cantly limited. How then does the correct metal fi nd the correct site within the cell? In the case of copper, this apparently dilemma is resolved by metallochaperones, factors that escort the metal and control the kinetics of metal ion transfer reactions. Unfortunately similar factors are not yet known for zinc. This talk will address the cytoplasmic chemistry of the cytoplasmic N-terminal domain of ZntA. This domain exhibits a variation on the structural scaffold of the copper chaperone Atx1, and the Menkes disease proteins, a variation that allows ZntA to mediate Zn(II) traffi cking hemistry. The function and coordination chemistry of this domain can be contrasted with the recent structural characterization of the zinc-binding domain of the ZntR metalloregulatory protein. The spectroscopy, energetics and structure of this metal-responsive switch provide new insights into how the cell monitors changes in zinc chemistry. Finally, to test emerging models for the zinc chemistry of the cell, a family of new fl uorescent zinc probes will be reported. These fl uorescent compounds can permiate the cell wall and are being used to delineate cytoplasmic and vesicular zinc chemistry in eukaryotic single cell organisms as well as neuronal tissues. 76 Journal of Inorganic Biochemistry 96 (2003)

The role of zinc in function of the Rhodobacter sphaeroides anti-sigma factor ChrR

Timothy J Donohue, University of Wisconsin-Madison, United States Jennifer R Anthony, University of Wisconsin-Madison, United States Jack Newman, University of Wisconsin-Madison, United States

We are studying the transcriptional control of the periplasmic electron carrier, cytochrome c2, in the facultative phototrophic bacterium, Rhodobacter sphaeroides. In this bacterium, transcription of the cytochrome c2 gene (cycA) is directed by 3 differentially-regulated promoters. A newly discovered zinc-metalloprotein, ChrR, inhibits σE, which is an alternative sigma factor that recognizes the cycA P3 promoter. Active preparations of ChrR and the σE-ChrR complex each contain stoichiometric amounts of zinc. Two N-terminal cysteine residues of ChrR in an N-terminal HX3CX2C motif appear to be critical to zinc binding since they are inaccessible to thiol-specifi c reagents unless the metal is removed from this protein. Zinc is required for ChrR function since the activity of ChrR as an inhibitor of σE in an in vitro transcription system and its ability to form a stable complex with this sigma factor is dependent on zinc binding. For example, single amino acid substitutions in ChrR that abolish zinc binding or treatment of the σE-ChrR complex with zinc-specifi c chelators destroy ChrR activity, suggesting that metal binding is critical to its function as an anti-sigma factor. The amino acid sequence surrounding the presumed N-terminal zinc-binding site of ChrR is conserved in other potential inhibitors of σE homologs in a variety of bacteria. This suggests that many other cells contain a previously unrecognized family of zinc-dependent anti-sigma factors. Experiments to determine the role of zinc in the ChrR function, how zinc is coordinated to ChrR, and the biological role of this signal transduction pathways are underway.

Intracellular Zinc(II) Visualization using the Selective Probe Zinquin A, and its Involvement in Zinc(II) Ternary Complexation

Stephen F Lincoln, Department of Chemistry, The University of Adelaide, Australia Kym M Hendrickson, Department of Chemistry, The University of Adelaide, Australia Jason P Geue, Department of Chemistry, The University of Adelaide, Australia Oska Wyness, Department of Chemistry, The University of Adelaide, Australia David A Ward, Department of Chemistry, The University of Adelaide, Australia

