Journal of Inorganic Biochemistry 96 (2003) 143

CW and Pulsed EPR Spectroscopy Reveal a New Structural Motif for the Active Site Mo Centre and an Unusual [4Fe-4S]+ Cluster in Dimethylsulfi de Dehydrogenase.

Graeme R Hanson, Centre for Metals in Biology and Centre for Magnetic Resonance, The University of Queensland, Australia Christopher J Noble, Centre for Metals in Biology and Centre for Magnetic Resonance, The University of Queensland, Australia Christopher A McDevitt, Centre for Metals in Biology and School of Molecular and Microbial Sciences, The University of Queensland, Australia Alastair G McEwan, Centre for Metals in Biology and School of Molecular and Microbial Sciences, The University of Queensland, Australia

Variable temperature CW EPR spectroscopy has identifi ed multiple redox centres (Mo(V), [3Fe-4S]+, [4Fe-4S]+) in ‘as isolated’ Rhodovulum sulfi dophilum dimethylsulfi de dehydrogenase (DMSDH).[1] A pH dependent EPR study of the Mo(V) 1 2 centre in H2O and H2O reveals the presence of three Mo(V) species in equilibrium, Mo(V)-OH2 (predominates between pH 6 and 8.2), Mo(V)-X (X, probably Cl-) and Mo(V)-OH. Comparison of the rhombicity and anisotropy parameters for these species with Mo(V) centres in other molybdoenzymes showed that they were most similar to the low pH nitrite species from E. coli nitrate reductase (NarGHI). Phylogenetic studies have shown that DMSDH, selenate reductase and NarGHI form a distinct class (Clade 2) of oxomolybdenum enzymes within the dimethylsulfoxide reductase family.[2] Whilst CW EPR studies have shown that the Mo ion is coordinated by four thiolate sulfur atoms from two pterins, and an aqua ligand, sequence homology suggests the protein side chain ligand is either Ser195, Thr214, or His220. Orientation selective pulsed HYSCORE spectra show unambiguously that His220 is ligated to the Mo ion. This represents the fi rst example of an oxomolybdenum enzyme with Histidine (nitrogen) coordination. A [4Fe-4S]+ cluster with an unusual g matrix (2.0158, 1.8870, 1.8620) which is very similar to that found for the minor conformation of Centre 1 in NarH [3] has also been identifi ed.[1] The two conformations in NarH may arise from an equilibrium involving the coordination/dissociation of a fi fth ligating atom (N or O) to an Fe atom in the cluster. The minor conformation corresponds to the cluster in which the fi fth ligand is coordinated. 1. McDevitt C.A.; Hanson, G.R.; Noble C.J.; Cheesman M.R.; McEwan A.G. Biochemistry, 2002, 41, 15234. 2. McDevitt C.A.; Hugenholtz P.; Hanson G.R.; McEwan A.G. Mol. Micro., 2002, 44, 1575. 3. Guigliarelli, B.; Asso, M., More, C.; A3.C.; Augher, V.; Blasco, F.; Pommier, J.; Giodano, G.; Bertrand, P. Eur. J. Biochem. 1992, 307, 63. 144 Journal of Inorganic Biochemistry 96 (2003)

Direct immobilization of yeast cytochrome c on gold via Cys102

Hendrik A Heering, Delft University of Technology, Kluyver Laboratory for Biotechnology, The Netherlands Frank GM Wiertz, Delft University of Technology, Kluyver Laboratory for Biotechnology, The Netherlands Cees Dekker, Delft University of Technology, Department of Nanoscience, The Netherlands Simon de Vries, Delft University of Technology, Kluyver Laboratory for Biotechnology, The Netherlands

Iso-1-cytochrome c from the yeast Saccharomyces cerevisiae (YCC) contains a surface cysteine residue (cys102) near the C-terminus, located opposite the docking site for enzymes such as cytochrome c peroxidase (CCP) [1]. We used the reactivity of this surface thiol to directly immobilize YCC on a bare gold electrode, with an orientation favorable for electron relay to enzymes in solution. Neither a spacer nor modifi cation of the gold surface is required, in contrast to the method of Haas et al. [2]. Cyclic voltammetry shows long-lived, reversible, and very fast electron transfer at potentials close to that of YCC in solution. The immobilized YCC is fully functional as electron donor to a variety of enzymes. Reductive catalytic turnover current is observed in the presence of its native redox partner CCP with hydrogen peroxide as substrate. Catalytic currents are also observed with the cd1 nitrite reductase (NIR) from Paracoccus denitrifi cans with nitrite, and the membrane-bound

NO-reductase (cNOR) from the same organism with either NO or O2 as substrate. The latter two enzymes offer the interesting opportunity to rebuild part of the denitrifi cation pathway [3] on the electrode. On gold micro-electrodes we are able to detect voltammetric reduction and oxidation peaks of 12 to 15 fA at 20 mV/s, and sub-pA catalytic currents in the presence of NIR and nitrite. The peak areas compute to 0.5-0.8 attomole of YCC (~300.000 to 500.000 molecules), which are by far the smallest samples detected by direct protein fi lm voltammetry to date. [1] H. Pelletier, J. Kraut, Science 258 (1992) 1748. [2] A.S. Haas, D.L. Pilloud, K.S. Reddy, G.T. Babcock, C.C. Moser, J.K. Blasie, P.L. Dutton, J. Phys. Chem. B 105 (2001) 11351. [3] I.M. Wasser, S. de Vries , P. Mo’nne-Loccoz, I. Schröder, K.D. Karlin, Chem. Rev. 102 (2002) 1201.

Structural-functional Model of Cytochrome P450: A Stable Synthetic Heme Thiolate

Tsunehiko Higuchi, Nagoya City University, Japan Noriyuki Suzuki, The University of Tokyo, Japan Tetsuo Nagano, The University of Tokyo, Japan

A distinctive structural feature of cytochrome P450 is the unusual thiolate coordination to heme. We have succeeded in the design and the synthesis of the fi rst synthetic thiolato-iron porphyrin (SR complex) which retains its structure during catalytic oxidation. Experiments using SR complex and the other iron porphyrins have revealed several remarkable axial thiolate ligand effects in catalytic oxidations. It has been found that thiolate ligation enhanced heterolytic cleavage of the O-O bond by heme in highly hydrophobic media and also made the active intermediate on heme suffi ciently potent for alkane hydroxylation. Another heme thiolate complex with NH-S hydrogen bond has been prepared for observing the effect of the hydrogen bond on the chemical property of heme thiolate. Hydrogen bonding toward the sulfur atom of heme thiolate increased the bond order of Fe-S bond, and altered the oxidizing property of its active species. This paper describes pronounced effects of the thiolate ligand on the catalysis of iron porphyrin relevant to that of P450 and NO synthase by using the chemical models. Journal of Inorganic Biochemistry 96 (2003) 145

Synthesis, characterisation and anti bacterial testing of bismuth maltol

Francis E Hinds, University of Western Sydney, Australia Janice R Aldrich-Wright, University of Western Sydney, Australia Peter Leverett, University of Western Sydney, Australia Stuart L Hazell, University of Southern Queensland, Australia

The increasing frequency of Helicobacter pylori (H. pylori) strains resistant to nitroimidazoles and clarithromycin is of major clinical concern and requires the development of new anti-H.pylori agents. Long before the association of H. pylori with gastroduodenal disease was identifi ed, bismuth was used to treat ulcer disease. Bismuth compounds such as ranitidine bismuth citrate (RBC) or colloidal bismuth citrate (DeNol) are used in combination with antibiotics to eradicate H.pylori infection. There are no reports of H.pylori developing resistance to bismuth compounds. Bismuth appears to have a non- specifi c effect on H.pylori, acting on numerous cellular mechanisms and contributing to the effectiveness of quadruple therapies or rescue therapies used following failure of initial treatment. Currently, the exact antibacterial mechanisms of bismuth is unclear; however the activity of a bismuth has the potential for improvement by altering the coordinating ligand structure. In the present study a novel bismuth compound, bismuth maltol was synthesised and characterised, then tested for invitro activity against strains of H.pylori. 146 Journal of Inorganic Biochemistry 96 (2003)

Is the Zinc Ion of Dipeptidyl Peptidase III directly Involved in Catalyzing the Substrate ?

Junzo Hirose, Department of applied biological science, Fukuyama University, Japan Hiroshi Kamigakiuchi, Department of applied biological science, Fukuyama University, Japan Hiroyuki Iwamoto, Japan Kayoko M Fukasawa, School of Density, Matsumoto Dental University, Japan

Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH..E (residue 450-455, 508) motif as the zinc binding site, is classifi ed as a zinc metallopeptidase (1, 2). But the zinc binding motif (HExxxH (x : appropriate amino acid)) of DPP III is different from that of common zinc binding motif (HExxH) like thermolysin. The copper derivative of thermolysine or carboxypeptidse A, well known zinc-peptidase, has no or little peptidase activity, but the copper derivative of DPP III (Cu- DPP III) showed high peptidase activity for Arg-Arg-NA, the substrate (2). It is the big question whether the zinc ion of DPP III is directly involved in catalyzing the substrate or not. To answer this question, EPR spectra of Cu-DPP III were measured in the presence of the competitive peptide inhibitor, Hisprophen (His-Pro-Phe-His-Leu-d-Val-Tyr). The EPR spectra of the adduct with Hisprophen was completely different from that of Cu-DPP III itself. This result clearly indicates that the copper ion in Cu-DPP III directly interacts with the competitive peptide inhibitor. To clarify the importance of the motif part (HELLGH) in DPP III, the recovery of the enzyme activity of apo-Leu453 deleted DPP III, which has the motif (HELGH) like thermolysin, and apo-DPP III were measured in the presence of cupric ions. The enzyme activity of apo-Leu453 deleted DPP III could not be recovered by the addition of cupric ions, while apo-DPP III could be easily reactivated by the addition of cupric ions. These observations indicate that the metal is directly involved in the expression of the enzyme activity and the motif part in DPP III infl uences the expression of the enzyme activity. References : 1) Fukasawa, K.M., Hirose, J., et al. Biochemistry 38, 8299 (1999) ; 2) Hirose et al, Biochemistry 39, 11860 (2001) Journal of Inorganic Biochemistry 96 (2003) 147

Short Peptide Alpha Helices Induced by Multiple Metal Clips

Huy N Hoang, Chemistry Department, The University of Queensland, Australia Gavin K Bryant, Chemistry Department, The University of Queensland, Australia Micheal J Kelso, Chemistry Department, The University of Queensland, Australia Renee L Beyer, Chemistry Department, The University of Queensland, Australia Trevor G Appleton, School of Molecular and Microbial Sciences Chemistry, The University of Queensland, Australia David P Fairlie, Australia

Alpha helices are key structural components of proteins and important recognition motifs in biology. New techniques for stabilizing short peptide helices could be valuable for studying protein folding, modeling proteins, creating artifi cial proteins, and may aid the design of inhibitors or mimics of protein function. We previously reported2 that 5-15 residue peptides, corresponding to the Zn-binding domain of thermolysin, react with 7 2+ [Pd(en)(ONO2)2]in DMF-d and 90% H2O 10% D2O to form a 22-membered [Pd(en)(H*ELTH*)] macrocycle that is helical in solution and acts as a template in nucleating helicity in both C- and N- terminal directions within the longer sequences in DMF. In water, however, there was less a-helicity observed, testifying to the diffi culty of fi xing intramolecular amide NH...OC H-bonds in competition with the H-bond donor solvent water. To expand the utility of [Pd(en)(H*XXXH*)]2+ as a helix- promoting module in solution, we now report the result that Ac-

H*ELTH*H*VTDH*-NH2(1), Ac-H*ELTH*AVTDYH*ELTH*-

NH2 (2) and Ac-H*AAAH*H*ELTH*H*VTDH*-NH2 (3) react with multiple equivalents of [Pd(en)(ONO2)2] to produce 7 15 1 exclusively 4-6 respectively in both DMF-d and water (90% H2O 10% D2O). Mass spectrometry, N- and 2D H- NMR spectroscopy, and CD spectra were used to characterise the structures 4-6, and their three dimensional structures were calculated from NOE restraints using simulated annealing protocols. Results demonstrate (a) selective coordination of metal ions at (i, i+4) histidine positions in water and DMF, (b) incorporation of 2 and 3 a turn-mimicking modules [Pd(en)(HELTH)]2+ in 10-15 residue peptides, and (c) facile conversion of unstructured peptides into 3- and 4- turn helices of macrocycles, with well defi ned α-helicity throughout and more structure in DMF than in water. (1) Scholtz, A.; Baldwin, R. L. Annu. Rev. Biophys. Biomol. Struct. 1992, 21, 95. (2) Kelso, M. J.; Hoang, H. N.; Oliver, W.; Sokolenko, N.; March, D. R.; Appleton, T. G.; Fairlie, D. P. Angew. Chem. Int. Ed. Engl. 2002, 42, 421. 148 Journal of Inorganic Biochemistry 96 (2003)

Applying databases of small molecule crystal structures to understanding the interactions about biologically relevent metal centres

Rosalie K Hocking, Sydney University, Australia Trevor W Hambley, Sydney University, Australia

Traditionally, the study of chemical bonding and the properties of transition-metal systems have not been associated with crystal structure data. It is generally viewed that the error on a single crystal structure is too great to be of any practical use in understanding chemical bonding phenomena. However, recently[1-3] we have successfully applied large databases of crystal structures to give us important information regarding the chemical bonding of transition metals. In this study we have expanded this work to consider a variety of biologically relevant metal-ligand interactions. The carboxylate ligand is an important ligand biologically, its interactions form an important component of the functions of many metalloenzymes particularly those of Zinc, Copper and Iron. The application of database analysis techniques has enabled us to gain a measure metal-carboxylate covalency based on the structural changes to the carboxylate ligand observable across databases of small molecule crystal structures, Figure 1. In addition to measures of carboxylate bonding the database techniques have proved applicable to a number of other biologically relevant ligands including CO, N2 and O2, providing new insights into the bonding of these ligands and a range of metal centres. References [1] Hocking R. K. and Hambley T. W. Inorg. Chem. 2002, 21(10) 2660-2666 [2] Hocking R. K. and Hambley T. W. Inorg. Chem. 2003 in press. [3] Hocking R. K. and Hambley T. W. Chem. Comm. Submitted. Journal of Inorganic Biochemistry 96 (2003) 149

ENDOR Characterization of Intermediates in the Activation of Dioxygen by Fe Enzymes

Brian M Hoffman, Department of Chemistry, Northwestern University, United States Roman Davydov, Northwestern University, United States

Dioxygen activation by heme enzymes is perhaps the most widely studied bioinorganic reaction: during the 1990’s, on average roughly one paper was published on cytochromes P450 alone, every fi ve hours of every day (Prof. Martin Newcomb, private communication). It had long been thought that heme hydroxylations involved the intermediates in Scheme 1, with the rate-limiting reduction of the dioxygen-bound ferriheme generating the peroxo-ferriheme, which then accepts one proton to generate the hydroperoxoferri-heme, followed by delivery of the second proton and heterolytic O-O cleavage to produce Compound I, the reactive, hydroxylating intermediate. However, all three intermediates are potentially capable of reacting with a substrate, and there are substantial reports that different combinations of enzyme/substrate involve reaction by a different intermediate. We therefore initiated a program designed to characterize the intermediates in heme hydroxylations. It employs cryogenic irradiation of an oxy-ferrous enzyme to inject the electron that initiates the catalytic process. Subsequent stepwise annealing stages permit an enzyme to traverse its catalytic cycle, through to product formation, with a combination of EPR and ENDOR measurements providing an optimal means of characterizing catalytic intermediates. We have applied this approach to studies of heme oxygenase in collaboration with Prof. Masao Ikeda-Saito and coworkers, studies of P450cam in collaboration with Prof. Steve Sligar and coworkers, and of nitric oxide synthase in collaboration with Profs. Bettie Sue Siler Masters, John Dawson, and coworkers. The status of this work will be presented. Dioxygen activation by mononuclear nonheme-Fe enzymes likewise plays an important role, and is involved in many processes of environmental, pharmaceutical, and medical signifi cance. Studies of nonheme Fe enzymes such as napthalene dioxygenase and superoxide reductase may be presented as well. 150 Journal of Inorganic Biochemistry 96 (2003)

Conformation of the Tyrosyl Radical in Escherichia coli Ribonucleotide Reductase R2

Martin Högbom, Department Biochemistry and Biophysics, Stockholm University, Sweden Marcus Galander, Max-Volmer-Laboratory, Technical University Berlin,PC14, Germany Martin Andersson, Department Biochemistry and Biophysics, Stockholm University, Sweden Matthias Kolberg, Department of Biochemistry, University of Oslo, N-0316, Norway Pär Nordlund, Department Biochemistry and Biophysics, Stockholm University, Sweden Friedhelm Lendzian, Max-Volmer-Laboratory, Technical University Berlin,PC14, Germany Pär Nordlund, Department Biochemistry and Biophysics, Stockholm University, Sweden Friedhelm Lendzian, Max-Volmer-Laboratory, Technical University Berlin,PC14, Germany

The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions but no structural information has been available for the active radical containing state of the protein. We have generated the tyrosyl radical Y122* in single crystals of E. coli R2 and performed high-fi eld EPR experiments on the radical containing crystals. A full rotational pattern of the spectra was collected, and the orientation of the radical g-tensor axes was determined relative to the crystal unit cell axes. This result directly yields the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42 Å X-ray structure of the met form of the protein. Comparison of the orientation of the radical Y122* obtained from high-fi eld EPR with that of the reduced tyrosine Y122-OH reveals a signifi cant rotation of the tyrosyl sidechain, away from the diiron center, in the active radical state. This may be one explanation for the radical’s unusual stability. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 will be discussed. Ref. Högbom, M. Galander, M. Andersson, M. Kolberg, M. Hofbauer, W. Lassmann, G. Nordlund, P. and Lendzian, F. PNAS 100 (6): 3209-3214, MAR 18, 2003 Journal of Inorganic Biochemistry 96 (2003) 151

The Effect of Oxidation State on Inhibition of Nitrate Reductase

Kevin R Hoke, Inorganic Chemistry Laboratory, Oxford University, United Kingdom Sean J Elliott, Boston University, United States Fraser A Armstrong, Oxford University, United Kingdom

Using protein fi lm voltammetry, we are exploring the effects of inhibitors on the cyclic voltammetry of a nitrate reductase, NaRGHI from E. Coli. Under turnover conditions, the uninhibited form of the enzyme gives cyclic voltammograms that exhibit an initial phase of activity at low driving force, with an additional increase at higher driving force (1). This complex behavior can result from enhanced affi nity of the substrate for Mo (V) over Mo (IV) forms of the enzyme. This preference results in a modulation of the amount of enzyme-substrate complex with applied driving force, and in turn, results in the observed voltammetric response. When azide is used as a competitive inhibitor (2), we see a simplifi cation of the shape of the catalytic voltammetry to that of a single sigmoid. From a Lineweaver-Burk analysis of the current at different potentials, we can infer that the apparent Km value of the Mo(V) state is increased greatly in the presence of azide, becoming nearly equal to that of Mo(IV). This indicates preferred binding of azide to Mo(V) over Mo(IV), out-competing the similar preference exhibited by nitrate itself. We are actively characterizing this behavior, and that for other inhibitors, with the goal of using changes in voltammetric shape to probe the inhibition of metalloenzymes. Such changes give an additional dimension in enzymology -- the role of specifi c oxidation states in inhibitor and substrate binding. 1. Anderson, L. J., Richardson, D. J., and Butt, J. N. (2001) Biochemistry 40, 11294-11307. 2. Butler, C. S., Charnock, J. M., Bennett, B., Sears, H. J., Reilly, A. J., Ferguson, S. J., Garner, C. D., Lowe, D. J., Thomson, A. J., Berks, B. C., and Richardson, D. J. (1999) Biochemistry 38, 9000-9012. 152 Journal of Inorganic Biochemistry 96 (2003)

The Synthesis of 2,3-Diamino-2,3-dideoxy-α-D-glucose, 3,4-Diamino-6- hydroxymethyl-tetrahydro-pyran-2,5-diol and their Chiral platinum Compounds

Marc van Holst, College of Science, Technology and Environment, School of Science, Food and Horticulture, University of Western Sydney, Australia David Jaramillo, College of Science, Technology and Environment, School of Science, Food and Horticulture, University of Western Sydney, Australia Janice R Aldrich-Wright, College of Science, Technology and Environment, School of Science, Food and Horticulture, University of Western Sydney, Australia

A small number of transition metal complexes containing amino sugars have been synthesized, isolated and characterised. Their interactions are of importance in the study of carbohydrate-mediated biological processes, which may lead to the development of carbohydrate-based therapeutics.Tsubomura et al1 have synthesised and fully characterised cisplatin-type complexes of amino sugars, which have displayed in vivo antitumor activity. 2,3-Diamino-2,3-dideoxy-α-D-glucose and 3,4-Diamino-6-hydroxymethyl-tetrahydro-pyran-2,5-diol differ by the chirality of one chiral centre, and are made using literature methods1. Upon coordination the structure of the complexes have been modelled and should be substantially different. Subsequently, it was of interest to produce platinum(II) complexes containing these related diamino sugars to investigate the effect of chirality on the biological activity of these platinum complexes. 1 Tsubomura, T.; Yano, S.; Kobayashi, K.; Sakurai, T.; Yoshikawa, S. J. Chem. Soc., Chem. Commun., 1986, 459

The electrochemistry of cytochrome P450. What are we actually measuring?

Michael J Honeychurch, Australia

The cytochrome P450 family of monooxygenases have been much studied over the last 30 years. Electrochemical measurements are typically performed to determine fundamental parameters such as the redox potential of the enzyme or to study the electron transfer between the enzyme and various electrodes either directly or through the use of mediators. In addition to fundamental studies electrochemical studies of P450 are of great interest due to the possibility of developing applications such as biosensors for analyte detection and bioelectrochemical catalysts for product synthesis. The electrochemical behaviour of enzymes is more complicated than that of smaller molecules that have traditionally been the subject of studies by electrochemists. The most important complication is the possibility of conformation changes within the enzyme occurring during the measurement process. How do we know that an electrochemical response is due to the functioning enzyme and not some altered conformation of the enzyme in which its activity is reduced or eliminated altogether? To date, in the published work, no systematic set of control experiments appear to have been carried out in order to eliminate the electrochemical response as being due to eg. cytochrome P420 or displaced heme. This paper will present a series of control experiments that may be used to eliminate the possibility that any electrochemical response obtained is due to a conformationally altered protein. Journal of Inorganic Biochemistry 96 (2003) 153

In search for anticancer compounds; new bis(2-phenylazopyridine) ruthenium(II) complexes

Anna CG Hotze, Leiden University, The Netherlands Jaap G Haasnoot, Leiden University, The Netherlands Jan Reedijk, Leiden University, The Netherlands

The α-isomer of the dichlorobis(2-phenylazopyridine) ruthenium(II) complexes, α-[Ru(azpy)2Cl2] (α indicates the coordinating pairs Cl, N(py), N(azo) are cis, trans, cis, respectively), shows a remarkably high cytotoxicity against a series of tumor-cell lines.[1,5] In order to investigate structure-activity relationships, research has been focussed on the investigation of DNA model base coordination[2-4] and synthesis of related complexes by variation of the azpy ligands and anions. The water solubility of the parent compound has been improved by changing the nature of the anions. A series of new water-soluble complexes α-[Ru(azpy)2X] with X=(nitrate)2, oxalato, malonato and 1,1-cyclobutanedicarboxylato (cbdca) (see fi gure) has been designed. The cytotoxicity and cellular uptake of these water-soluble complexes have been evaluated in several human tumor cell lines.[5] These complexes display a very high cytotoxicity, comparable to the activity of the anticancer drug cisplatin and even better than the second generation drug carboplatin. Moreover, the high cytotoxicity in A2780cisR, a cisplatin resistant cell line, shows that this class of ruthenium complexes might not be infl uenced by the multifactorial resistance mechanism that affect platinum anticancer agents. References: [1] A.H. Velders, H. Kooijman, A.L. Spek, J.G. Haasnoot, D. de Vos, J. Reedijk, Inorg. Chem. 2000, 39, 2966-2967. [2] A.C.G. Hotze, A.H. Velders, F. Ugozolli, M. Biagini-Cingi, A.M. Manotti-Lanfredi, J.G. Haasnoot, J. Reedijk, Inorg. Chem. 2000, 39, 3838-3844 [3] A.C.G. Hotze, M.E.T. Broekhuisen, A.H. Velders, K. v.d. Schilden, J. G. Haasnoot, J. Reedijk, Eur. J. Inorg. Chem. 2002, 369-376 [4] A.C.G. Hotze, M.E.T. Broekhuisen, A.H. Velders, H. Kooijman, A.L. Spek, J. G. Haasnoot, J. Reedijk, J. Chem. Soc., Dalton Trans.,. 2002, 2809-2810 [5] A.C.G. Hotze, M. Bacac, A.H. Velders, B.A.J. Jansen, H. Kooijman, A.L. Spek, J.G. Haasnoot, J. Reedijk, J. Med. Chem. 2002, submitted

DNA binding studies of ruthenium(II) dipyridotetrahydrophenazine complexes

Warren A Howard, School of Science, Food and Horticulture, University of Western Sydney, Australia Janice R Aldrich-Wright, School of Science, Food and Horticulture, University of Western Sydney, Australia

Over the past three decades there has been considerable interest in the DNA binding properties of ruthenium(II) metal complexes. However, despite many publications in the fi eld, studies on specifi c binding data has remained limited. 2+ A series of ruthenium(II) polypyridyl complexes of the type [Ru(L-L)2dpqC] , where L-L = 1,10-phenanthroline (phen),

2,9-dimethyl-1,10-phenanthroline (Me2phen), 3,4,7,8-tetramethyl-1,10,-phenanthroline (Me4phen), 2,2’-dipyridyl (bipy) or 4,4’-dimethyl-2,2’-dipyridyl (Me2bipy), have been investigated in this work. The equilibrium binding constants (Kb) and binding sites (n) were determined by fl uorescence and UV-Vis spectroscopy (See Figure). Derivations were conducted following the McGhee and von Hippel method and calculated using non-linear regression (Sigma PlotTM software). Figure: A) Fluorescence titration spectra; 3.93 x 10-6 M 2+ [Ru(bipy)2dpqC] in 3 mL Phosphate buffer (10 mM sodium phosphate, 100 mM sodium chloride, pH 7.0). 154 Journal of Inorganic Biochemistry 96 (2003)

The Chemistry of the Vanadium Complexes in a S-rich Coordination Environment

Hua-Fen Hsu, Chemistry Department, National Cheng Kung University, Taiwan, Province of China Wei-Cheng Chu, Taiwan, Province of China Chen-Hsiung Hung, Chemistry Department, National Changhua University of E, Taiwan, Province of China

In our effort to understand V-nitrogenase, an enzyme catalyzing the reduction of dinitrogen, we have explored the vanadium chemistry in a sulfur-rich ligation environment. The enzyme has not been understood as thoroughly as its analog, Mo- nitrogenase. However, the spectroscopic studies have shown that the vanadium in the enzyme has the S-rich coordination sphere. To comprehend the role of the vanadium in the enzyme, we have been aiming on obtaining vanadium complexes binding to the substrates, intermediates or products of the nitrogen fi xation. By utilizing tristhilatophosphine ligand system, PS3, PS3' and PS3", we have obtained several novel V(III) complexes, [V(PS3”)(N2H4)3] (1), [V(PS3')(Im)3] (2), [V(PS3í)(Py)] (3). The vanadium centers in compunds 1 and 2 adopt 7-coordinate geometry by binding to the tetradentate title ligand and three coligands, hydrazines and imidazoles, respectively. In contrast, compound 3 embraces 5-coordinate structure with the same title ligand and one pyridine completing the coordination. Compounds 1 and 2 are the fi rst examples of a 7-coordinate V(III) complex in a sulfur-rich ligation environment. Compound 2 is one of the few vanadium complexes binding to hydrazine, an intermediate of the nitrogen fi xation. Our results demonstrate the diversity of the V(III) coordination chemistry. In addition, we have also obtained some high valent vanadium complexes such as [V(IV)(PS3)2] - - and [V(IV)2(PS3")2(CH3O )3] .

Interaction of Metal-Thiolate Cluster with Polypeptide and Domains Transformation in Metallothioneins

Zhong-Xian Huang, Department of Chemistry, Fudan University, China Wen-Hao Yu, Department of Chemistry, Fudan University, China Qi Zheng, Department of Chemistry, Fudan University, China Yuan Gao, Department of Chemistry, Fudan University, China Bin Cai, Department of Chemistry, Fudan University, China

Metallothioneins (MTs) are a family of small protein, having high content of cysteines and metal ions. Metallothioneins may play multiple functions in vivo including regulation of zinc and copper homeostasis, detoxifi cation of heavy metals and scavenging of oxidative agents, but its certain function is still elusive. Mammalian metallothioneins consist of a b-domain with Cd3S9 cluster and an a-domain with Cd4S11 cluster. The possible infl uence of cysteine residues on the binding and stability of MT domains have been examined by domain transformation (from b-a domain to a-a or b-b domain by mutation). It shows differences in properties and reactions are closely related to their distinct, conservative cysteine number and position. Furthermore, MT3 is a brain-specifi c isoform of MTs, which is functionally unique in inhibiting the neurotrophic activity of brain. In comparison of MT1/2, MT3 also contains 20 cysteines at conserved position, comprising two metal-thiolate clusters. However, two additional inserts (Thr5 and an acidic hexapeptide EAAEAE at position-55) in the MT3 show prominent difference from MT1/2. The functional difference between MT3 and MT1/2 implies the uniqueness of MT3 in structure and property. To reveal the essence of these additional residue and loop, a series of mutation have been designed and their properties and reactivity have been studied. Our detailed studies revealed obviously, that the EAAEAE insert is essential to the property of MT3. Meanwhile, alteration at Thr5 makes no signifi cant effect on the properties of MT3. References (1) Uchida, Y. et al., (1991) Neuron, 7, 337-347. (2) Huang, Z.-X. et al., J. Inorg. Biochem. 2002, 92, 183-192. (3) Vallee, B. L. Methods Enzymol. 1991, 205, 3-7. This project was supported by NSF of China Journal of Inorganic Biochemistry 96 (2003) 155

EPR- and UV-vis-spectroscopical evidence for an altered respiratory chain in Halobacterium salinarum due to iron limitation

Dirk Hubmacher, University of Luebeck, Institute for Biochemistry, Ratz, Germany Berthold F Matzanke, University of Luebeck, Isotope Laboratory TNF, Ratzebur, Germany Stefan AneMüller, University of Luebeck, Institute for Biochemistry, Ratz, Germany

H. salinarum belongs to the Archaea, the third domain of life and thrives in hypersaline environments. Growth of H. salinarum depends on at least 2.5 M NaCl and the demand on other nutrients like amino acids is very high. Energy requirements are fullfi lled by respiration, the use of bacteriorhodopsin under low oxygen conditions or by arginine fermentation under strictly anaerobic conditions. The components of the respiratory chain were studied and revealed c-, b-, a- and o-type cytochromes as well as iron-sulphur cluster containing proteins (1). To our knowledge, nothing is known on the regulation of the aerobic electron transport chain by environmental factors like iron- availability. For our investigations, we used H. salinarum JW5 which is defective in carotinoid-biosynthesis and thus can only convert energy by aerobic respiration. We could previously show, that H. salinarum can be grown in iron-limited cultures for months (2). Our work presented here revealed, that iron-starved membranes show an altered cytochrome pattern compared to membranes obtained from iron replete cells. Furthermore, we provide EPR-spectroscopical evidence for a strong reduction of proteins, containing iron-sulphur-cluster in iron starved membranes. However, the respiratory rates we determined indicate, that the respiratory chain is still active in cell-membranes grown under iron limitation. In conclusion, the ratio between NADH-dehydrogenase, belonging to type-II in H. salinarum (containing only FAD as cofactor) and the succinate-dehydrogenase (harbouring three FeS-cluster as cofactors) seems to be altered. Under iron limitation the electrons are fed in the respiratory electron transport chain mainly by NADH-dehydrogenase, type-II and to a lesser extend by succinate-dehydrogenase which seems to be highly affected by iron limitation. (1) Skulachev, V. P. (1993) in ‘The biochemistry of archaebacteria’, eds. Kates, M., Kushner, D. J., & Matheson, A. T. (Elsevier), 25-40 (2) Hubmacher, D., Matzanke, B. F. & Anemuller, S., (2002) Biochem. Soc. Trans. 30, 710-712. 156 Journal of Inorganic Biochemistry 96 (2003)

Insights into Catalysis by Flavocytochrome c3 using Protein Film Voltammetry Janette M Hudson, Inorganic Chemistry Laboratory, University of Oxford, United Kingdom Katy R Halliwell, Inorganic Chemistry Laboratory, University of Oxford, United Kingdom Emma L Rothery, Department of Chemistry, University of Edinburgh, United Kingdom Stephen K Chapman, Department of Chemistry, University of Edinburgh, United Kingdom Fraser A Armstrong, Inorganic Chemistry Laboratory, University of Oxford, United Kingdom

The catalytic mechanism of fl avocytochrome c3, a soluble fumarate reductase from the Shewanella genus, has been investigated using protein fi lm voltammetry. The enzyme has been adsorbed onto a graphite electrode in a catalytically active form, allowing it to perform the two electron, two proton reduction of fumarate to succinate with the electrode as a direct electron donor. The four heme groups act as an ‘electron wire’ to allow very rapid communication between the buried fl avin and the electrode. The cooperative two-electron fl avin peak is clearly visible above the four one-electron heme groups in cyclic voltammetry up to scan rates of 100 V/s (see fi gures). The ability of fast scan protein fi lm voltammetry1 to reveal kinetic and thermodynamic data simultaneously has been utilised to study the electron-transfer rates over a range of conditions. These studies shed light on the question: what is the rate determining step? 1 Jones, A. K., Camba, R., Reid, G. A., Chapman, S. K. and Armstrong, F. A. J. Am. Chem. Soc. 122, 6494-6495 (2000).

Vitamin C causes a much-pronounced damage to DNA in the presence of Cd2+ but not in the presence of Zn2+ or Ag+. Why?

Fazlul Huq, School of Biomedical Sciences, University of Sydney, Australia Zahed Hossain, Australia

Recently it has been reported that 1:1 mixture of cadmium(II) acetate and ascorbate caused a much greater damage to DNA than that caused by either ascorbate or cadmium(II) acetate alone. Similarly it was found that 1:1 mixture of silver(I) acetate caused a much greater damage to DNA than that due to either silver(I) acetate or ascorbate. However, it was found that 1:1 mixture of cadmium(II) acetate and ascorbate was more damaging to DNA than 1:1 mixture of silver(I) acetate and ascorbate. SOD and Rhodamine assays carried out in the present study show that superoxide ions and hydroxyl radicals were produced from the interaction between molecular oxygen and 1:1 mixture of cadmium(II) acetate and ascorbate or 1:1 mixture of silver(I) acetate and ascorbate. Molecular modeling analysis combined with pH measurement show that in presence of Cd2+, ascorbic acid essentially acts as a strong polyprotic acid whereas in presence of Ag+, it acts as a monoprotic acid. It is believed that it is the covalent binding of ascorbate with the metal ions Cd2+ and Ag+ that causes it molecular activation such that it is more susceptible to attack by molecular oxygen. Reactive oxygen species produced from the interaction between activated ascorbate species and molecular oxygen damage DNA. Journal of Inorganic Biochemistry 96 (2003) 157

A kinetic study of catalytic CO2 hydration by water-soluble model compound of carbonic anhydrase and an anion inhibition effect on CO2 hydration Kazuhiko Ichikawa, Grad. School of Environmental Earth Science, Hokkaido University, Japan Kou Nakata, Grad. School of Environmental Earth Science, Hokkaido University, Japan Naomi Shiina, Grad. School of Environmental Earth Science, Hokkaido University, Japan Mitunori Izumi, Research Institute of Higher Education Programs, Hokkaido Tokai University, Japan

We have been for the latest ten years concerned with the small weight zinc-model compounds mimicking each active site of zinc enzymes and in vitro exerting biofunction.

A kinetic study of CO2 hydration was carried out using the water-soluble zinc model complex with water-soluble - - nitrilotris(2-benzimidazolylmethyl-6-sulfonate) L1S, [L1SZn(OH2)] , in the presence and absence of anion inhibitor NCS - 2 3 3 -1 -1 or Cl . The obtained rate constants kcat for CO2 hydration were 5.9 x 10 , 1. 7 x 10 , and 3.1 x 10 M s at 5, 10, and 15°C, 4 -1 -1 respectively ; kcat = ca. 10 M s extrapolated towards 25°C has been the largest among the literature data of kcat for zinc - - model complexes. It was also revealed that NCS , Cl and acetazolamide play a role of inhibitors by the decrease of kcat ; 7 x 2 3 -1 -1 - - - 10 and 2 x 10 M s for NCS and Cl at 15°C, respectively. The sequence of their magnitudes in kcat is Cl ~ acetazolamide > NCS -. The crystal structure of anion-binding zinc model complex with water-insoluble tris(2-benzimidazolylmethyl)amine 2+ L1, [L1Zn(OH2)]0.5[L1ZnCl]0.5 (ClO4)1.5, was revealed by X-ray crystallography. The geometry around Zn was tetrahedrally - coordinated by three benzimidazolyl nitrogen atoms and one oxygen atom of H2O, or Cl .