The widespread nature of Zn2+ in biology renders its visualization in cellular systems of signifi cant interest. Consequently, we developed a range of Zn2+ specifi c fl uorophores exemplifi ed by [2-methyl- 8-(4-toluenesulfon-amido)-6-quinolyl-oxy]acetic acid, Zinquin A, 1LH2, and its ethyl ester, Zinquin E, both of which fl uoresce when coordinated by Zn2+ and have been widely used in intracellular studies [1] (Figure 1). Most intracellular Zn2+ is bound by proteins and other ligands with very little existing in the fully hydrated state. As all reported studies of intracellular Zn2+ using Zinquin A and E show fl uorescence consistent with their coordination by Zn2+, it is probable that fl uorescent ternary Zn2+ complexes are formed where 1L2- is one of the ligands. Therefore, it is of interest to establish the extent to which 1L2- coordinates Zn2+ in ternary complexes and its fl uorescence is modifi ed by the presence of other ligands. Accordingly, we have studied the formation of ternary Zn2+ complexes of 1L2- and several well-characterized multidentate ligands by potentiometric and fl uorescence methods. The fl uorescence quantum yields vary signifi cantly as the 1L2- coordination environment changes in the ternary complex. These observations are in accord with a major proportion of the fl uorescence of 1L2- observed in intracellular studies arising from 1L2- in ternary complexes formed by Zn2+ with proteins and other ligands. The stoichiometry of the ternary complexes will vary with the ability of 1L2- to compete for coordination sites and raises the possibility that different Zn2+ probes may detect different ensembles of Zn2+ complexes in intracellular studies [2]. [1] K. M. Hendrickson et al, J. Chem. Soc., Dalton Trans., 1997, 3879. [2] K. M. Hendrickson, J. P. Geue, O. Wyness, S. F. Lincoln and A. D. Ward, J. Am. Chem. Soc., in press. Journal of Inorganic Biochemistry 96 (2003) 77

Cellular imaging of zinc ion by novel fl uorescent probes: ZnAFs

Kazuya Kikuchi, The University of Tokyo, Japan

Fluorescence probes for Zn2+, ZnAF-1 and ZnAF-2, were developed based on photo-induced electron transfer (PET) and their experimental utility to detect Zn2+ from living cells was examined. ZnAFs were designed, utilizing the TPEN moiety as the acceptor for Zn2+, linked to aminofl uorescein as a fl uorophore. ZnAFs showed high sensitivity and selectivity for Zn2+ against other cation species. Moreover, since there was nearly no basal fl uorescence, ZnAFs were suitable for biological applications. ZnAF-2 was applied to synaptic terminals of rat hippocampal mossy fi ber (MF) and it was visualized that Zn2+ was released by MF synaptic terminals in an activity-dependent manner, and thereafter diffuses extracellularly into the neighboring neurons to inhibit NMDA receptor activities. We also synthesized the fl uorescence probes (ZnAF-Rs) that make it possible to detect Zn2+ ratiometrically, thereby eliminating most or all of the possible variability due to differences in instrument effi ciency and content of effective dye. ZnAF-R2 was designed and synthesized by conjugating N,N-bis(2- pyridylmethyl) ethylenediamine and 6-bromobenzofuran derivatives. When these molecules formed complexes with Zn2+, the wavelengths of the excitation maxima were blue-shifted, while the emission maxima remained essentially unchanged. Therefore, these molecules should be useful for studies on the biological functions of Zn2+. 1 Hirano, T., Kikuchi, K., Urano Y. and Nagano, T. J. Am. Chem. Soc. 124, 6555-6562 (2002). 2 Ueno, S., Tsukamoto, M., Hirano, T., Kikuchi, K., Yamada, M.K., Nishiyama, N., Nagano, T., Matsuki, N. and Ikegaya Y. J. Cell Biology 158, 215-220 (2002). 3 Maruyama, S., Kikuchi, K., Hirano, T., Urano, Y. and Nagano T. J. Am. Chem. Soc. 124, 10650-10651 (2002).