CO Binding Study of Mouse Heme-regulated eIF-2α Kinase: Kinetics and Resonance Raman Spectra

Jotaro Igarashi, Institute of Multidisciplinary Research for Advanced Materials, Japan Akira Sato, Center for Integrative Bioscience, Okazaki National Research Institute, Japan Teizo Kitagawa, Okazaki National Research Institute, Japan Ikuko Sagami, Institute of Multidisciplinary Research for Advanced Materials, Japan Toru Shimizu, Institute of Multidisciplinary Research for Advanced Materials, Japan

Heme-regulated eukaryotic initiation factor (eIF)-2α kinase (HRI) regulates the synthesis of globin chains in reticulocytes with heme availability. Heme defi ciency causes HRI to become active and phosphorylate the α-subunit of eIF-2. The phosphorylated eIF-2 tightly binds to eIF-2B and inhibits its guanine nucleotide exchanging activity needed for protein synthesis. HRI, potentially, has two distinct heme binding domains, one is an N-terminal domain where the heme is bound in 6-coordinated fashion, the other is a kinase insert domain, in which the heme is reversibly and weakly bound. Carbon monoxide (CO) and nitric oxide (NO), exogenous axial ligands to the heme, affect HRI activity. Since the N-terminal domain of HRI, but not the holoenzyme nor the kinase insert domain, has fi rmly bound heme, detailed spectroscopic analysis of the N-terminal domain of HRI is a useful way of characterizing the role of heme in catalysis. In the present study, CO binding kinetics to the 6-coordinated Fe(II) heme of the N-terminal domain of mouse HRI and resonance Raman spectra of the Fe(II)CO complex are examined to probe the character of the heme environment. í -1 -1 -1 The CO association rate constant, k on, and CO dissociation rate constant, koff, were 0.0029 µM s and 0.003 s , respectively. These values are very slow compared with those of mouse neuroglobin and sperm whale myoglobin, while -1 the koff value of HRI was close to those of the 6-coordinated hemoglobins (Hb) from Chlamydomonas trHb (0.0022 s ) and barley Hb (0.0011 s-1). The dissociation rate constant of an endogenous ligand, which occurs prior to CO association, was 18.3 s-1, which was lower than those of Chlamydomonas trHb (197 s-1) and barley Hb (47 s-1). Resonance Raman spectra suggest that the Fe-C-O adopts an almost linear and upright structure and that the bound CO interacts only weakly with nearby amino acid residues. 158 Journal of Inorganic Biochemistry 96 (2003)

The role of lysine residue in cytochrome c3 on the intermolecular interaction with hydrogenase

Shin Iida, Department of Bioengineering, Tokyo Institute of Technology, Japan Noriyuki Asakura, Department of Bioengineering, Tokyo Institute of Technology, Japan Toshiaki Kamachi, Department of Bioengineering, Tokyo Institute of Technology, Japan Ichiro Okura, Department of Bioengineering, Tokyo Institute of Technology, Japan

Cytochrome c3 from Desulfovibrio vulgaris (Miyazaki) is a redox protein containing four hemes in the molecule. Cytochrome c3 acts as an electron donor and an acceptor for hydrogenase, in vivo. In this reaction, enzyme-substrate complex formation is caused by the electrostatic interaction, because cytochrome c3 and hydrogenase are a basic and an acidic protein, respectively.

As cytochrome c3 involves 20 lysine residue in 108 amino acids, some of these lysine residues probably participate in the electrostatic interaction with hydrogenase. In this study, the role of lysine residues in the cytochrome c3 in the intermolecular interaction with hydrogenase was studied. One lysine residue in cytochrome c3 was modifi ed with succinic anhydride. By this modifi cation, positive charge of lysine residue was converted to negative charge. Various types of succinylated cytochrome c3 were obtained. Succinylated cytochrome c3,of which one lysine residue around heme I was modifi ed, was purifi ed by positive ion exchange chromatography. Hydrogen evolution reaction and hydrogen uptake reaction of hydrogenase with succinylated cytochrome c3 were carried out. In the case of succinylated cytochrome c3, of which one lysine residue around heme I was modifi ed, both these reaction rates was low (Figure). These results show the specifi c lysine residue around heme I plays an important role in the intermolecular interaction with hydrogenase

Synthesis and Spectroscopic Characterization of Copper(I)-diazene Complexes

Yoko Ishikawa, Department of Chemistry, University of Tsukuba, Japan Nicolai Lehnert, Institut für Anorganische Chemie, Christian-Albrechts-U, Germany Tetsuya Ono, Department of Chemistry, University of Tsukuba, Japan Yoshitaro Miyashita, Department of Chemistry, University of Tsukuba, Japan Ken-ichi Okamoto, Department of Chemistry, University of Tsukuba, Japan Kiyoshi Fujisawa, Department of Chemistry, University of Tsukuba, Japan

We have reported the synthesis and characterization of side-on peroxo binuclear Cu(II) complexes using hydrotris( pyrazolyl)borate ligands. These complexes showed many similarities to oxyhemocyanin and oxytyrosinase in their spectroscopic properties. And recently, the corresponding side-on disulfi do binuclear Cu(II) complex has been synthesized which is structurally analogous to the side-on peroxo binuclear Cu(II) complexes. Although the disulfi de ion is very similar to the peroxide ion, it has different chemical properties. In this work, we explored the reaction of the precursors

([L1Cu(OH)2CuL1] and [L3Cu(OH)] (L1 = HB(3,5-iPr2pz)3 anion, L3 = HB(3-tBu-5-iPrpz)3 anion)) with hydrazine and/ or 1,2-dimethylhydrazine. In these reactions, hydrazine and 1,2-dimethylhydrazine functioned as the reducing agent and Cu(I) complexes were obtained. We investigated the structural and spectroscopic properties of these complexes using X-ray structural analysis, and resonance Raman, electronic absorption, NMR, IR and Far-IR spectroscopies. X-ray analysis and resonance Raman spectroscopy revealed that the complexes with L1 were binuclear, two Cu(I) ions bridged by diazene and/or dinitrogen and trans-dimethyldiazene, respectively ([L1Cu(HN=NH)CuL1] (1a) and/or [L1Cu(N≡N)CuL1] (1b) and [L1Cu(MeN=NMe)CuL1] (2)). On the other hand, the complex with L3 is a mononuclear Cu(I)-hydrazine complex

([L3Cu(H2N-NH2)] (3)). The electronic absorption spectra also suggested that the Cu(II) ions in the precursors were reduced to Cu(I) by treatment with the hydrazines. In the complexes with L1, the absorption bands from -* transitions of N2 and charge transfer bands from Cu(I) to N2 were observed. NMR spectra showed the sharp proton peaks of these complexes. From IR and Far-IR spectroscopies, additional vibrations (NH ~3300 cm-1, Cu-N ~500 cm-1) could be identifi ed. These results indicate the possibility of dinitrogen fi xation by Cu(I) ions in biological systems. In further studies, we will attempt the reaction of these precursors with other hydazines. Journal of Inorganic Biochemistry 96 (2003) 159

Highly selective monooxygenation of hydrocarbons by a diiron(III) complex of a dinucleating ligand having two 2-aminoacetate groups connected by a 1,2-dipyridylethane spacer

Motoharu Itoh, Doshisha University, Department of Molecular Science and Technology, Japan Masahito Kodera, Doshisha University, Department of Molecular Science and Technology, Japan Koji Kano, Doshisha University, Department of Molecular Science and Technology, Japan

A diiron(III) complex of a dinucleating carboxyl containing ligand L, [Fe2(O)(CH3COO)2(L)] (1), (L = 1,2-bis(N-benzyl- 2-aminomethyl-6-pyridyl)ethane N',N'-diacetic acid) has been synthesized and characterized by FT-IR, FAB-MS, UV-vis, and 1H NMR spectroscopies and by an X-ray analysis (Fig 1). The crystal structure of 1 shows that each iron(III) ion is coordinated by a pyridyl nitrogen, a tertiaryamine nitrogen, three carboxylate oxygens, and an oxo-bridge oxygen to assume a distorted octahedral geometry. Two oxygen atoms of two pendant acetate groups, both of which coodinate to the iron atoms, are involved in hydrogen bonding interactions with a water molecule in 1. The Fe1···Fe2 distance, two Fe-Ooxo bond distances, and the Fe-O-Fe bond angle are 3.12, 1.77, 1.78 Å,and 123.0o. Compound 1 catalyzes the monooxygenation of alkanes to alcohols using m-chloroperbenzoic acid ( mCPBA ) as an oxidant. Selectivities in the monooxygenation of hydrocarbons catalyzed by 1 are 10 and 13.6 for the formation of alcohol/ketone and for the monooxygenation of tertiary/secondary carbon, respectively. The high selectivity of 1 may be due to the carboxylate donors.

Biochemical and Spectroscopic Investigations of Different Forms of BioB Under Single Turnover Conditions

Guy N L Jameson, Department of Physics, Emory University, Atlanta, GA 30, United States Michele Mader Cosper, Department of Chemistry and Center for Metalloenzyme Studies, United States Michael K Johnson, Department of Chemistry and Center for Metalloenzyme Studies, United States Boi Hanh Huynh, Emory University, United States

Biotin is an essential vitamin that is synthesized by micro-organisms and plants. In all organisms, biotin is a necessary cofactor in enzymes which are involved in the transfer of CO2 during carboxylation, decarboxylation and trans-carboxylation reactions. The fi nal step of the biotin biosynthetic pathway is catalyzed by the bioB gene product, termed BioB or biotin synthase, and involves the insertion of sulfur into dethiobiotin at the C6 and C9 positions in a S-adenosyl-L-methionine (SAM)-dependent reaction. Although there is general consensus that as-purifi ed, recombinant biotin synthase contains [2Fe- 2S]2+ clusters, the number and type of the Fe-S clusters present in the functional wild-type enzyme in vivo still remains to be established. The isolated protein is a 78-kDa homodimer and is able to hold only [2Fe-2S], only [4Fe-4S] or a mixture of [2Fe-2S] and [4Fe-4S] clusters. To determine the functional form of BioB we have used a combination of biochemical and spectroscopic techniques to investigate the activity of the different forms under single turnover conditions. The results of these experiments and their interpretation will be presented. 160 Journal of Inorganic Biochemistry 96 (2003)

The Structure-Activity Relationship of Chiral Square-Planar Platinum(II) Metallointercalators

David Jaramillo, University of Western Sydney, Australia J Grant Collins, Department of Chemistry, University College, (UNSW), Australia Peter Junk, School of Chemistry, Monash University, Victoria 3800, Australia Janice R Aldrich-Wright, University of Western Sydney, School, Australia

Four platinum(II) complexes of 1,10-phenanthroline (phen) and 3,4,7,8-tetramethyl-

1,10-phenanthroline (Me4phen) with the ancillary ligands (1R,3S) and (1S,3R)-1,3- diamino-1,2,2-trimethylcyclopentane (tmcp) have been synthesized and characterized. The complexes have been screened for antibacterial and cytotoxic effects in vitro against A. tumefaciens and the L1210 cell lines, respectively, and a relationship between structure and biological activity is reported here. [Pt(Me4phen)(R,S-tmcp)]Cl2 and [Pt(Me4phen)(S,R-tmcp)]Cl2 showed antibacterial properties greater than their non- methylated counterparts, [Pt(phen)(R,S-tmcp)]Cl2 and [Pt(phen)(S,R-tmcp)]Cl2. No differences in growth inhibitory potency between enantiomers were observed. However, enantioselectivity was present in the in vitro cytotoxicity, with compounds having the

(R,S) confi guration showing higher IC50 values against the L1210 cells. The binding affi nity, cytotoxicity and specifi c mode of binding were determined through Circular

Dichroism (CD) studies, IC50 experiments against L1210 cell lines, and DNA-NMR titrations, respectively. Intercalation and partial intercalation, are the suggested binding modes, where the extent of insertion between the base pairs of DNA and any interaction with the helix backbone, is governed by the substituents on the aromatic moiety and the confi guration of the chiral ancillary ligands, respectively. Complexes that incorporated 3,4,7,8-tetramethyl-1,10- phenanthroline (Me4phen) exhibited an increase in biological activity over the parent compound [Pt(phen)(tmcp)]Cl2.

Electrochemical Studies on Purifi ed Rhus Vernicifera Laccase

Daniel L Johnson, Flinders University of South Australia, Australia Janene L Thompson, Flinders University of South Australia, Australia Sandra Brinkmann, Flinders University of South Australia, Australia Kathryn A Schuller, Flinders University of South Australia, Australia Lisandra L Martin, Flinders University of South Australia, Australia

The simplest members of the multi-copper oxidase family are laccases, capable of catalysing the four-electron reduction of oxygen to water. Recently, we have simplifi ed the purifi cation method for Rv laccase using hydrophobic interaction chromatography, to yield a single band on SDS-PAGE. During the past decade, alkanethiol monolayers (SAM’s) have been extensively used to modify gold surfaces for electrostatic and covalent protein immobilisation. However, quality reports of direct protein electrochemistry have been limited; cytochrome c and azurin being notable exceptions. We have successfully immobilised laccase purifi ed by the above method onto a gold electrode using a 3-mercapto propionic acid SAM followed by activation with EDC/NHS. Using this technique, amines on the laccase form a covalent amide bond with carbonyl groups previously immobilised on the electrode surface. This has enabled us to undertake a comprehensive study of the electrochemical properties of laccase. Our study has highlighted a concerted 4-electron transfer at slow scan rates (v) under anaerobic conditions, involving all four copper atoms within the protein, as assessed by peak current ratios (ipa/ipc). Addition of micromolar concentrations of the known laccase inhibitors N3- and F- resulted in a signifi cant reduction in peak current ratio, indicative of binding to the type 2 (T2) or type 3 (T3) copper atoms. This study suggests that azide binds to two of the copper atoms at the triangular active site, probably one T3 copper atom and the T2 copper atom. In the presence of a potential difference, the complexed copper atoms are lost from the protein, resulting in a second redox wave at lower potential. Under unbuffered conditions, catalytic currents were observed in the presence of the biological substrate oxygen, emphasising the retention of in vivo function throughout the immobilisation process. Journal of Inorganic Biochemistry 96 (2003) 161

Modelling Protein-Protein Electron Transfer in Cross-linked Dimers of Azurin

Thyra E de Jongh, Leiden Institute of Chemistry, Leiden University, Gorla, The Netherlands Miguel Prudencio, Leiden Institute of Chemistry, Leiden University, Gorla, The Netherlands Marcellus Ubbink, Leiden Institute of Chemistry, Leiden University, Gorla, The Netherlands Gerard W Canters, Leiden Institute of Chemistry, Leiden University, Gorla, The Netherlands

Electron transport (ET) between proteins is extremely important in many biochemical processes such as photosynthesis and oxidative phosphorylation. A good understanding of the parameters involved in electron transfer is therefore of great importance. The electron self-exchange reaction in the Type I blue copper protein azurin represents a simple model system for the study of ET between proteins in relation to factors such as structure and surface properties.

By introduction of certain point mutations in the protein and the use of several different cross-linking agents, both covalent and non-covalent dimers of azurin can be formed. Such dimers can help gaining a better insight into the relation between molecular orientation and rates of electron transfer. By introduction of a surface exposed cysteine in the hydrophobic patch of azurin and coupling through a bifunctional cysteine-reactive linker, a homodimeric covalent complex has been formed that displays very fast electron self-exchange within the dimer (1). On the basis of this fi nding, we are now creating a pH dependent ET switch, by the introduction of a protonatable group in the dimer interface. As an alternative to covalent cross- linking, it is also possible to form non-covalent dimers by the use of bifunctional ‘hotwires’ that act as a ligand for the active site (2). Depending on the length of the wire, the dimer may have different conformations. The structure of dimers is being studied with a new approach using long-range distance information obtained via attachment of a paramagnetic probe to the surface of one monomer of the dimer. (1) van Amsterdam IMC, Ubbink M, Einsle O, Messerschmidt A, Merli A, Cavazzini D, Rossi GL, Canters GW, Nat. Struc. Biol. 2002, 9(1), 48-52 (2) vanPouderoyen G, denBlaauwen T, Reedijk J, Canters GW, Biochemistry 1996, 35 (40), 13205-13211

A Theoretical Study on the Mechanism of Camphor Hydroxylation by Compound I of Cytochrome P450

Takashi Kamachi, Institute for Fundamental Research of Organic Chemistry, Japan Kazunari Yoshizawa, Institute for Fundamental Research of Organic Chemistry, Japan

Mechanistic and energetic aspects for the conversion of camphor to 5-exo-hydroxycamphor by the compound I iron-oxo species of cytochrome P450 are discussed from B3LYP DFT calculations. This reaction occurs in a two-step manner along the lines that the oxygen rebound mechanism suggests. The activation energy for the fi rst transition state of the H-atom abstraction at the C5 atom of camphor is computed to be more than 20 kcal/mol. This H-atom abstraction is the rate- determining step in this hydroxylation reaction, leading to a reaction intermediate that involves a carbon radical species and the iron-hydroxo species. The second transition state of the rebound step that connects the reaction intermediate and the product alcohol complex lies a few kcal/mol below that for the H-atom abstraction on the doublet and quartet potential energy surfaces. This energetic feature allows the virtually barrierless recombination in both spin states, being consistent with experimentally observed high stereoselectivity and brief lifetimes of the reaction intermediate. The overall energetic profi le of the catalytic mechanism of camphor hydroxylation particularly with respect to why the high activation energy for the H-atom abstraction is accessible under physiological conditions is also considered and calculated. According to a proton source model involving Thr252, Asp251, and two solvent water molecules (Biochemisty 1998, 37, 9211), the energetics for the conversion of the iron-peroxo species to compound I is studied. A signifi cant energy over 50 kcal/mol is released in the course of this dioxygen activation process. The energy released in this chemical process is an important driving force in alkane hydroxylation by cytochrome P450. This energy is used for the access to the high activation energy for the H-atom abstraction. 162 Journal of Inorganic Biochemistry 96 (2003)

Application of electrochemical quartz crystal microbalance

technique on direct monitoring of cytochrome c3 function as the electron pool during intermolecular electron transfer

Toshiaki Kamachi, Tokyo Institute of Technology, Japan Noriyuki Asakura, Tokyo Institute of Technology, Japan Ichiro Okura, Tokyo Institute of Technology, Japan

In biological intermolecular electron transfer, redox proteins containing metal center act as an electron mediator, and an accepted electron is relayed to the redox partner molecule. It is speculated that some of the redox proteins have the strict partner selectivity, which means that electron relay is regulated according to its redox state. The regulation of the intermolecular electron transfer is called as an electron pool or electron gate. To clarify the function of redox protein is of importance for understanding the biological electron transfer systems. In this study, the cytochrome c3 function as the electron pool was monitored directly by electrochemical quartz crystal microbalance technique. Figure shows EQCM response accompanied by CV. V2+ and V+· represents viologen dication form and viologen radical cation form, respectively. ‘Ox’ and ‘Red’ show the cytochrome c3 redox state. At initial state, the immobilized viologen dication and the oxidized cytochrome c3 associate (State 1). During reduction, the stationary association (State 1) turns into the electron transfer complex between viologen radical cation and the oxidized cytochrome c3 (State 2), so that the distance between cytochrome c3 and the viologen radical cation surface became short and frequency decrease (mass gain) was observed. As soon as cytochrome c3 is reduced, the reduced cytochrome c3 repels from the viologen radical cation, and change to an stationary state (State 3), which was shown by the frequency increase. In anodic scan, similar QCM responses were observed. These results illustrate the principle advantages of this measurement. Journal of Inorganic Biochemistry 96 (2003) 163

Copper ligand infl uences in synthetic model reactivity studies of the cytochrome c oxidase binuclear active site

Kenneth D Karlin, Johns Hopkins University, United States Eunsuk Kim, Johns Hopkins University, United States Reza A Ghiladi, Johns Hopkins University, United States Matthew E Helton, Johns Hopkins University, United States Jason Shearer, Johns Hopkins University, United States Shen Lu, OGI School of Science and Engineering at Oregon Health, United States Hong-wei Huang, OGI School of Science and Engineering at Oregon Health, United States Pierre Moenne-Loccoz, OGI School of Science and Engineering at Oregon Health, United States

Cytochrome c oxidase (CcO) is the terminal respiratory membrane protein complex which catalyzes the reduction of dioxygen to water. The active site of the enzyme contains a binuclear center composed of heme a3 and CuB, where O2 binding and reduction occur. To contribute to a fundamental understanding of O2-reactivity of heme/Cu centers relevant to II I O2-reduction chemistry in CcO, we have synthesized a series of (P)Fe /Cu (P = porphyrinate) model complexes utilizing a tetrakis(2,6-difl uorophenyl)porphyrinatate and various copper chelates. Upon low-temperature oxygenation, the reduced II I III 2- II + Fe /Cu complexes form bridged peroxo intermediates, [(P)Fe -(O2 )-Cu ] , characterized by various spectroscopic methods, including UV-Vis, resonance Raman, multinuclear NMR, Mössbauser, EXAFS spectroscopies as well as stopped-

fl ow kinetics, O2-uptake manometry, and mass spectrometry. They are all S = 2 spin systems with strong magnetic coupling between the iron and copper centers, yet there are striking differences in the nature of peroxo moiety, depending on the presence of a tridentate versus tetradentate Cu chelate. The former is shown to dramatically lower ν(O-O) values. The µ- peroxo complexes thermally transform to µ-oxo complexes [(P)FeIII-O-CuII]+ whose structures are also found to be infl uenced by copper ligand environment. This study adds signifi cantly to ongoing investigations focusing on how heme-copper centers react with dioxygen, with particular consideration of the important infl uence of copper ligand denticity.

1 Kim, E., Helton, M. E., Wasser, I. M., Karlin, K. D., Lu, S., Huang, H.-w., Mo’nne-Loccoz, P., Incarvito, C. D., Rheingold, A. L., Honecker, M., Kaderli, S. and Zuberb¸hler, A. D., Proc. Natl. Acad. Sci. USA, 2003, 100, 3623. 2 Ghiladi, R. A., Hatwell, K. R., Karlin, K. D., Huang, H.-w., Mo’nne-Loccoz, P., Krebs, C., Huynh, B. H., Marzilli, L. A., Cotter, R. J., Kaderli, S., and Zuberb¸hler, A. D., J. Am. Chem. Soc., 2001, 123, 6183. 3 Ghiladi, R. A., Ju, T. D., Lee, D.-H., Mo’nne-Loccoz, P., Kaderli, S., Neuhold, Y.-M., Zuberb¸hler, A. D., Woods, A. S., Cotter, R. J., and Karlin, K.D., J. Am. Chem. Soc., 1999, 121, 9885. 164 Journal of Inorganic Biochemistry 96 (2003)

Crystal structures of naphthalene dioxygenase along the reaction pathway

Andreas Karlsson, Swedish University of Agricultural Sciences, Uppsala, Sweden Hans Eklund, Swedish University of Agricultural Sciences, Uppsala, Sweden S Ramaswamy, The University of Iowa, Iowa City, United States Juanito V Parales, The University of Iowa, Iowa City, United States Rebecca E Parales, The University of Iowa, Iowa City, United States David T Gibson, The University of Iowa, Iowa City, United States

We have determined structures of naphthalene dioxygenase that show a molecular oxygen species bound to the mononuclear iron in a side-on fashion. While computational and spectroscopic results have suggested side-on binding to metal proteins, only end-on binding of oxygen to iron has earlier been observed in crystal structures. The series of NDO complex structures with oxygen, substrate, substrate and oxygen and product represent states along a reaction pathway. In a complex with substrate and dioxygen, the dioxygen molecule is lined up for an attack on the double bond of the aromatic substrate. The arrangement of the oxygen atoms in the ternary complex positions the oxygen atoms almost parallel to those observed in the product complex, albeit closer to the iron. This arrangement of atoms is ideal for the reaction, with the oxygen atoms having van der Waals interactions with the substrate carbons to be hydroxylated. These structures provide the basis for a reaction mechanism that explains the high stereospecifi city of the reaction catalyzed by naphthalene dioxygenase.

The Gln95His mutant of mavicyanin has a type-1.5 copper site

Kunishige Kataoka, Department of Chemistry, Faculty of Science, Kanazawa University, Japan Mayuko Takata, Department of Chemistry, Faculty of Science, Kanazawa University, Japan Takeshi Sakurai, Department of Chemistry, Faculty of Science, Kanazawa University, Japan

Mavicyanin isolated from zucchini peelings is a member of the family known as cupredoxin or blue copper protein, although the physiological role of it in the plant is unknown [1, 2]. We previously synthesized a gene coding for the 109-amino acid, non-glycosylated form of mavicyanin and expressed it in Escherichia coli [3]. We also proved that the Gln95 is the axial ligand toward Cu ion in mavicyanin by the site-directed mutagenesis [3]. In this study, in order to modulate the copper center of mavicyanin, we have prepared a mutant that the axial ligand (Gln95) was replaced by His. To modulate the copper center of mavicyanin, His was substituted for Gln95 by site-directed mutagenesis. By the replacement the rhombic EPR signal changed to an axial one (g|| = 2.28, A|| = 11.8 mT) that is quite similar to that of corresponding M121H Alcaligenes denitrifi cans azurin with large EPR hyperfi ne coupling [4]. The electronic absorption spectrum of the mutant consisted of two peaks at 437 (e = 4,000) and 575 nm (e = 1,600 M-1cm-1) in the visible region at pH

7.0, being comparable to that of the M121H- (type-1.5) azurin. The midpoint potential of the mutant (E1/2 = +145 mV at pH

7.0) showed the negative shift of 68 mV compared with the recombinant mavicyanin (E1/2 = +213 mV, DEp = 68 mV). These differences in spectroscopic and electrochemical properties between wild-type and Q95H-mavicyanin are due to the changes in a ligand group, a coordination geometry, and hydrophobicity of copper site. 1. Marchesini, A., Minelli, M., Merkle, H., and Kroneck, P. M. (1979) Eur. J. Biochem. 101, 77-84. 2. Maritano, S., Marchesini, A., and Suzuki, S. (1997) J. Biol. Inorg. Chem. 2, 177-181. 3. Kataoka, K., Nakai, M., Yamaguchi, K., and Suzuki, S. (1998) Biochem. Biophys. Res. Commun. 250, 409-413. 4. Kroes, S. J., Hoitink, C. W. G., Andrew, C. R., AI, J., Sanders-Loehr, J., Messerschmidt, A., Hagen, W. R., and Canters, G. W. (1996) Eur. J. Biochem. 240, 342-351. Journal of Inorganic Biochemistry 96 (2003) 165

Solution structures in acetonitrile of Xn+/Cys-X-Y-Cys with NH•••S hydrogen bond

Masahiro Kato, Graduate School of Science, Osaka University, Japan Taka-aki Okamura, Graduate School of Science, Osaka University, Japan Hitoshi Yamamoto, Graduate School of Science, Osaka University, Japan Norikazu Ueyama, Graduate School of Science, Osaka University, Japan

Cys-X-Y-Cys is an amino acid sequence that is often found in the active center of metalloenzyme such as Ada protein, rubredoxin and Zinc fi nger as thiolate chelating ligands, and thiol-protein oxidoreductase such as thioredoxin and thiol transferase as nucleophile. The crystallographic analysis of these enzymes have been suggested that the regulation of the catalytic activities is associated with the NH•••S hydrogen bond between the S atom of cysteine residue and the amide NH n+ n+ of peptide main chain. We report the synthesis of X /Z-Cys(1)-Pro-Leu-Cys(2)-OMe (X =NEt4, Na, Pt(bpy)) complexes and the relations between the NH•••S hydrogen bond and the secondary structure. Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum(II) (cisplatin) and inhibited intracellular activity of 2+ the thioredoxin system. Therefore Pt /Cys-X-Y-Cys complex is important to support the solution of mechanism. NEt4 and t t Na complexes were synthesized by the reaction of Z-Cys-Pro-Leu-Cys-OMe with (NEt4)(S Bu) or Na(S Bu), respectively. 1 The solution structures in acetonitrile of the thiol state and the NEt4 complex were determined by H NMR using a simulated annealing method. The only thiolate anion states form a folded hairpin turn structure with an intramolecular NH•••S hydrogen bond between the Cys(1) S atom and the amide NH of Leu and Cys(2). The 1H NMR spectra of the thiol and monothiolate anion states in Na complex indicate clear amide NH signals in acetonitrile, but the dithiolate anion state does not exhibit any amide signals. Intermolecular NH•••S hydrogen bond between Cys(2) sulfur and other amide NH presumably leads to the broadness of amide NH signals with these small T1 values. Pt(II) complex was synthesized by the reaction Pt(bpy)Cl2 with - - (NEt4)2(Z-Cys(S )-Pro-Leu-Cys(S )-OMe). Formation of a ternary complex has been confi rmed by the ESI mass spectra, which exhibited signals at m/z 955.2 [Pt(bpy)(Z-Cys-Pro-Leu-Cys)-OMe + Na+]. 166 Journal of Inorganic Biochemistry 96 (2003)

Studies of the Interaction of Streptomyces avermitilis (4-hydroxyphenyl)pyruvate Dioxygenase with the Specifi c Inhibitor NTBC

Michael Kavana, University of Wisconsin-Milwaukee, United States Vincent M Purpero, University of Wisconsin-Milwaukee, United States Graham R Moran, University of Wisconsin-Milwaukee, United States

4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is a non-heme Fe(II)enzyme that catalyzes the conversion of (4- Hydroxyphenyl)pyruvate (HPP) to homogentisate as part of the tyrosine catabolism pathway. Inhibition of HPPD by the triketone, NTBC, is used to treat Type I tyrosinemia, a rare but fatal defect in tyrosine catabolism. Although triketones have been used for many years as HPPD inhibitors for both medical and herbicidal purposes, the mechanism of inhibition is not well understood. The following work provides mechanistic insight into NTBC binding. The tautomeric population of NTBC in aqueous solution is dominated by a single enol, shown by NMR spectroscopy. NTBC preferentially binds to HPPD.Fe(II) as evidenced by a visible absorbance feature centered at 450 nm. The rate of binding of NTBC to HPPD.Fe(II) was measured on a stopped-fl ow spectrophotometer at 450nm and shown to have a -1 hyperbolic dependence on NTBC concentration. (Kntbc = 1.25±0.08 mM, klimit=7.9±0.3 s ) There is an isotope effect on the limiting rate when D2O is used as the solvent. (kh/kd = 1.3) It is therefore proposed that the Lewis-acid assisted conversion from iron(II) bound enol to enolate is the irreversible step klimit. Although the native enzyme without substrate reacts with molecular oxygen, HPPD.Fe(II).NTBC does not. The binding feature does not decrease over the course of two days when exposed to atmospheric oxygen. This means that not only does the HPPD.Fe(II).NTBC complex not oxidize, but also that the off rate for NTBC is essentially zero as any HPPD.Fe(II) would readily oxidize in the presence of oxygen. The NTBC bound enzyme also doesn’t form a complex with the oxygen mimic, nitric oxide. EPR spectroscopy has shown that only two percent of HPPD.Fe(II).NTBC forms an NO complex as compared to the holoenzyme. Journal of Inorganic Biochemistry 96 (2003) 167

Synthesis, Characterization, and Toxicity Studies of New Ruthenium Maltolato and Imidazole Complexes against Human Breast Cancer Cells and Hypoxic Cells

David C Kennedy, University of British Columbia, Canada Brian R James, University of British Columbia, Canada Brian O Patrick, University of British Columbia, Canada

Ru-imidazole complexes have been investigated as potential antitumour agents,1 radiosensitizers and hypoxia imaging agents.2 Complexes of Ru with maltolato (I) and various imidazole ligands have now been synthesized and shown to have toxicity toward human breast cancer cells as well as selective toxicity toward hypoxic cells. Ru(III) complexes of the form trans-[Ru(maltolato)2(L)2][CF3SO3] (II) and [Ru(maltolato)(L)4][CF3SO3]2 have been synthesized and characterized (L = imidazole, 1-methylimidazole, 2-methylimidazole and 4(5)-methylimidazole, 2-nitroimidazole, metronidazole (III)). The paramagnetic 1H NMR spectra and reduction potentials of these complexes will be presented. MTT assays3 using human breast cancer cells (HTB-129) show that the complexes can exhibit IC50 values considerably lower than that of cisplatin (40 µM). Trans-

[Ru(ethylmaltolato)2(2-methylimidazole)2][CF3SO3] exhibits an

IC50 value of 0.5 µM, and is also selectively toxic toward Chinese Hamster Ovary cells under hypoxic conditions at 100 µM for 3 h. 1 B.K. Keppler, W. Rupp, U.M. Juhl, H. Endres, R. Niebl, and W. Balzer, Inorg. Chem, 26, 4366 (1987). 2 P.K.L. Chan, B.R. James, and K.A.Skov, Int. J. Radiat. Biol., 52, 49 (1987); I.R. Baird, K.A. Skov, B.R. James, S.J. Rettig, and C.J. Koch, Syn. Commun., 28, 3701 (1998). 3 M.C. Alley, D.A. Scudiero, A. Monks, M.L. Hursey, M.J. Czerwinski, D.L. Fine, B.J. Abbott, J.G. Mayo, R.H. Shoemaker and M.R. Boyd, Cancer Res., 48, 589 (1988).

Reactive Intermediates in Epoxidation and cis-Dihydroxylation

by Non-Heme Iron Catalysts with H2O2 Jinheung Kim, Changwon National University, Korea Ju Y Ryu, Ewha Womans University, Korea Soonyoung Heo, Changwon National University, Korea Wonwoo Nam, Ewha Womans University, Korea Lawrence Que, Jr, University of Minnesota, United States

The oxygenation of carbon-carbon double bonds by iron enzymes generally results in the formation of epoxides and cis- diols (Rieske dioxygenases). Here, we present a systematic study of olefi n oxidations with H2O2 catalyzed by a group II 2+ of nonheme iron complexes, i.e. [Fe (BPMEN)(CH3CN)2] (BPMEN = N,N’-dimethyl-N,N’-bis(2-pyridylmethyl)-1,2- II 2+ diaminoethane) and [Fe (TPA)(CH3CN)2] (TPA = tris(2-pyridylmethyl)amine) and their derivatives. Olefi n epoxidation and cis-dihydroxylation are demonstrated to be different facets of the reactivity of a common FeIII-OOH intermediate, whose spin state can be modulated by the electronic and steric properties of the ligand environment. Highly stereoselective epoxidation is favored by catalysts with no more than one 6-methyl substituent, which give rise to low spin FeIII-OOH species. On the other hand, cis-dihydroxylation is favored by catalysts with more than one 6-methyl substituent, which afford high spin FeIII-OOH species. For catalysts with the low spin intermediate, 18 18 both the epoxide and the cis-diol product incorporate O from H2 O. These esults implicate a cis-H18O-FeV=O species derived from O-O 18 III bond heterolysis of a cis-H2 O-Fe -OOH intermediate. In contrast, 18 catalysts with the high spin intermediate incorporate both oxygen atoms from H2 O2 into the dominant cis-diol product, via a putative FeIII-OOH species. Thus a key feature of the catalysts in this family is the availability of two cis labile sites. We also demonstrate that Fe(TPA) complexes can catalyse the oxidation of olefi ns to cis-diols under conditions of limiting substrate with high conversion effi ciency. 168 Journal of Inorganic Biochemistry 96 (2003)

A Model for Mn Lipoxygenase

Sanne Kjéregaard-Knudsen, University of Southern Denmark, Odense, Denmark Martin N Mortensen, University of Southern Denmark, Odense, Denmark Christine J McKenzie, University of Southern Denmark, Odense, Denmark

Using a mononegative pentadentate ligand system we have characterised reactive Mn species as models for the biological H atom abstracting Mn(III)-OH species proposed for Mn-Lipoxygenase.[1] These rare examples of mononuclear [Mn(III)-OH] species were generated by oxidising dinuclear Mn(II) complexes in hydroxylic solvent. The same reaction in non-hydroxylic solvent gives mono-oxo-bridged di-manganese(III) and di-oxo-bridged di-manganese(IV) and complexes in a stepwise manner. The oxo-bridged dinuclear complexes may be relevant models for the Oxygen Evolving Center. All the species of

higher Mn valency than +2 are unstable. [1] C. Su, E. H. Oliw, J. Biol. Chem. 1998, 273, 13072-13079; M. Hamberg, C. Su, E. Oliw, J. Biol. Chem., 1998, 273, 13080-13088. L. Hörnsten, C. Su, A. E. Osbourne, U. Hellman, E. H. Oliw, Eur. J. Biochem. 2002, 269, 2690-2697.