Modeling the Structure of the NifEN Complex, a Putative Scaffold for the Biosynthesis of the FeMo-cofactor of Nitrogenase

Bruce A Averill, University of Toledo, United States Patricia A Eldredge, University of Toledo, United States Yanli Wang, University of Toledo, United States Xiche Hu, University of Toledo, United States Dong Xu, Oak Ridge National Laboratory, United States

Homology-based modelling and energy minimization methods have been used to predict the structure of the nifEN complex from Azotobacter vineladii, a putative molecular scaffold for the biosynthesis of the FeMo-cofactor of nitrogenase (FeMo- co). Special emphasis has been placed on analyzing the predicted structure to determine what the predicted environment of the FeMo-co precursor cluster in nifEN can tell us about the assembly of FeMo-co in vivo. The overall structure predicted for the nifEN complex is very similar to that of the nifDK complex (MoFe-protein), with some key differences in both the P- cluster and FeMo-co precursor sites. In particular, three of the cysteine residues that coordinate to the P-cluster of nitrogenase are absent in nifEN, leading to the conclusion that the nifEN complex contains a single 4Fe-4S cluster in the P-cluster site. The FeMo-cofactor precursor binding site is a cylindrical hydrophobic cavity that is 11 A x 8 A and lacks potential ligands that could coordinate to potential precursor clusters. Thus, biosynthesis of FeMo-co via a cluster condensation process seems unlikely. Inclusion of the homocitrate ligand of FeMo-co made it impossible to minimize the structure due to numerous unfavorable steric overlaps. This suggests that homocitrate is added to an FeMo-co precursor cluster after insertion of Mo and export of the precursor cluster from nifEN. These results suggest a radically new strategy for the synthesis of FeMo-co and synthetic analogs thereof. 78 Journal of Inorganic Biochemistry 96 (2003)

Disulfi de reduction mediated by site-specifi c [4Fe-4S] cluster chemistry

Michael K Johnson, University of Georgia, United States Elizabeth M Walters, University of Georgia, United States Guy N Jameson, Emory University, United States Boi Hanh Huynh, Emory University, United States Peter Schurmann, University of Neuchatel, Switzerland

Thioredoxin-mediated light regulation in plant chloroplasts involves a unique class of disulfi de reductases that catalyze disulfi de reduction in two one-electron steps using a [2Fe-2S] ferredoxin as the electron donor and an active-site comprising a [4Fe-4S] cluster and a redox-active disulfi de. Spectroscopic studies of ferredoxin:thioredoxin reductase (FTR) from spinach and Synechocystis have identifi ed a S = 0 [4Fe-4S]2+ cluster in both the as prepared (disulfi de-intact) and two-electron- reduced (disulfi de-cleaved) forms. However, freeze-quench EPR studies have identifi ed a transient, slow-relaxing resonance, g = 2.11, 2.00, 1.98, corresponding to a one-electron-reduced catalytic intermediate. Stable analogs of this intermediate with identical EPR characteristics have been prepared by selectively modifying (NEM FTR) or mutating (C57A FTR) the cysteine of the active-site disulfi de that forms a heterodisulfi de intermediate with the thioredoxin substrate. Detailed spectroscopic characterization of NEM FTR using absorption, EPR, ENDOR, VTMCD, resonance Raman and Mossbauer spectroscopies indicates that the one-electron reduced catalytic intermediate involves two-electron disulfi de reduction coupled with one- electron cluster oxidation to yield a unique type of S = 1/2 [4Fe-4S]3+ cluster with two cysteine residues ligated at a specifi c Fe site. Moreover, Mossbauer studies show that the one-electron oxidation of the cluster occurs predominantly at the unique Fe site. The results indicate a novel mechanism for disulfi de cleavage in two one-electron steps involving site-specifi c cluster chemistry. A similar mechanism is proposed for direct [4Fe-4S]-mediated cleavage of the CoM-S-S-CoB heterodisulfi de in methanogenic archaea by heterodisulfi de reductases. The potential relevance of this new type of site-specifi c cluster chemistry for understanding the mechanism of Fe-S cluster assembly and of reductive cleavage of S-adenosylmethionine by a [4Fe-4S]2+,+ cluster in the radical-SAM family of Fe-S enzymes will be discussed. Journal of Inorganic Biochemistry 96 (2003) 79