Metal binding in CopC from P.syringae pathovar tomato

Melissa Koay, The University of Melbourne, Australia Zhiguang Xiao, The University of Melbourne, Australia Anthony G Wedd, The University of Melbourne, Australia

The structure and function of copper-binding proteins has been investigated in numerous organisms. The copper chaperones predominantly coordinate copper(I) via cysteinyl sulfur ligands. Recently, the transport of copper has been studied in aerobic bacteria. CopC from Pseudomonas syringae pathovar tomato is a copper-binding protein thought to be associated with intracellular copper tolerance.1 Recently, an NMR solution structure of CopC was reported (see Figure).2 The protein appears to bind both Cu(I) and Cu(II) in different sites.3 The nature of the metal binding sites are currently under investigation. Using L → M charge-transfer bands as a probe, CopC has been shown to bind one equivalent of Cu(II) and one equivalent of Hg(II) simultaneously or, alternatively, two equivalents of Hg(II). Chemical modifi cation, proteolysis and protein mapping techniques have been employed to defi ne the nature of the binding sites. References: 1 Cha J. and Cooksey D., Proc. Natl. Acad. Sci. USA, 88, 8915-8919 (1991). 2 Arnesano F., Banci L., Bertini I. and Thompsett A.R., Structure, 10, 1337-1347 (2003). 3 Arnesano F., Banci L., Bertini I., Mangani S. and Thompsett A.R., Proc. Natl. Acad. Sci, 100, 3814-3819 (2003). Journal of Inorganic Biochemistry 96 (2003) 169

Large acceleration of hydrolysis of phosphate esters through binding to a dizinc(II) complex which have hydroxyethyl pendant groups

Masahito Kodera, Department of Molecular Science and Technology, Doshish, Japan Yoshihito Umeda, Department of Molecular Science and Technology, Doshish, Japan Naoko Ono, Department of Molecular Science and Technology, Doshish, Japan Koji Kano, Department of Molecular Science and Technology, Doshish, Japan

Dinuclear zinc(II) complexes [Zn2(H2L1)(AcO)4](PF6)2 (1), [Zn2(H2L2)(AcO)4](PF6)2 (2), and [Zn2(H2L3)(AcO)4](PF6)2 (3) have been synthesized by template reactions of 1,2-bis(6-formyl-2-pyridyl)ethane with N,N-dimethyl ethylenediamine,

N-hydroxyethyl ethylenediamine, and N-hydroxypropyl ethylenediamine in the presence of Zn(OAc)2, respectively, and mononuclear zinc(II) complexes [Zn(L4)(AcO)2] (4) and [Zn(HL5)(AcO)2] (5) have been obtained by a similar template reaction of 6-methyl-2-formylpyridine in place of 1,2-bis(6-formyl-2-pyridyl)ethane with N,N-dimethyl ethylenediamine and N-hydroxyethyl ethylenediamine, respectively. Crystal structures of 1-3 and 5 have been determined by X-ray analysis. Each zinc ion in 1-3 and 5 assumes distorted square-pyramidal geometry. The Zn—Zn distances of 1, 2, and 3 are 7.35, 7.71, and 7.71 Å, respectively, and the zinc ions are not bridged by the acetate ligands. The hydrolytic activity of 1-5 is examined by kinetic studies for hydrolysis of tris(p-nitrophenyl)phosphate (TNP). The activity of 2 having hydroxyethyl pendant groups is ca. 20 times higher than 1. On the other hand, the hydrolytic activity of 3 having hydroxypropyl pendant groups is almost the same as that of 1. The reaction rates for the hydrolysis of TNP with 2 or 3 are saturated at high concentrations of the zinc complexes. The 1H NMR studies show that monophenyl phosphate ester reacts with 2 in a 1:1 ration to form a phosphate-bridged complex. The kinetic and spectral data demonstrate that 2 and 3 accelerate the hydrolysis of the phosphate ester through binding at the dizinc site, and furthermore the hydroxyethyl pendant group is specifi cally effective for the hydrolysis of phosphate esters.

Weak Non-Covalent Interaction between a Coordinated Imidazole and an Aromatic Ring Induces New Insights into Protein Function in the Met16 Pseudoazurin Mutants

Takamitsu Kohzuma, Ibaraki University, Japan Masaya Seki, Ibaraki University, Japan Tomotake Niizeki, Ibaraki University, Japan

Weak non-covalent interactions in biological molecules including proteins, nucleic acids, and lipids are very important for introducing specifi c functions and structures. Weak non-covalent interaction in the second coordination sphere of metallo-proteins and inorganic model compounds have been discussed. The biological signifi cance of such a stacking interaction between a copper coordinated imidazole ring and the phenyl ring of a phenylalanine residue has been found in a blue copper protein, plastocyanin from fern leaves, which clearly refl ects the properties as demonstrated in the inorganic model complexes.In the structure of a blue copper protein, pseudoazurin, a methionine, M16 is located in the vicinity of the Cu2+-coordinated H81 residue within the van der Waals interaction. Very recently, we succeeded to obtain the weak non- covalent interaction induced mutant of pseudoazurin, and here we report that the M16 mutation in pseudoazurin indicates the characteristic features and importance of the weak non-covalent interaction in the second coordination sphere of metallo- proteins. 170 Journal of Inorganic Biochemistry 96 (2003)

Proton-coupled electron transfer in a Ru(II)-pterin complex

Takahiko Kojima, Department of Chemistry, Kyushu University, Japan Taisuke Sakamoto, Department of Chemistry, Kyushu University, Japan Kei Ookubo, Osaka University CREST, Japan Shunichi Fukuzumi, Osaka University CREST, Japan Yoshihisa Matsuda, Department of Chemistry, Kyushu University, Japan

Pterins are redox-active fused heteroaromatics and play indispensable roles in many kinds of oxido-reductases in the presence of various metal ions, such as phenylalanine hydroxylase (non-heme Fe and Cu), nitric oxide synthase (heme-Fe), and all oxo-molybdenum enzymes including DMSO reductase. Important reactivity of pterins is proton-coupled electron transfer (PCET) to accept and release four protons and four electrons maximally in the course of catalytic events. We will present synthesis and characterization of a novel Ru(II) complex of a pterin derivative with tris(2-pyridylmethyl)amine (TPA),

[Ru(dmdmp)(TPA)]ClO4 (1), and its redox behaviour including PCET. X-ray crystallography on 1 indicated that the pterin binds to the Ru(II) center in as a monoanionic N,O-bidentate ligand in an imidate form with stereoselectivity, in which the imidate oxygen coordinates to the position trans to the axial pyridine of TPA ligand. The complex 1 was revealed to show two reversible redox processes in cyclic voltammetry in acetonitrile. The fi rst 1-e oxidation took place at the Ru(II) center to form a Ru(III) species as detected by EPR spectroscopy, followed by the oxidation of dmdmp- ligand to form a Ru(III)-coordinated pterin radical. The complex 1 also underwent reversible + two-step protonation (pKa = 5.21 and 0.45). At the second protonation, the H2dmdmp ligand could be reversibly reduced to form a Ru(II)-coordinated dihydropterin radical (g = 2.0008), whose unpaired electron density delocalised in the PCET region of the pyrazine ring based on analysis of its 8-line EPR signal. The PCET behaviour of 1 is summarized in Scheme 1. Those observations are suggestive of PCET process observed for pterins and important in the light of understanding of their characteristics. Journal of Inorganic Biochemistry 96 (2003) 171

A Cisplatin Analogue with 2,2í-Dipyriylketone Ligand: Cytotoxicity and Interaction with DNA Constituents

Bernt Krebs, Institute of Inorganic and Analytical Chemistry, West Fäclische Wilhelms, Universität, Münster, Germany Michael J Rauterkus, Institute of Inorganic and Analytical Chemistry, University, Germany

Cisplatin is one of the most successful anticancer agents. Its antitumor activity arises from its binding to genomic DNA. The majority of cisplatin-DNA adducts (> 90 %) are intrastrand 1,2-d(GpG) and 1,2-d(ApG) crosslinks, in which the two exchangeable chlorine atoms of cisplatin are replaced by the N7 atoms of adjacent purine bases. These cisplatin-DNA complexes bend and unwind the duplex at the lesion site. To get more information about the interaction of platinum-based antitumor agents with DNA we focused on the synthesis of complexes with tertiary amine ligands. These third generation cisplatin analogues exhibit signifi cant antitumor activity despite the lack of primary amine ligands. Here we present a novel platinum(II) compound with the chelating ligand 2,2'-dipyridylketone (dpk). The in vitro activity of [Pt(dpk)Cl2] was examined in three glioma cell lines and one human fi broblast cell line using standard MTT 2+ tests. Its cytostatic activity was found to be within the range of cisplatin. Reaction products of [Pt(dpk)(H2O)2] with DNA constituents have been characterized by X-ray structure analyses. When reacting with the nucleoside thymidine

(Thd) dinuclear complexes are being formed, representing possible models for drug-DNA interactions in vivo. [Pt2(dpk)( dpkOH2)(Thd)2](PF6)2 • 6 H2O (1) crystallizes in the monoclinic space group C2 (no. 5). The platinum centers are bridged by two thymidines in a head-to-head orientation. One chelating ligand was hydrolyzed by adding a water molecule to its ketone group, leading to 2,2'-dipyridylketone-hydrate (dpkOH2). An intramolecular platinum-platinum distance of 2.931 Å was determined. To our knowledge the thymidine adduct presented is the fi rst dinuclear platinum complex with bridging nucleosides. In addition, this complex is the fi rst adduct of a cisplatin analogue with tertiary amine ligands bound to a greater DNA constituent, which could be characterized by X-ray diffraction. 172 Journal of Inorganic Biochemistry 96 (2003)

Catalytic Oxidation of 3,5-Di-tert-butylcatechol by a Series of Mononuclear Manganese Complexes: Synthesis, Structure, and Kinetic Investigation

Bernt Krebs, Department of Inorganic and Analytical Chemistry, Westfälische Wilhelms – Universität Münster, Germany Michael U Triller, Institute of Inorganic and Analytical Chemistry, Westfälische Wilhelms – Universität Münster, Germany Daniel Pursche, Department of Inorganic and Analytical Chemistry, Westfälische Wilhelms – Universität Münster, Germany Wen-Yuan Hsieh, Department of Chemistry, University of Michigan, Ann Arbor, United States Vincent L Pecoraro, Department of Chemistry, University of Michigan, Ann Arbor, United States Annette Rompel, Department of Inorganic and Analytical Chemistry, Westfälische Wilhelms – Universität Münster, Germany

The series of mononuclear compounds [Mn(bpia)(OAc)(OCH3)](PF6) (1), [Mn(bipa)(OAc)(OCH3)](PF6) (2),

[Mn(bpia)(Cl)2](ClO4) (3), [Mn(bipa)(Cl)2](ClO4) (4), [Mn(Hmimppa)(Cl)2]•CH3OH (5), and [Mn(mimppa)(TCC)]∑2CHCl3 (6), (bpia = bis(picolyl)(N-methylimidazole-2-yl)amine; bipa = bis(N-methylimidazole-2-yl)(picolyl)amine; Hmimppa = ((1- methylimidazole-2-yl)methyl)((2-pyridyl)methyl)-2-hydroxyphenylamine; TCC = tetrachlorocatechol) were synthesized and characterized by various techniques such as X-ray crystallography, mass spectrometry, IR, EPR and UV/Vis spectroscopy, cyclic voltammetry and elemental analysis. 1 and 2 crystallize in the triclinic space group P1bar (no. 2), 4 and 6 in the monoclinic space group P2(1)/n (no.14) and 5 in the orthorhombic space group Pna2(1). Complexes 1 – 4 are good structural models exhibiting an N4O2 donor set (1 and 2) or N4Cl2 donor set (3 and 4) for the proposed active site of the manganese dependent extradiol cleaving catechol dioxygenase and additionally show high catalytic activity regarding the oxidation of 3,5 di-tert-butylcatechol. They exhibit saturation -1 kinetics at high substrate concentrations. The turnover numbers kcat = (86 ± 7) h (1), -1 -1 -1 kcat = (101 ± 4) h (2), kcat = (230 ± 4) h (3), kcat = (130 ± 4) h (4) were determined from the double reciprocal Lineweaver-Burk plot. A clear correlation between the redox potential and the catalytic activity was revealed. Cyclic voltammetric data show that the substitution of oxygen donor atoms with chloride causes a shift of redox potentials to more positive values. To our knowledge 5 is the fi rst mononuclear Mn(II) compound featuring an N3OCl2 donor set. Compounds 5 and 6 can be regarded as structural models for intradiol cleaving catechol dioxygenases. Though there are no manganese dependent enzymes of this class known to date, there are many examples of enzymes existing both as iron and manganese containing forms.

Thiaether Ligands as Unique Copper Chelators

Chandrika P Kulatilleke, Baruch College-The City University of New York, United States

Copper is an essential trace element for living beings. The search for selective copper chelating agents has recently received renewed attention as a result of mounting evidence that copper is essential to the growth of cancerous tumors. It has long been recognized that copper levels are abnormally high in solid tumors. It is now suspected that the increased copper level is associated with the development of a capillary network (angiogenesis) that is essential in sustaining tumor growth. Of the several compounds identifi ed as angiogenic promoters, all bind copper strongly, suggesting that copper may be essential to their function. Furthermore, copper depletion therapy has shown encouraging results in inhibiting angiogenesis and/or tumor growth. The copper chelation drugs tested so far has poor selectivity for copper and have posed serious side effects. A series of tetrathiaether ligands, we are studying, show promise as selective copper chelators. One criterion for an effective copper chelator is its selectivity towards copper with respect to other trace metal ions. Our preliminary studies on chelation of these tetrathiaether ligands with Cu(II) and Ni(II) showed that, they bind Cu(II), ten to the power 9 times stronger than Ni(II). Crystal structure data clearly explain the rationale for the preference. Studies are underway to estimate the selectivity of these ligands with respect to other essential metal atoms such as Fe(II), Cr(II), Mn(II) and Zn(II). We are studying both the solution and crystal structures of these metal complexes for correlation purposes. Journal of Inorganic Biochemistry 96 (2003) 173

Interaction between Sensor Domain and Histidine Kinase Domain of Sensory Histidine Kinase in the Two-Component Signaling System

Hideyuki Kumita, RIKEN Harima Institute/SPring-8, Japan Seiji Yamada, RIKEN Harima Institute/SPring-8, Japan Hiro Nakamura, RIKEN Harima Institute/SPring-8, Japan Yoshitsugu Shiro, RIKEN Harima Institute/SPring-8, Japan

The two-component signaling system composed of a sensory histidine kinase and its cognate response regulator is widespread in bacteria, fungi and plants. The rhizobial FixL protein, one of the sensory histidine kinases, is a heme-based O2 sensor for regulating the transcription of nitrogen-fi xation genes in plant root nodules. The association/dissociation of O2 to/from the heme iron of the sensor domain of FixL modulates ATP-dependent autophosphorylation in its kinase domain, followed by the phosphoryl transfer to the transcriptional activator FixJ. In this context, FixL is the most useful model for studying an interdomain signaling in the sensory histidine kinase because the kinase-inactive (O2-bound) and -active (O2-free) forms of FixL are spectroscopically distinguishable. To demonstrate the interaction between the sensor domain and the kinase domain, we prepared fi ve chimeric sensory histidine kinases (CskAs), in which the sensor domain of S. meliloti FixL was fused with the histidine kinase domain from a hyperthermophile, T. maritima, each at a different position, and enzymatically and spectroscopically compared them with the wild-type FixL. Although CskAs and FixL shared the secondary and heme environmental structures, all CskAs did not exhibit O2-dependent regulation of kinase activity. Some of CskAs contained high kinase activities, similar to those of the truncated histidine kinase domain, and the others exhibited dramatic decrease in kinase activity. Furthermore, in case of these CskAs with low kinase activity, the O2-binding affi nity of the sensor domain is also reduced by protein fusion with the kinase domain. Such a reverse action from the kinase domain is also observed in wild-type FixL, that the binding of ADP to a catalytic site of the kinase domain can reduce the O2-binding affi nity. It is, therefore, likely that the interdomain interaction present in CskAs would be related to the O2-dependent regulatory interaction of FixL. The present study demonstrated that the interaction in the native sensory histidine kinase would be strictly and fi nely controlled to mediate the signal of ligand-binding state to the kinase domain, and will cast a new insight into the mechanism of signal transduction. 174 Journal of Inorganic Biochemistry 96 (2003)

A Non-Heme Iron Nitric Oxide Reductase that Protects Against Nitrosative Stress in Acetogenic Bacteria

Donald M Kurtz, Jr, University of Georgia, United States Radu Silaghi-Dumitrescu, University of Georgia, United States Amaresh Das, University of Georgia, United States Guy N L Jameson, Emory University, United States Lars G Ljungdahl, University of Georgia, United States Boi Hanh Huynh, Emory University, United States

Non-denitrifying bacteria have systems for removal of nitric oxide generated either endogenously from nitrate reduction or exogenously, as a response to colonization or infection. Acetogenic bacteria, which are non-denitrifying and strictly anaerobic, typically colonize the mammalian intestine. Some acetogens can use nitrate as terminal electron acceptor. We report here the characterization of a non-respiratory nitric oxide reductase (NR-NOR) from acetogenic bacteria. This NR-NOR consists of two non-heme iron fl avoproteins, Hrb and FprA, that together catalyze reduction of NO to N2O by NADH (1). Hrb was found to contain an N-terminal fl avin binding domain and a C-terminal rubredoxin-like [Fe(SCys)4] domain. The FprA polypeptide contains a C-terminal fl avodoxin domain and an N-terminal diiron-binding domain that is distinct from those of other non-heme diiron proteins (2). Mössbauer spectroscopy showed that the irons of the diferric site of FprA are antiferromagnetically coupled, implying a single-atom, presumably solvent, bridge. NADH was unable to directly reduce either the fl avin or the diiron site in FprA, whereas Hrb was found to function as an effi cient NADH:FprA oxidoreductase. FprA, as expected by analogy to an E. coli homolog (3), was found to function as the terminal component of the NR-NOR. Substitution of zinc for iron in FprA completely abolished NOR activity. The initial rate vs [NO] data were accuratelly fi t using a rate law derived from a mechanism in which two NOís react at each FprA active site in the committed step. Under appropriate growth conditions, nitric oxide (micromolar) or nitrate (millimolar) were found to induce expression of FprA in two different acetogens. These results, together with the previously reported co-transcription of their genes (2), strongly implicate Hrb and FprA as a novel NR-NOR in acetogenic bacteria. 1 Silaghi-Dumitrescu, R., Coulter, E. D., Das, A., Ljungdahl, L. G., Jameson, G. N. L., Huynh, B. H., Kurtz, D. M., Jr., Biochemistry 2003, 42, 2806-2815. 2 Das, A., Coulter, E. D., Kurtz, D. M., Jr., Ljungdahl, L. G. J. Bacteriol. 2001, 183, 1560-1567. 3 Gardner, A. M., Helmick, R. A., and Gardner, P. R. J. Biol. Chem. 2002, 277, 8172-8177.

The Electrochemical Interconversions Between the Active and Inactive States of a [NiFe]-Hydrogenase; Implications for the Development of a Bio-Fuel Cell

Sophie E Lamle, Department of Chemistry, University of Oxford, United Kingdom Louise M Halliwell, Department of Chemistry, University of Oxford, United Kingdom Simon P Albracht, Swammerdam Institue for Life Sciences, Biochemistry, The Netherlands Fraser A Armstrong, Department of Chemistry, University of Oxford, United Kingdom

Further investigations of the electrochemical interconversions between active and inactive states of the [NiFe]-hydrogenase [1] from A.vinosum have been studied using protein fi lm voltammetry on a pyrolytic graphite ‘edge’ electrode [2]. The inactive states of the enzyme contain a bridging ligand (thought to be an oxo or hydroxo species) between the Ni and Fe in * the active site; this ligand is removed upon activation [3]. This process is fast from the Nir state (the ready state, formed * under anaerobic conditions) and slow from the Niu state (the unready state, formed under aerobic conditions).