NifS-Mediated Assembly of [4Fe-4S] Clusters on NifU and Homologous Scaffold Proteins

Boi Hanh Huynh, Emory University, United States Guy N L Jameson, Department of Physics, Emory University, Atlanta, United States Archer D Smith, IV, Department of Chemistry, University of Georgia, Athens, United States Michael K Johnson, Department of Chemistry, University of Georgia, Athens, United States Jeverson Frazzon, Department of Biochemistry, Virginia Tech, Blacksburg, United States Dennis R Dean, Department of Biochemistry, Virginia Tech, Blacksburg, United States Carsten Krebs, Department of Biochemistry and Molecular Biology, Pennsylvania, United States

Although Fe-S clusters are among the most ubiquitus and functionally diverse prosthetic groups in biology, biosynthesis of Fe-S clusters has only recently become the subject of extensive genetic, biophysical and biochemical investigations. These intensive activities followed the initial discovery that two nif gene products, NifS and NifU, of the nitrogen fi xing bacterium Azotobacter vinelandii were essential for optimal assembly of Fe-S clusters for the nitrogenase proteins, and the subsequent detection of homologous of nifS and nifU, termed iscS and iscU, respectively, that are part of a widely conserved prokaryotic operon involved with general Fe-S cluster biosynthesis (1). Both NifS and IscS catalyze the desulfuration of L-cysteine to yield L-alanine and elemental sulfur, thus providing the source of sulfur for Fe-S cluster assembly (1). NifU is a homodimeric protein containing a redox-active [2Fe-2S] cluster and additional site(s) that serve as a scaffold for the NifS-mediated assembly of Fe-S clusters (2). In this study, UV-Vis absorption and Mössbauer spectroscopies were used to investigate this complex cluster assembly process on NifU. The results show that each dimeric NifU can assemble up to two [4Fe-4S] clusters. Analysis of the time-dependent spectra suggests that oxidation of ferrous ion and reduction of the NifS- bound cysteine persulfi de are coupled to form an initial transient [2Fe-2S] cluster, followed by reductive coupling of the [2Fe-2S] clusters to form the [4Fe-4S] clusters. Comparison of results obtained for other scaffold proteins suggest that the coupled delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur, observed in NifU, is a common mechanism used by scaffold proteins. In addition, the ability to assemble [4Fe-4S] clusters was found to be a common attribute of all the NifU homologous investigated in our laboratories. 1 Zheng et al. (1998) J. Biol. Chem. 273, 13264, and references therein. 2 Yuvaniyama et al. (2000) Proc. Natl. Acad. Sci. USA 97, 599