These reactions have been studied over a range of pH, partial pressures of H2 and other gases (e.g. O2, CO) and temperature using electrochemical techniques such as cyclic voltammetry and chronoamperometry. This approach provides precise potential control and yields kinetic and thermodynamic information relating to the mechanism of these interconversions.

The implications of these results will be discussed in relation to the feasibility of a H2/O2 bio-fuel cell based upon a [NiFe]- hydrogenase. [1] For recent reviews on hydrogenases see papers by Maroney et al., Siegbahn et al. and Fan et al. in J.Biol.Inorg.Chem., 6 (2001) 453-456. [2] H.R.Pershad, J.L.C.Duff, H.A.Heering, E.C.Duin, S.P.J.Albracht and F.A.Armstrong, Biochemistry 38 (1999) 8992. [3] M.Carepo, D.L.Tierney, C.D.Brondino, T.C.Yang, A.Pamplona, J.Telser, I.Moura, J.J.G Moura and B.M.Hoffman J.Am.Chem.Soc. 124 (2002) 281-286 Journal of Inorganic Biochemistry 96 (2003) 175

New non-heme iron model compounds as catalysts in the oxidation of olefi ns

Carlos Lopez de Laorden, Universitaet Heidelberg, Germany Michael Merz, Universitaet Heidelberg, Germany Peter Comba, Universitaet Heidelberg, Germany

Olefi ns can be oxidized in the presence of various iron catalysts. Under mild conditions (diluted solutions of H2O2), the main products are alcohols and epoxides. We have performed the reaction under aerobic and anaerobic conditions to study the infl uence of oxygen as oxidant. All ligands used are based on the rigid 3,7-diazabicyclononane backbone. The heteroaromatic substituents R1 and R2 are widely varied to investigate steric as well as electronic infl uences on electronic properties and reactivities. The immediate oxidation product is an iron(III) intermediate, and its stability, reactivity and electronic properties are studied in detail, and correlated with the ligand structure and donor groups. A detailed knowledge of the reaction mechanism will allow us to tune the reactivity and selectivity of the catalyst, and to gain some insight in the reaction mechanism of biologically active analogs.

Dioxygen and water activation by dimanganese complexes

Frank B Larsen, University of Southern Denmark, Odense, Denmark Kenneth B Jensen, University of Southern Denmark, Odense, Denmark Christine J McKenzie, University of Southern Denmark, Odense, Denmark

Multi-electron redox processes, like O2 binding, activation and formation are often supported by more than one transition metal ion in both biological and synthetic systems. A dimanganese(II) complex of a dinucleating ligand

(see crystal structure in fi gure) reacts with O2 in solution and the solid state. In solution, the nature of the oxidized products depends on whether or not solvent oxidation occurs. In acetone, the formation of a co-crystallised product containing formate, and acetate bridging groups, can be explained by an oxidative cleavage of acetone to give the one and two carbon atom precursors required. Trace water is necessary for the reaction. In ‘dry’ solvents a series of dinuclear and tetranuclear complexes, whose topology and total oxidation state is apparently controlled by adventitious water coordination, have been characterized. 176 Journal of Inorganic Biochemistry 96 (2003)

DNA Cleavage with Dinuclear Metal Complexes of Ligands with Varying Flexibility

Geoffrey A Lawrance, University of Newcastle, Australia Paul V Bernhardt, University of Queensland, Australia Geoffry N De Iuliis, University of Newcastle, Australia

It is now well known that divalent metal ions play a key role in the catalytic centre of restriction endonucleases, which cleave DNA with exceptional sequence specifi city. These nucleases usually feature metal ions in close proximity in the active site, and there is good evidence that multi-metal cooperativity is important in the hydrolytic cleavage mechanism, with metal ions appearing to have a role both in positioning of the substrate and providing the water that acts as a nucleophile and is also activated for attack via its coordination. As a result of such observations, it is not surprising that development of metal complexes as artifi cal nucleases that hydrolytically cleave both DNA and RNA has been the subject of growing interest. The use of dinuclear metal complexes, which may present two adjacent sites for the separate tasks of substrate binding and delivery of a nucleophile, is one particular focus of attention. We have been developing and examining potentially dinucleating ligands that offer different levels of fl exibility when incorporating two metal ions. The cleavage of supercoiled plasmid DNA by metal(II) (mainly copper) complexes of these ligands has subsequently been probed. Highly fl exible amine ligands of the family L1 can bind prefentially one or two copper ions depending on the chain length, and DNA cleavage is seen to vary with chain length, refl ecting this behaviour. The less fl exible aminothioether L3 has been shown to be far superior at DNA cleavage than a mononuclear close analogue. The relatively rigid ‘side- by-side’ dinucleating macrocycle L2 is inactive in square- planar metal complexes where no axial water molecules for exchange binding and nucleophile supply exist. Aspects of the preparation, structures and DNA cleavage capacity of these systems will be presented.

Iron- Nitrosyl Sulfi nate Complexes: Relevance to the Fe-Containing Nitrile Hydratases

Chien-Ming Lee, Department of Chemistry, National Tsing Hua University, Taiwan, Province of China Chien-Hong Chen, Department of Chemistry, National Tsing Hua University, Taiwan, Province of China Chung-Hung Hsieh, Department of Chemistry, National Tsing Hua University, Taiwan, Province of China Amitava Dutta, Department of Chemistry, National Tsing Hua University, India Gene-Hsiang Lee, Instrumentation Center, National Taiwan University, Taiwan, Province of China

- Iron-NO complex containing monosulfi nate [(NO)Fe(S,SO2-C6H4)(S,S-C6H4)] (3) has been isolated from sulfur oxygenation - - of complex [(NO)Fe(S,S-C6H4)2] (2) which is obtained from addition of NO molecule to [(C4H8O)Fe(S,S-C6H4)2] (1), in organic solvents. In contrast, complex 1 does not initiate any O2 activation to yield iron-sulfi nate/-sulfenate complex. This result reveals that binding of NO to the iron center is required in promoting oxygenation of iron-bound dithiolates by dioxygen to yield iron-sulfi nate-nitrosyl species. Analysis of the bond angles for complexes 2 and 3 reveals that iron is best described as existing in a distorted trigonal bipyramidal coordination environment surrounded by one NO, three thiolates, and one sulfi nate in complex 3, whereas the distorted square pyramidal geometry is adopted in complex 2. Complex 3 - further reacts with molecular oxygen in the presence of [NO2] to produce dinuclear bis(sulfi nate) [PPN]2[(NO)Fe(SO2,SO2-

C6H4)(S,S-C6H4)]2 (4). Complex 3 showed reaction with PPh3in THF/CH2Cl2 to yield complex 2 and Ph3PO. Extrusion of [O] atoms from complex 3 was also observed on photolysis of CH2Cl2 solutions of complex 3 under N2 at ambient temperature. Obviously, complex 3 is thermally quite stable but is photochemically active toward [O] release. Journal of Inorganic Biochemistry 96 (2003) 177

Novel Mononuclear Fe(III) Mono(terpyridine) Complexes Having Labile Solvent Ligands: A Comparison of Homegeneous vs. Heterogeneous Catalytic Activity

Jin-Kyu Lee, School of Chemistry, Seoul National University, Korea Cheal Kim, Department of Fine Chemistry, Seoul National University, Korea Dong-Woo Yoo, School of Chemistry, Seoul National University, Korea Sang-Kun Yoo, Department of Fine Chemistry, Seoul National University, Korea

We have been exploring to fi nd the new active mononuclear nonheme iron(III) complexes having at least one open coordination site that is vacant or occupied by a solvent molecule to allow the room for exogenous ligands to bind during the catalytic reaction. Although a handful of examples have been recently isolated as mononuclear nonheme Fe(III)-OH complexes by introducing sterically bulky substituents, electrochemically well studied Fe poly(pyridyl) complexes have been hardly investigated for catalytic applications probably due to the lack of synthetic methods for preparing coordinatively unsaturated one. A new polymer-bound Fe(III)-terpyridine complex (PCD-Fe(tpy)) (1), which may have three additional water and/or methanol ligands, was obtained from the reaction of polymer supporter PCD, potassium carbonate and 2,6-bis(2’-pyridyl)-4’-(p-hydroxyphenyl)pyridine. This polymer-supported catalyst 1 has shown an excellent catalytic activity on the ring-opening reaction of various epoxides by water and alcohol under mild and neutral conditions to give stereospecifi c (trans-1,2-diol or trans-1,2-diol mono-ether) and relatively regioselective products where the nucleophile is incorporated preferentially to the more substituted carbon center in epoxide ring. The catalytic method appears to be an effi cient, mild, and simple method for the alcoholysis of epoxides and to be useful to prepare more substituted-ether alcohol, which might be exploited immediately for the practical synthesis of a wide range of interesting 1,2-diol mono-ethers. Moreover, the catalyst 1 could be easily recovered by a simple fi ltration and be used repeatedly without any signifi cant change from original catalytic activity even when used consecutively over 20 times. Furthermore, the catalytic system has the advantages such as availability and environment-friendly nontoxicity of the reagent, ease of workup and catalyst recycling, and the excellent conversion of epoxide into 1,2-diol mono-ether could make it as a promising candidate for a useful addition to the present methodologies in organic synthesis. We will also discuss a comparison of homogeneous vs. heterogeneous catalytic activity for [(terp)FeIII(S)3] and catalyst 1.

Thioether Ligand Containing Copper Complexes; Modeling PHM and DβH Proteins

Yunho Lee, Department of Chemistry, The Johns Hopkins University, United States Dong-Heon Lee, Department of Chemistry, Chonbuk National University, Korea Kenneth D Karlin, Department of Chemistry, The Johns Hopkins University, United States Yoon-Jung Mah, Department of Chemistry, Chonbuk National University, Korea

The involvement of a sulfur atom as a ligand in copper complexes is currently of considerable interest in bioinorganic chemistry. Of special interest are the active sites of PHM (peptidylglycine α-hydorxylating monooxygenase) and DβH (Dopamine β-hydroxylase) containing a methionine residue as one of the ligands of a central catalytic copper. As synthetic models, we have prepared CuI and CuII complexes with a series of novel mono- or dinucleating ligands containing a thioether sulfur atom (Figure 1). Ligands LNNS-1 and LNNS-2 (LNNS-1 = Methyl-(2-phenethylsulfanyl-propyl)-(2-pyridin-2- yl-ethyl)-amine, LNNS-2 = (2-Benzylsulfanyl-propyl)-methyl-(2- pyridin-2-yl-ethyl)-amine) were designed to possess a potential internal ligand-derived benzylic substrate. In order to more closely mimic the active sites of the enzymes we also prepared methionine amino-acid based mono- (LMET-PY1) and dinucleating (LHis-Met) ligands. The copper complexes were characterized by various spectroscopic techniques, such as UV-Vis, NMR, EPR and Mass spectroscopy, as well as elemental analysis and X-ray crystallography. Oxygenation of CuI complexes with LNNS-1 and LNNS-2 leads to sulfoxidation and their CuII complexes leads to sulfonation. These results will be presented. 178 Journal of Inorganic Biochemistry 96 (2003)

Reactive Intermediates Formed During the Reactions of Chromium(VI) with Glutathione: Which Species are Responsible for the DNA Damage?

Aviva Levina, University of Sydney, Australia Christina Ludwig, University of Sydney, Australia Peter A Lay, University of Sydney, Australia

Glutathione (g-Glu-Cys-Gly, GSH), the most abundant of biological reductants, is directly involved in cellular metabolism of genotoxic and carcinogenic Cr(VI) compounds (a comprehensive recent review is given in the references [1,2]). Incubation of isolated DNA with the Cr(VI) + GSH reaction mixture (but not with the Cr(III) products of this reaction), as well as exposure of mammalian cells to Cr(VI), lead to potentially genotoxic lesions, such as strand breaks and GSH-Cr(III)-DNA adducts [1,2]. We performed the fi rst structural studies of the reactive intermediates, formed during the Cr(VI) + GSH reaction in aqueous solutions, using a combination of X-ray absorption spectroscopy, electrospray mass spectrometry, and analytical techniques. Conditions for quantitative generation of Cr(VI)- and Cr(V)-GSH complexes in solutions have been developed, and the structures of these complexes have been assigned as I and II, respectively [3,4]. The species I represents the fi rst known example of reversible H2O binding to a Cr(VI) complex. Contrary to some suggestions in the literature, there was no evidence for the formation of detectable amounts of Cr(IV)-GSH intermediates. Kinetics of decomposition of I and II in neutral aqueous solutions have been studied. These data allowed for the design of experiments, where DNA damage was tested in the presence of only one of the intermediates (either I or II). Complex II, unlike for I, induces the formation of single strand breaks in plasmid DNA. Studies will also be reported on the roles of I and II in the formation of Cr(III)-DNA adducts. Acknowledgment. Financial support of this work was provided by ARC and ASRP grants. References [1] Codd, R.; Dillon, C. T.; Levina, A.; Lay, P. A. Coord. Chem. Rev. 2001, 216-217, 533-577, and references therein. [2] Levina, A.; Codd, R.; Dillon, C. T.; Lay, P. A. Progr. Inorg. Chem. 2003, 51, 145-250, and references therein. [3] Levina, A.; Zhang, L.; Lay, P. A. Inorg. Chem. 2003, 42, 767-784. [4] Levina, A.; Lay, P. A. To be submitted. Journal of Inorganic Biochemistry 96 (2003) 179

Three dimensional structures and topology of the transmembrane domain 4 of divalent metal transporter (DMT1) in membrane-mimetic environments

Hongyan Li, Department of Chemistry and Laboratory of Chemical Biology, Hong Kong Fei Li, Department of Chemistry and Laboratory of Chemical Biology, Hong Kong Qing-Yu He, Department of Chemistry and Laboratory of Chemical Biology, Hong Kong Zhong-Ming Qian, Department of Applied Biology and Chemical Technology, Hong Kong Hongzhe Sun, Department of Chemistry and Laboratory of Chemical Biology, Hong Kong

DMT1 is a newly discovered gene, which encodes the transporter responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues.1,2 Integral DMT1 consists of 561 amino acids with 12 putative transmembrane domains. It has an unusually broad substrate specifi city, ranging from essential divalent metals such as Fe2+, Zn2+, Mn2+ and Cu2+ to toxic metals such as Pb2+ and Cd2+, and features a proton-coupled process of metal transport.1 The transmembrane domain 4 (TM4) of DMT1 was shown to be crucial for its biological function, with disease-causing mutations (e.g. G185R) locating in this domain.3 The secondary structure of the TM4 peptide in membrane mimetic environments had been studied previously in our laboratory.4 In the present study, we characterized the three dimensional structure and topology of a synthetic peptide, corresponding to the sequence of the TM4, by NMR spectroscopy in membrane-mimetic environments. The structures of the TM4 peptide are similar in TFE and SDS micelles, with fl exible N- and C-termini fl anking an ordered helical region. The folding of the C-termini is highly pH-dependent and becomes well-structured only at low pH values. The effects of spin-label (12-doxylPC) on NMR signal intensities demonstrated that the peptide is embedded into the interior of SDS micelles, with the C-terminus surface-exposed. Fast amide proton exchange and signifi cant broadening of NMR signals observed in both the C-terminus and the buried helical region from paramagnetic metal ions (Mn2+) suggested a possibility of the formation of a water-fi lled pore or channel, which allows metal ions to be transported. The gate of the channel is likely closed at low pH values. These results provide an informative insight into the structural-function relationship for the integral protein in vivo. 1. Gunshin H, Mackenzie B, et al. (1997) Nature 388, 482-488. 2. Fleming MD, Romano, MA, et al. (1998) Proc. Natl. Acad. Sci. USA 95, 1148-1153. 3. Su MA, Trenor CC, et al. (1998) Blood 92, 2157-2163 4. Li H, Li F, Sun H & Qian ZM (2003) Biochem. J., in press We thank the AoE of UGC, the RGC (HK), Hong Kong Polytechnic University and the University of Hong Kong for their support. 180 Journal of Inorganic Biochemistry 96 (2003)

Syntheses, Structures and Spectroscopic Studies of Non-heme Iron Nitrosyl Complexes

Lijuan Li, California State University, Long Beach, United States Eric B Sundberg, California State University, Long Beach, United States Ximeng Wang, California State University, Long Beach, United States

Nitric oxide (NO) has become a fascinating entity in biological chemistry over the past few years. This molecule is responsible for controlling blood pressure, preventing platelet aggregation, killing invading microorganisms and a number of other important bodily functions. These recent discoveries have spawned a great interest in the development of transition metal complexes containing NO. Among them are non-heme iron nitrosyl complexes that have been identifi ed as products after biosynthetic evolution of nitric oxide. Synthesizing non-heme iron nitrosyl with ligands that mimic biologically active complexes provides evidence that compounds of analogous structures may be involved in the physiological processes of NO storage and liberation. Currently, few non-heme iron nitrosyl have been isolated and little is known about their structure and electronic properties. Our research aims at producing and examining the structure of particular non-heme iron nitrosyl complexes that mimic biologically active compounds by using ligands similar to the amino acid histidine.

Complex of Fe(NO)2(MeIm)2, where MeIm is 1-methylimidazole, was synthesized and single crystals were isolated from diethyl ether. X-ray crystallographic analysis of Fe(NO)2(MeIm)2 reveals that the coordination about Fe is tetrahedral in which the nitrosyls are linear in geometry. This compound is the fi rst isolated g = 2.03 family of iron dinitrosyl complexes coordinated to biological ligands.

However, Mass Spectrometry studies of the reactions between Fe(NO)2(CO)2 and a series of substituted imidazoles and benzimidazoles show the formation of clusters in the solution. By carefully modifying reaction conditions, we recently isolated a novel cyclic ring compound, [Fe(NO)2(Im)]4. The EPR, NMR, IR, electrochemistry, and X-ray crystallographic studies of these complexes will be discussed. Journal of Inorganic Biochemistry 96 (2003) 181

Fast Replacement of Methionine from [Pt(L-Met-S,N)2] by Glutathione and Formation of Polynuclear Pt(II) Adducts

Qin Liu, State Key Laboratory of Coordination Chemistry, China Jun Lin, State Key Laboratory of Coordination Chemistry, China Haiying Wei, State Key Laboratory of Coordination Chemistry, China Zijian Guo, State Key Laboratory of Coordination Chemistry, China

Sulfur-containing biomolecules such as methionine and glutathione are believed to play important roles in the metabolism and mechanism of platinum based anticancer drugs. Pt(II)-methionine adducts, such as [Pt(Met-S,N)2]have been isolated from metabolite of patients or rats dosed with cisplatin.[1] In this work, the reactions of [Pt(Met-S, N)2] with GSH and L-cysteine were studied by 1H NMR , ESMS and HPLC at different pH values. The S, N-chelated L-methionine can be readily replaced by GSH or L-cysteine (L-Cys) giving rise to free L-MetH. The bi-, tri-, tetra- and pentanuclear complexes such as [Pt2(η-GS-S)2(Met-S,N)2], [Pt3(η-GS-S)4(Met-S,N)2], [Pt4(η-GS-S)6(Met-S,N)2] and [Pt5(η-GS-S)8(Met-S,N)2] have been detected from the reaction of [Pt(Met-N,S)2] with GSH. With the increase of pH values, the substitution reaction rate increased but the extent of polymerization lowered although the bi- and tri-nuclear complexes could still be observed at pH 8.5. For the reaction of L-Cys, only bi- and tri-nuclear platinum complexes were detected due to the lower solubility of polymeric Pt-L-Cys. The HPLC experiment showed that the cis-isomer is more reactive than the trans isomer towards GSH.

Figure: The tetra-nuclear Pt(II)-GS complex and its isotope distribution of the three-negatively-charged ion (a) determined (zoom scanned), (aí) calculated (isopro 3.0). Acknowledgements This work is supported by the National Natural Science Foundation of China (No.s 29925102, 20231010). References [1] C. M. Riley, L. A. Sternson, A. J. Repta, and S. A. Slyter, Anal. Biochem.,1983, 130, 203-214. 182 Journal of Inorganic Biochemistry 96 (2003)

The mode of complexation and non-covalent interaction in the systems of Cu(II), Cd(II) and Hg(II) ions,adenosine 5’-diphosphate and biogenic amines

Lechoslaw Lomozik, A.Mickiewicz University, Faculty of Chemistry, Poland Anna Gasowska, A.Mickiewicz University, Faculty of Chemistry, Poland Romualda Bregier-Jarzebowska, A.Mickiewicz University, Faculty of Chemistry, Poland

Biogenic amines: putrescine (Put), spermidine (Spd) and spermine (Spm), present in cells of all living organisms, play a signifi cant role in genetic information transfer. Results of the potentiometric and spectral studies have proved the occurrence of non-covalent interactions between di- or triamines and adenosine 5’-diphosphate (ADP) and formation of molecular complexes in the pH range in which the polyamine is protonated and the nucleotide deprotonated, similarly as it has been observed earlier for adenosine 5’-monophosphate system [1]. The effi ciency of the adduct formation with the nucleotide depends on the number of the protonated amine groups and on the length of the polyamine chain. The interaction centres of the bioligands in adducts are the potential metallation sites, and thus, the metal ion should be treated as an agent interfering in the non-covalent interactions. The binary complexes of Cu(II), Cd(II) and Hg(II) with ADP reveal the coordination dichotomy (chromophores {N(7),O} and {N(1),O}). Introduction of the polyamine into the system changes the pH range of the dichotomy. In the system with Cu(II), the presence of amines and triamines changes the metallation sites of the nucleotide and leads to disappearance of the coordination dichotomy. In the protonated species of Cu(ADP)H(Put), Cu(ADP)H2(Spd) there is the chromophore {N(7),O}, while in the species Cu(ADP)(Put), Cu(ADP)(Spd) the main centre of metallation is the phosphate group of the nucleotide. In the systems with Cd(II) and Hg(II), the presence of the polyamine extends the pH range of the coordination dichotomy. The formation of the chromophores {N(7),O} and {N(1),O} is observed in the MLL’H as well as in MLL’ species, e.g. Hg(ADP)H(tn), Cd(ADP)H(Put), Hg(ADP)(Put) and Cd(ADP)(Spd). [1] A.Gasowska, L.Lomozik, R.Jastrzab, J.Inorg.Biochem., 78 (2000) 139.

Introducing Unnatural Amino Acids into Protein Metal-binding Sites and its Role in Metalloprotein Design and Engineering

Yi Lu, University of Illinois at Urbana-Champaign, United States Steven M Berry, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Mathew D Gieselman, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Martina Ralle, Department of Biochemsitry and Molecular Biology, Orego, United States Donald W Low, Gryphon Sciences, United States Wilfred A van der Donk, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Ninian J Blackburn, Department of Biochemsitry and Molecular Biology, Orego, United States

Over the past two decades, site-directed mutagenesis and loop-directed mutagenesis have been powerful tools to probe the role of certain residues, to fi ne-tune the activity of proteins, and to design new proteins.1 A limitation of this approach has been the accessibility of only a restricted number of functional groups through the 20 amino acids in the genetic code. Therefore, often more than one factor, such as sterics, geometry, and electronic interactions, is changed in one experiment. It is desirable to perform mutagenesis by changing as few parameters as possible. Recently, we have signifi cantly expanded the bioinorganic chemists’ ability to design and fi ne-tune metalloprotein structure and function by reporting the fi rst successful incorporation of unnatural amino acids into the metal-binding site of a protein at a quantity that is amenable to biophysical studies using the expressed protein ligation method. For example, by replacing the cysteine ligand with a selenocysteine, we probed the role of this ligand, such as its contribution to the covalency of the site, without interference from other parameters, such as sterics, hydrophobicity, or charge.2 Furthermore, by replacing Met121 in azurin with a series of iso-structural amino acids, selenomethionine and norleucine, we were able to deconvolute different factors affecting the reduction potentials of the blue copper center.3 A careful analysis, including the plot of observed reduction potentials of the WT azurin and its variants obtained in this work with the corresponding hydrophobicity of the axial ligand side chain, revealed hydrophobicity as the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands. 1 Lu, Y. et al., Chem. Rev., 101, 3047 (2001); 2. Berry, S. M. et al., J. Am. Chem. Soc.,124, 2084 (2002); 3. Berry, S. M. et al., J. Am. Chem. Soc. in press. Journal of Inorganic Biochemistry 96 (2003) 183

Biochemical and Biophysical Study of Transition Metal Ion-dependent DNAzymes

Yi Lu, University of Illinois at Urbana-Champaign, United States Amdrea K Brown, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Juewen Liu, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Jing Li, 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

Like proteins and RNA molecules, many DNA molecules have now been shown to catalyze a variety of reactions and are thus called DNAzymes. With limited building blocks, DNAzymes need to recruit cofactors in order to match other enzymes in terms of reaction diversity and catalytic effi ciency. Several unique properties make transition metal ions an ideal cofactor for DNAzymes.1 We have obtained new DNAzymes that bind transition metal ions with high affi nity and selectivity through the use of a combinatorial biology tool called in vitro selection.2 The work makes it possible to obtain different classes of metallo-DNAzymes in the laboratory within a short period of time. It also offers a rare opportunity to compare and contrast structural and functional properties of metal-binding sites in proteins and in DNAzymes. A detailed biochemical and mechanistic study3 of a DNAzyme showed that metal-binding affi nity of the DNAzymes is in the order of Pb2+ > Zn2+ > Mg2+. While Mg2+ and Zn2+ catalyze only a transesterifi cation reaction with formation of a product containing a 2′,3′-cyclic phosphate, Pb2+ catalyzes a transesterifi cation reaction followed by hydrolysis of the 2′,3′-cyclic phosphate. Although this two-step mechanism has been shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the fi rst time that this DNAzyme may also use the same mechanism. Furthermore, we have used FRET to study the transition metal ion-dependent folding of the DNAzymes, and developed a novel strategy of labeling each branch of DNAzyme with a fl uorophore so that the folding branch can be monitored independently.4 We showed that the system is simple and yet powerful in studying complicated biomolecular structure and dynamics, and is capable of revealing new sophisticated structural changes that may have functional implications. 1 Lu, Y. Chem. Euro. J. 8, 4588 (2002); 2. Li, J. et al., Nucleic Acids Res. 28, 481-488 (2000); 3. Brown, A. K. et al., Biochemistry, in press; 4. Liu, J. and Lu, Y., J. Am. Chem. Soc. 122, 15208 (2002). 184 Journal of Inorganic Biochemistry 96 (2003)

The Role of Copper and Protons in Heme-Copper Oxidases: Kinetic Study of an Engineered Heme-Copper Center in Myoglobin

Yi Lu, University of Illinois at Urbana-Champaign, United States Jeffrey A Sigman, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Hyeon K Kim, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Xuan Zhao, Department of Chemistry, University of Illinois at Urbana-Champaign, United States James R Carey, Department of Chemistry, University of Illinois at Urbana-Champaign, United States

Heme-copper oxidases (HCO) are a superfamily of terminal oxidases that contain a heteronuclear heme-copper center where O2 binding, activation and reduction to water occur. To probe the roles of the CuB center and protons in HCO, we made 1 model HCO proteins by engineering a CuB center in yeast cytochrome c peroxidase and sperm whale myoglobin.2 Our work allows a direct comparison of Mb (an oxygen carrier) or CcP (a peroxidase) with HCO (a group of oxidases) in the same protein framework and provides simple model protein systems for study of more complex enzymes such as HCO. Recently we have performed kinetic studies on 3, 4 the model HCO protein in myoglobin, called CuBMb. In the absence of metal ions, the engineered CuB center in CuBMb decreases the O2 binding affi nity of the heme. However, addition of Ag(I), a redox-inactive mimic of Cu(I), increases the

O2-binding affi nity. More importantly, a copper ion in the CuB center is essential for O2 reduction, as no O2 reduction can be observed in copper-free, Zn(II), or Ag(I) derivatives of CuBMb. Instead of producing a ferryl-heme as in HCO, the CuBMb generates verdoheme because the engineered CuBMb may lack a hydrogen bonding network that delivers protons to promote the heterolytic O-O cleavage necessary for the formation of ferryl-heme. Reaction of oxidized CuBMb with H2O2, a species - equivalent in oxidation state to 2e reduced O2 but possessing the extra protons, resulted in ferryl-heme formation, as in HCO.

The results showed that the CuB center plays a critical role in O2 binding and reduction, and proton delivery during the O2 reduction is important to avoid heme degradation and to promote the HCO reaction. 1 Sigman, J. A. et al., J. Am. Chem. Soc.. 122, 8192 (2000); 2. Sigman, J. A. et al., J. Am. Chem. Soc.. 121, 8949 (1999); 3 Sigman, J. A. et al., J. Am. Chem. Soc.. 123, 6945 (2001); 4 Sigman, J. A. et al., Proc. Natl. Acad. Sci. USA, in press.

Beta-alanine synthase the terminal enzyme in the pyrimidine catabolic pathway

Stina Lundgren, Medical Biochemistry and Biophysics, Sweden Doreen Dobritzsch, Medical Biochemistry and Biophysics, Sweden

Beta-alanine synthase (BAS), also known as N-carbamyl-beta-alanine amidohydrolase or beta-ureidopropionase, is the third and fi nal enzyme in the catabolic pathway of pyrimidines. The fi rst enzyme dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of uracil and thymine to 5,6-dihydrouracil and 5,6-dihydrothymine, respectively. This is the rate-limiting step of the pathway. Thereafter dihydropyrimidinase opens up the pyrimidine ring and depending on the dihydropyrimidine either N-carbamyl-beta-alanine or N-carbamyl-aminoisobutuyric acid is formed. These are subsequently degraded to the corresponding beta-amino acids by BAS. In mammals, this is the only pathway resulting in the biosynthesis of beta-alanine identifi ed so far. Microorganisms can form beta-alanine also by direct alpha-decarboxylation of L-aspartate or degradation of polyamines. The role of beta-alanine in mammals is still not completely understood. Based on the chemical similarity to gamma-amino-eta-butyric acid (GABA), it has been indicated that beta-alanine functions as a neurotransmitter. One case of BAS defi ciency has been described for a four-year-old girl, suffering of hyper-beta-alaninemia in connection with severe neural dysfunction and seizures. We have solved the structure of BAS from S. kluyveri to a resolution of 2.8≈ by MAD methods using SeMet-substituted protein. BAS is a homodimer with each subunit consisting of 455 amino acids. The monomer folds into two domains, a catalytic domain and a dimerization domain, and shows overall structural similarity to Streptomyces griseus aminopeptidase. The catalytic domain contains two zinc ions, which are most likely involved in the enzymatic reaction. An alignment of available BAS sequences from different organisms shows clear diferences between them and makes it possible to divide them into groups. In the group that includes S. kluyveri BAS the zinc-coordinating residues are conserved. Journal of Inorganic Biochemistry 96 (2003) 185

Direct electrochemistry of blue-copper proteins at a polycrystalline boron-doped diamond electrode

James P McEvoy, Oxford University, United Kingdom John S Foord, Oxford University, United Kingdom

Boron-doped diamond (BDD) electrodes hold great promise in the spectroelectrochemistry of redox proteins. There are two reasons for this: 1) BDD is transparent over a wide electromagnetic frequency range, including the far-IR; 2) the material has some excellent electrochemical properties. There have been two recent reports of direct, quasi-reversible electrochemistry obtained from cytochrome c on BDD electrodes.1, 2 It is important to investigate further the electroactive interaction between proteins in solution and BDD. This poster presents, for the fi rst time on BDD, quasi-reversible, diffusional voltammetric results from two blue copper electron-transfer proteins, azurin from ‘Pseudomonas aeruginosa’ and plastocyanin from parsley. Good signals are obtained only when the electrode surface is oxidized, either by anodic pretreatment or by abrasion in air with an alumina slurry. X-ray photoelectron spectroscopy has been used to demonstrate the oxidizing effect of such abrasion. Successful plastocyanin electrochemistry further requires the presence of a positively charged ëpromoterí such as neomycin, 3- 3+ a polyamine. The simple inorganic ions, Fe(CN)6 and Ru(NH3)6 , have been used to probe the electrostatic character of the BDD surface at various pHs, along with a variety of electrochemical techniques (cyclic and square-wave voltammetry, rotating-disk electrodes). Both protein and inorganic ion results are rationalized according to their electrostatic requirements on the BDD surface. (1) Marken, F.; Paddon, C. A.; Asogan, D. Electrochemistry Communications 2002, 4, 62-66. (2) Haymond, S.; Babcock, G. T.; Swain, G. M. Journal of the American Chemical Society 2002, 124, 10634-10635.

Measuring the Reduction Potentials of Trp and Tyr Radicals in Azurin

Michele A McGuirl, University of Montana, United States D Elias Lankford, University of Montana, United States Lindsay Orr, University of Montana, United States

Amino acid radicals are vital to many biological processes, including nucleic acid biosynthesis, DNA repair, water oxidation, and respiration. Additionally, amino acid radicals may serve as ‘sinks’ for positive charges during oxidation, thus facilitating long range electron transfer in proteins via multi-step tunneling (hopping). Yet despite the signifi cance of amino acid radicals in biology, their properties are not well defi ned. We seek to elucidate fundamental structure/function questions with studies of Trp and Tyr radicals formed within azurin, the prototypical blue-copper protein. Using electrochemical techniques, we will measure the reduction potentials of Trp and Tyr radicals incorporated at positions of varying solvent accessibilities (local protein environments). We will also incorporate non-natural analogs of Trp and Tyr into these positions. This novel approach will extend the range of potentials from -0.4V to +0.15V compared with the standard amino acids at pH 7. These data will provide a basis for future kinetics studies on the rates of electron transfer via amino acid radicals in azurin. 186 Journal of Inorganic Biochemistry 96 (2003)

Structural Determinants of Electronic Differences among Classes of [2Fe-2S] Ferredoxins: Insights from Paramagnetic NMR and Electronic Structure Calculations

Timothy E Machonkin, University of Wisconsin, United States William M Westler, University of Wisconsin, United States John L Markley, University of Wisconsin, United States

We have carried out detailed NMR investigations of one member of each of the three major classes of [2Fe-2S] ferredoxins: plant-type (Anabaena PCC7120 vegetative ferredoxin), vertebrate-type (human ferredoxin), and clostridial-type (Aquifex aeolicus ferredoxin). Preliminary goals were to resolve and assign signals from the cysteines ligating the iron atoms and from other residues in the loops near the Fe-S clusters of these proteins in their oxidized and reduced states and to determine their 1 T1 relaxation rates. Owing to the slow electronic relaxation rates in these proteins, traditional H-detected NMR experiments could not be used. The experimental strategy involved stable isotope labeling of the proteins (uniform labeling with 13C and 15N and selective labeling with 2H, 13C, and 15N) combined with direct detection of 2H, 13C, and 15N NMR signals. Next, starting with available three-dimensional structural models of the oxidized states of the three ferredoxins, hybrid density functional methods were used to calculate hyperfi ne shifts and T1 relaxation rates for both oxidation states. These theoretical values were compared with experimental values. The results (both theoretical and experimental) are discussed in terms of the geometric structures of the [2Fe-2S] clusters, exchange coupling between the metal centers, and differences in hydrogen bonding to the clusters. Site-directed mutagenesis is being explored as a way to test hypotheses concerning the structural origins of the observed differences in hyperfi ne shifts and relaxation rates.

Supported by NIH Grant R01 GM58667; T.E.M. is the recipient of an NIH Postdoctoral Fellowship F32 GM20497.

Structural Variations In Dinuclear Metal Complexes As Model Hydrolases

Azad H Mahdy, University College Dublin, Ireland David A Brown, University College Dublin, Ireland T erence J Kemp, University of Warwick, United Kingdom Guy Clarkson, University of Warwick, United Kingdom W illiam K Glass, University College Dublin, Ireland Noel J Fitzpatrick, University College Dublin, Ireland Hassan Nimir, University College Dublin, Ireland

Dinuclear metallohydrolases are an important group of metalloenzymes, which catalyze the hydrolysis of a range of peptide and phosphate ester bonds and include the amidohydrolases, amidinohydrolases and peptide hydrolases. A common structural feature of these metallohydrolases is a dinuclear metal active site featuring Zn(II), Ni(II), Co(II) and Mn(II) and carboxylate bridges which occur respectively in Leucine Aminopeptidase (LAP), Urease, Methionine Aminopeptidase (MAP) and Arginase. Hydroxamic acids are potent inhibitors of urease and the structure of acetohydroxamate inhibited Cys319Ala variant of Klebsiella aerogenes urease (KAU) shows the deprotonated hydroxyl oxygen of the hydroxamic acid bridging the nickel atoms in contrast to normal chelation of a metal ion. We have previously modelled this mode of bonding by complexation of hydroxamic acids with the model hydrolases [M2(OAc)4(µ-H2O)L2(L`4)] where L= tmen (tetramethylethylenediamine). In this contribution we report the effect of structural variations in the above system with

M = Ni(II), Co(II) and Mn(II); L` = Imidazole e.g. [Co2(OAc)4(µ-H2O)(Im)4] * and replacement of acetate (MeCOO) by pivalate (Me3CCOO), e.g. [Ni2(Me3C2O2)4(µ-H2O)(tmen)2], [Co2(Me3C2O2)4(µ-H2O)(tmen)2], [Mn2(Me3C2O2)4(µ-

H2O)(tmen)2]. * The hydroxamic acids were prepared in high purity: acetohydroxamic acid(AH), benzohydroxamic acid (BHA),

N-phenylacetohydroxamic acid (NPhAHA) and glutarodihydroxamic acid (GluH2A2) and a series of compounds prepared from the reaction of the dinuclear Ni(II), Co(II) and Mn(II) hydrolase models and these hydroxamic acids and in many cases characterized by crystal structure determinations. For example, glutarodihydroxamic acid forms [Co2(OAc){(OC)2N(O)(

CH2)3}(Im)4][OTf]2 with a penta coordination mode which mimics the enzyme (MAP) isolated from several mammalian and microbial sources in nature, [Ni2(OAc)(NPhAA)2(tmen)2][PF6] * which mimics the inhibited urease enzyme and

[Mn2(OAc)3(AA)(tmen)2] * which mimics the inhibited arginase enzyme. ( * ) Manuscript in preparation. Journal of Inorganic Biochemistry 96 (2003) 187

Structural studies on the selenate reductase from Thauera selenatis.