Thioredoxin-like [2Fe-2S] ferredoxins

Jacques Meyer France, CEA-Grenoble, France

Most low-potential ferredoxins (Fds) belong to either the [2Fe-2S] plant and mammalian type, or the [3Fe-4S]/[4Fe-4S] bacterial type. The existence of a third but more sparsely distributed family has long been known (1), but its thioredoxin-like fold has only recently been unveiled in a crystal structure (2). A remarkable property of these Fds is the presence near the [2Fe- 2S] cluster of a fl exible loop (ca. 15 residues) along which displacements of one of the four cysteine ligands are allowed (3). The data show that this loop can undergo large (> 10 A) movements driven by the affi nity of the [2Fe-2S] cluster for cysteine ligands. The possibility of Fe-S cluster-driven polypeptide chain movements of signifi cant amplitude is thereby established. A recent high resolution (1.5 A) structure of the wild type thioredoxin-like Fd from Aquifex aeolicus (4) has revealed that the [2Fe-2S] cluster differs by pronounced distortions from the canonical active site of plant- and mammalian-type Fds. Yet higher resolution structures, 1.25 and 1.05 A, respectively, have been obtained for the Cys55Ser and Cys59Ser variants of the A. aeolicus Fd (4). The data confi rm serine ligation to the [2Fe-2S] cluster, and show that both the cluster and surrounding protein matrix change in subtle ways to accommodate the ligand substitution. The structures also suggest leads toward understanding the occurrence of valence-delocalized states (S=9/2) in the reduced [2Fe-2S]+ level of these serine-ligated clusters (5,6). Thioredoxin-like Fds thus illustrate in various ways the combination of stabilizing and dynamic interactions that rules essential structural and functional properties of Fe-S proteins at large. 1 J. Meyer (2001) FEBS Lett. 509, 1. 2 A.P. Yeh et al. (2000) J. Mol. Biol. 300, 587. 3 M.-P. Golinelli et al. (1998) Biochemistry 37, 10429. 4 A.P. Yeh et al. (2002) J. Biol. Chem. 277, 34499. 5 B.R. Crouse et al. (1995) J. Am. Chem. Soc. 117, 9612. 6 C. Achim et al. (1996) J. Am. Chem. Soc. 118, 8168. 80 Journal of Inorganic Biochemistry 96 (2003)

Allosteric DNA and ultrafast electron transfer of drug with DNA

Xiaogang Qu, Changchun Institute of Applied Chemistry, Chinese Acade, China

DNA is polymorphic and exists in a variety of distinct conformations. It is well known that metal ions can modulate DNA conformations. Here we report the anticancer agent daunorubicin/WP900 enantiomeric pair can discriminate between right- and left-handed DNA, and that each compound can act as an allosteric effector to convert DNA to a conformation that binds each ligand with higher affi nity. These results open a new avenue for the rational design of potential anticancer agents that target polymorphic DNA. For the drug dynamic studies, we fi nd ultrafast electron transfer can take place between drug and DNA after photoactivation, and the direct involvement of molecular oxygen and DNA base-drug charge separation. The rates for the reduction of the drug and dioxygen indicate the crucial role of drug/base/O2 in the effi cient and catalytic redox cycling. These dynamical steps, and the subsequent reactions of the superoxide product(s), can account for the photoenhanced function of the drug in cells, and potentially for the cell death. The author thanks Dr. J. B. Chaires(UMC) and Dr. A. H. Zewail(Caltech) for all the supports.

Kinetically-Controlled Metal Binding to DNA: Inspiration for New Antitumor Drugs

Jan Reedijk, Leiden Institute of Chemistry, The Netherlands

Cancer treatment deals with either: surgical excision, high-energy irradiation or chemotherapy. In chemotherapy killing the tumor cells without causing too much harm to healthy cells is crucial. Most anticancer drugs act on DNA in one way or another, and therefore the interaction pathways of such compounds with nucleic acids are of interest. Many metal compounds known to bind to DNA, such as certain Pt and Ru compounds, exhibit anticancer properties. The fact that such compounds often have ligand-exchange kinetics of the same order of magnitude as tumor cell division, provides a clue to explain their activity [1,2,3]. The DNA-binding process of such compounds will be discussed with special attention to cisplatin derivatives and new Ru compounds, and to the cellular binding sites and processes of such compounds. Even though one generally accepts that platinum antitumor drugs eventually end up on the DNA, it remains a challenge to understand how (fast) they reach the DNA inside the cell nucleus, and how (fast) they are removed and/or repaired. The kinetics of ligand exchange around platinum and ruthenium appears to play a crucial role here, and especially the possible role of other ligands as intermediates is of great interest in the case of Pt compounds. If a potential active compound with the right geometric and electronic properties does not stay long enough coordinated to the DNA no activity will be the result. Fine-tuning of the compounds with substituents that have steric, electronic, and/or H-bonding implications for DNA binding, provides a great potential for new compounds. Finally, details of a new platinated DNA structure will presented and discussed in the light of the mechanistic studies of metal antitumor compounds, where kinetics and secondary hydrogen bonding play key roles. References: 1 Reedijk, J. (1996) Chem. Commun., 801-806 2 Reedijk, J. (1999) Chem. Rev. 99, 2499-2510 3 Reedijk, J. (2003), Proc. Natl. Acad. Sc.(USA),100, in press Journal of Inorganic Biochemistry 96 (2003) 81

Remarkable pKa shifts of adenine and cytosine nucleobases into the physiological pH range following metal binding

Bernhard Lippert, Universität Dortmund, Germany Marta Garijo Anorbe, Universität Dortmund, Germany Michael Roitzsch, Universität Dortmund, Germany Marc Sven Lüth, Universität Dortmund, Germany Jens Müller, Universität Dortmund, Germany Partick Lax, Universität Dortmund, Germany Helmut Sigel, University of Basel, Department of Chemistry, Inorganic, Switzerland

The common nucleobases (G, C, A, T) have pKa values in the range 4 > pKa > 9, hence values that are well outside the physiological pH range. There is increasing evidence, in particular with RNA’s, that perturbations of nucleobase acidities or basicities are possible, resulting in near-neutral pKa values. This can be the consequence of a particular microenvironment of the functional group of the nucleobases or of a chemical modifi cation, and can lead to acid/base catalysis.[1] For example, the Hepatitis Delta Virus (HDV) ribozyme appears to use a H+ transfer mechanism for catalysis,[2] and peptide bond formation in the ribosomal machinery is related to the action of an adenosine having a pKa of 7.6 (2).[3] Here we discuss scenarios in which transition metal binding to adenine and cytosine nucleobases leads to signifi cant pKa perturbations and shifts into the physiological pH range. Examples for the following cases will be discussed: (i) Metal binding to exocyclic amino groups of A and C (and concomitant shifts of a proton to N1 of A or N3 of C, resulting in ‘metal- stabilized rare nucleobase tautomers’), (ii) twofold metal binding (N1, N7) to A, and (iii) combined methylation of and metal coordination to A. Particular attention will be paid to neighboring group effects.[4] The fi ndings will be discussed with regard to their possible biological relevance. Financial support from DFG, FCI, SFOES/COST D20 and SNSF is gratefully acknowledged. [1] (a) Legault, P.; Pardi, A. J. Am. Chem. Soc. 1997, 119, 6621 and refs. cited. (b) Oyelere, A. K.; Kardon, J. R.; Strobel, S. A. Biochemistry 2002, 41, 3667. [2] Ferre-D’Amare, A. R.; Zhou, K. H.; Doudna, J. A. Nature 1998, 395, 567. [3] Muth, G. W.; Ortoleva-Donnelly, L.; Strobel, S. A. Science 2000, 289, 947. [4] (a) L¸th, M. S.; Willermann, M.; Lippert, B. Chem. Commun. 2001, 2058. (b) Müller, J.; GlahÈ, F.; Freisinger, E.; Lippert, B. Inorg. Chem. 1999, 38, 3160.

Sequence and Structural Selectivity of Nucleic Acid Binding Ligands

Jinsong Ren, Changchun Institute of Applied Chemistry, Chinese Acade, China Jonathan B Chaires, University of Mississippi Medical Center, United States

A novel, thermodynamically rigorous competition dialysis assay was developed that permits rapid screening of the sequence and structural selective nucleic acid binding of small molecules. DNA structural forms included in the assay ranged from single-stranded forms, through a variety of duplex forms, to multistranded triplex and tetraplex forms. Left-handed Z-DNA, RNA, and a DNA-RNA hybrid were also represented. Results on over 160 compounds were obtained so far, revealing a wide range in sequence and structural selectivity. An overview of these results will be presented, followed by a more detailed look at compounds that selectively recognize triplex,tetraplex and DNA-RNA hybrid structures.