Megan J Maher, University of Sydney, Australia Joanne M Santini, La Trobe University, Australia Graham N George, Stanford Synchrotron Radiation Laboratory, Australia

Thauera selenatis, a member of the beta subclass of the Proteobacteria, was isolated from selenate-contaminated waste water in the San Joaquin Valley, California. It is an unique organism in that it can use the respiratory substrate acetate as the electron donor and carbon source when respiring with selenate according to the following equation:

- 2- - 2- + CH3COO + 4SeO4 → 2HCO3 + 4SeO3 + H The reduction of selenate to selenite is catalysed by the soluble, periplasmic enzyme selenate reductase. The enzyme, the fi rst of its type, has a native molecular mass of 160 kDa and is a heterotrimer (α1β1γ1; 96, 40 and 23 kDa, respectively). Analysis of the purifi ed enzyme for molybdenum, iron and acid-labile sulfur revealed the presence of 1, 13 and 8 mol per mol of enzyme, respectively. UV-visible absorption spectroscopy revealed the presence of the additional cofactor heme b. Selenate reductase has been cloned and sequenced and shown to have sequence similarity to members of the DMSO reductase subfamily of molybdenum oxotransferases, specifi cally the DMS dehydrogenase from Rhodovulum sulfi dophilum and the membrane-bound respiratory nitrate reductase from E. coli. We have crystallised selenate reductase and solved its structure by X-ray crystallography. In addition, we have characterised the active site structure of the enzyme by X-ray absorption spectroscopy. Results of these studies will be discussed.

Bioelectrochemistry of Metalloproteins

Lisandra L Martin, Flinders University, Australia

Electrochemistry of redox active metalloproteins and enzymes can contribute signifi cantly to the understanding of structure, mechanism and function. However, obtaining reliable and reproducible data has not always been easy. Electrostatic attachment of proteins has been particularly successful for elucidation of molecular mechanisms for proteins and transient or kinetically limiting processes such as, proton coupled electron transfer reactions assessed by cyclic voltammetry. Factors that determine the electrochemical response for a metalloproteins include; size, pI, temperature, salt strength, electrode material, metal ions and the co-adsorbing molecules. Covalent immobilisation of larger biomolecules enables larger proteins to be investigated and also led to preparation of electrochemical biosensors and devices. Our recent protein electrochemistry research has provided interesting and unexpected correlations especially focusing on the reactivity profi le for some proteins. Here we describe results from three studies where we have developed methods to examine the nature of electron transfer processes. Electrostatic immobilisation of small proteins, such as ferredoxin, adrenodoxin and plastocyanin to graphite and pyrolytic graphite electrodes, together with functionalised SAM’s (self-assembled monolayers) on gold electrodes for larger enzymes, such as laccase and P450’s, have been studied. The topography of the protein on the electrode surfaces has been probed using AFM (Atomic Force Microscopy). The application of these electrochemical studies for the preparation of biosensors or devices that provide assays for enzyme activity or function will be presented. 188 Journal of Inorganic Biochemistry 96 (2003)

The nature of the metal ion in the cobalt corrinoids (vitamin B12 derivatives), or, how does nature labilise Co(III)?

Helder M Marques, Molecular Sciences Institute, School of Chemistry, University of Witwatersrand, South Africa Christopher B Perry, Molecular Sciences Institute, School of Chemistry, University of Witwatersrand, South Africa Leanne Knapton, Molecular Sciences Institute, School of Chemistry, University of Witwatersrand, South Africa Manuel A Fernandes, Molecular Sciences Institute, School of Chemistry, University of Witwatersrand, South Africa Timothy J Egan, Department of Chemistry, University of Cape Town, Priva, South Africa

The function of the cobalt corrinoids seems confi ned to a handful of reactions catalysed by the B12-requiring enzymes. Why corrin? why Co(III)? and how is the inertness of Co(III) overcome? The rate of substitution of the axial H2O ligand in + aquacobalamin (B12a, H2OCbl ) is fast for Co(III). We believe that corrin transfers electron density to Co(III) and confers on it a softer, more labile character. The required electronic communication between the equatorial ligand and the axial coordination sites is revealed in a study of the electronic spectra of several cobalamins. The kinetic consequence of this phenomenon was probed by replacing the H at C10 by an electron withdrawing NO and resonance-donating Cl. The former deactivates the metal ion towards ligand substitution whereas the latter speeds up these reactions. We substituted H2O in B12a by four ambidentate nucleophiles and determined their structures. In the solid state, the SCN- in SCNCbl is coordinated through N, but 13C-NMR shows linkage isomerism in solution. - - 2- SeCN , NO2 and S2O3 are coordinated through the softer donor in each case (Se, N and S). This preference for the softer donor is not unusual for Co(III) complexes. The Co-N bond length in SCNCbl is marginally long, but the axial bond lengths of the other complexes are normal. Hence the putative transfer of electron density to Co(III) has no major effect on the ground state structures of its complexes. We used molecular mechanics and semi-empirical MO calculations to probe the contribution of a mechanochemical triggering effect on the enzymatic activation of the Co-C bond of coenzyme B 12 for homolysis. We fi nd this effect is unlikely to contribute more than a fraction of the observed catalysis. ZINDO/1 calculations on a version of mechanochemical triggering in which compression of the axial Co-N bond in the transition state for Co-C homolysis stabilises the transition state by increased Co-N orbital overlap showed that this mechanism can explain the observed enzymatic catalysis. Journal of Inorganic Biochemistry 96 (2003) 189

Preparation and characterization of manganese(III) and iron(III) complexes with Schiff base ligands and their reactions with active oxygen species t - ( BuOOH, H2O2 and O2 ) Takayuki Matsushita, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Hideyuki Asada, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Sei Negoro, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Kyohei Shitasue, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Naoya Kishida, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Kiyoto Hashizume, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Manabu Fujiwara, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan

The reactions of transition metal complexes with active oxygen species have been extensively investigated in relation to the metalloenzymes such as superoxide dimutases(SOD) and catalases(CAT). In this paper we describe the preparation and characterization of manganese(III) Schiff base complexes, Mn(salMedpt)X í and Mn(saldpt)X where X is Cl and OAc and diiron(III) complexes, [Fe(µ-X -salbn)Cl]2 (Fig.1). The Schiff base ligands, í í H2salMedpt, H2saldpt, and H2-X -salbn were obtained by the reactions between appropriate amines and salicylaldehydes (X t - = H, 5-Me, 5-Cl). The reactivities of the manganese(III)and diiron(III) complexes toward BuOOH, H2O2 and O2 have been investigated in DMSO or MeCN by measuring UV-vis spectra and cyclic voltammograms. - In the reactions of the manganese complexes with O2 , the central Mn(III) ions have been found to be reduced to Mn(II) - with evolving dioxygen molecule and then they were decomposed by the addition of excess amount O2 . However, the addition of pyrazole molecule into the reaction system has been found to retard the decomposition of the complex. The - diiron(III) complexes have been found to react with H2O2 and O2 to give predominantly the corresponding µ-oxo dimer, í t [Fe(µ-X -salbn)(µ-O)]2, whereas with BuOOH almost no reaction observed. This can be explained by the steric hindrance of tBuOOH which may not interact with the vacant coordination site of the iron ions surrounded by the two bridging salbn ligands. 190 Journal of Inorganic Biochemistry 96 (2003)

FhuF is the First Siderophore Reductase

Berthold F Matzanke, University of Luebeck, Ratzeburger Allee 160, D-23538 L, Germany Stefan AneMüller, Institute of Biochemistry, University of Luebeck, Germany Klaus Hantke, Eberhard-Karls-Universitaet, Instiute of Microbiology, Germany

Fe3+ siderophores are an important iron source for bacteria. In fact, it is known, that ferric iron complexed by the siderophore is reduced inside the cell and the deferrated siderophore is in most cases rapidly excreted. Siderophores have a much lower affi nity for Fe2+ than for Fe3+ and the kinetics for ligand exchange for high-spin Fe2+ are much faster than for Fe3+. Therefore, reduction of ferric siderophores accompanied by ligand exchange is an excellent mechanism for intracellular iron release. However, against the expectations of many scientists no specifi c Fe3+ siderophore reductases could be identifi ed so far. FhuF mutants of E. coli showed reduced growth on plates with ferrioxamine B as iron source, although no defect in siderophore uptake experiments was observed. Moreover, these growth conditions lead to a derepression of various Fur dependent iron transport systems. We have characterized the isolated protein by EPR- and Mossbauer spectroscopy uncovering an unusual [2Fe-2S]-protein. In this study we present a datailed analysis of the elctronic structure of the FhuF- metal cnter. Furthermore, the redox potential E1/2 of FhuF was determined by EPR redox titration. E1/2 is -310±25mV vs NHE. According to the Nernst equation, the effective range for a thermodynamically favorable redox reaction is ±59mV with respect to the corresponding reduction potential. If, in addition, the uncertainty of the experimental data is taken into account it is obvious that reduction of coprogen, ferrichrome and to lesser extent of ferrioxamine B is achievable,. In an in-vitro Mössbauer spectroscopic analysis we could prove, in fact, reduction of the ferrichrome type siderophore ferricrocin and re-oxidation of FhuF. Finally, removal of iron from various siderophores has been monitored by extraction of Fe3+ siderophores from cells of fhuF mutants and parent strains of E.coli. These experiments provide evidence for a signifi cant decrease of intracellular reductive iron-removal from a variety of hydroxamate-type siderophore complexes in vivo, i.e. in fhuF- mutants To our knowledge, this report presents the fi rst experimental evidence for a siderophore-specifi c reductase in microorganisms.

Spectroscopic Characterization of Iron Deposits in a Yeast Model(YFH1-) of Friedreich`s Ataxia

Berthold F Matzanke, Isotopenlabor TNF, University of Luebeck, Germany Emanuel Lesuisse, Institut Jacques Monod, Universites Paris 6 and 7, France Volker Schünemann, Institute of Physics, University of Luebeck, Germany Alfred X Trautwein, Institute of Physics, University of Luebeck, Germany Wolfram Meyer-Klaucke, EMBL Outstation(DESY), Germany

The YFH1 gene is the yeast homologue of the human FRDA gene encoding a protein named frataxin. Mutations of the frataxin gene lead to a decreased frataxin expression causing Friedreichís ataxia, the most common autosomal recessive neurodegenerative disease of Caucasians [1,2]. A defect in the yeast frataxin homologue leads to several S. cerevisiae phenotypes. Iron uptake is considerably higher compared to wild-type cells, with most of the iron being found in the mitochondria. These cells exhibit defective respiration, unstable mitochondrial DNA and hypersensitivity to oxidative stress. One major goal of our project was to uncover the role of yfh1 in mitochondrial iron metabolism of yeast and in particular, to identify the major mitochondrial iron components. For this purpose we analysed mitochondria of wild-type Saccharomyces and of a yfh1- mutant strain by means of in situ Mossbauer spectroscopy [3], EXAFS and biochemical methods. Mossbauer spectra of mitochondria purifi ed from the yfh1- mutant, measured at 77K, 4.3 K, 1.9 K and in a fi eld of 7 T are consistent with the presence of small and very amorphous nano-particles of iron in mitochondria. Various attempts to visualize these particles on PAGE failed. EXAFS data gave best fi ts for the fi rst shell with 6 oxygen atoms at a distance of 1.98(1) A. The second shell environment of iron is explained best with phosphorus. We conclude, therefore, that iron is essentially present in yfh1- mitochondria of Saccharomyces cerevisiae as nano-particles of ferric phosphate. A detailed analysis based on the above mentioned techniques will be presented. [1] Campuzano, V., Montermini, L., Molto, M. D., Pianese, L., Cossee, M., Cavalcanti, F., Monros, E., Rodius, F., Duclos, F., Monticelli, A., and et al. (1996) Science 271(5254), 1423-7. [2] Puccio, H., and Koenig, M. (2000) Hum Mol Genet 9(6), 887-92. [3] Matzanke, B.F. (1991). In: Handbook of Microbial Iron Chelates (Siderophores), G. Winkelmann ed., CRC Boca Raton, USA, 15 – 60 Journal of Inorganic Biochemistry 96 (2003) 191

A Thermodynamic and Spectroscopic Study on the Copper(ii) Complexes with Hexarepeats Fragments of the Avian Prion Protein

Diego La Mendola, Institute of Biostructures and Bioimages – CNR – Catania, Italy Raffaele P Bonomo, Department of Chemical Sciences-University of Catania, Italy Giuseppe Maccarrone, Department of Chemical Sciences-University of Catania, Italy Giuseppe Pappalardo, Institute of Biostructures and Bioimages – CNR – Catania, Italy Enrico Rizzarelli, Department of Chemical Sciences-University of Catania, Italy

Prion diseases are a group of neurodegenerative disorders, affecting both animals and humans, thought to be caused by the conformational transition of the native and predominantly a-helical prion protein (PrPC) to the β-sheet rich and protease resistant pathogenic isoform (PrPSc). Although avian species express prion protein, they seems to not suffer of such neurodegenerative disorders. The chicken prion has only 40% of similarity to human PrPC but conserves the essential features. In particular there is a tandem amino acid repeats containing histidine (PHGGGWGQ in mammals and PHNPGY in avian), followed by a highly conserved hydrophobic core. Many studies suggest that mammalian prion is a copper protein, and in particular copper binds to the octarepeat region of PrPC with affi nities ranging between 10-6-10-15 M. Conversely only few studies have been conducted on the N-terminal tandem repeat of avian prion. We have synthesized the single hexapeptide PHNPGY and the two-hexarepeat PHNPGYPHNPGY peptide sequences with both N and C-termini blocked by acetylation and ammidation respectively. Their copper(II) complexes have been charcterized by means of potentiometric measurements, UV-vis, Circular Dichroism, EPR and Electrospray Mass Spectrometry. The fi rst anchor group in the synthesised oligopeptide results to be the π-N nitrogen of imidazole chain of Histidine residues as expected. Furthermore the presence of two and four prolyl residues, in the hexapeptide and dodecapeptide respectively, induces a bent structure for the oligopeptides, leading to the stabilisation of species coordinated through either amide nitrogens of residues beyond the prolyl residue in addition to side chain donor centres. This allowed us to support performed previous data concerning the copper(II) interaction with avian prion, in contrast to report that denied the metal- protein interaction.

Synthesis of Some Metal Dye-Complexes as Antimicrobial Agents

Mohamed Abbas Metwally, Faculty of Science, University of Mansoura, Egypt

An enormous number of 4-arylazo-2-pyrazolin-5-ones has been reported in the literature as antimicrobial agents. The reaction of 4-arylazo-1-phenyl-3-(methyl or phenyl)-2-pyrazolin-5-ones (1) with copper and cobalt salts resulted in the formation of some new metal dye complexes (2). The importance of the newly synthesized complexes as antimicrobial agents has been discussed. References: 1 Amer F.A., Harhash A.H. and Metwally M.A., Z. Natuforsch., 32b,943(1977). 2 Fadda A.A., Metwally M.A. and Khalil A.M., Indian J. Text. 8, 82(1983). 3 Metwally M.A., Fadda A.A., Hassan H.M. and Afsah E., Org. Prep. Proc Int 17(3), 198(1985 ). 192 Journal of Inorganic Biochemistry 96 (2003)

First MCD Characterization of Intermediate X in Ribonucleotide Reductase: Insight into the Geometric and Electronic Structure Description of X

Natasa Mitic, Department of Chemistry, Stanford University, United States Lana Saleh, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, United States Martin J Bollinger, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, United States Edward I Solomon, Department of Chemsitry, Stanford University, United States

The intermediate, known as X, which is formed during the self-assembly reaction of the tyrosyl radical/µ-oxo bridged diferric cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) is responsible for the oxidation of Y122 to •Y122. Earlier spectroscopic studies (notably by stopped-fl ow, EPR, Mössbauer, ENDOR, and EXAFS) have all indicated that X is a spin-coupled Fe3+/Fe4+ center with at least one µ-oxo bridge present. In order to elucidate the electronic structure of intermediate X, a protocol has been developed to perform magnetic circular dichroism (MCD) measurements on a strain free optical sample generated using rapid-freeze-quench (RFQ). Data have been obtained on wt, W48A, and the Y122F/Y356F double mutant. While X has been reported to exhibit a broad absorption band at 365nm, there are at least ten electronic transitions observed at low temperature MCD. From C0/D0 ratios, the transitions of X can be divided into three regions: 1) the 16000-22000 cm-1 region involving spin allowed ligand fi eld transitions of the Fe4+, 2) the 23000-24000 cm-1 region of spin 4+ -1 forbidden, spin fl ip transitions on the Fe , and 3) the charge transfer (CT) region from 26000-32000 cm . The C0/D0 ratios from d →d and CT transitions strongly support signifi cant Fe4+ character coupled into the paramagnetic center. Ligand fi eld (spin allowed d →d region), nephelauxetic (spin forbidden d →d region) and optical electronegativity (CT region) analyses allow us to distinguish between the presence of (i) a bis-µ-oxo complex or (ii) a complex with one µ-oxo plus a monoanionic bridge for the active site structure of intermediate X. This provides the fi rst detailed insight into the molecular mechanism of active site formation in RNR.

The enzyme catalyzing the methane hydroxylation in cytoplasmic membrane of Methylosinus trichosporium OB3b

Akimitsu Miyaji, Tokyo Institute of Technology, Japan Toshiaki Kamachi, Tokyo Institute of Technology, Japan Ichiro Okura, Tokyo Institute of Technology, Japan

Methane-oxidizing bacteria metabolize methane as carbon and energy source. The fi rst step of methane metabolism in the bacteria is methane hydroxylation catalyzed by methane monooxygenase(pMMO) in cytoplasmic membrane of the bacteria. To investigate the enzyme(s) catalyzing the methane hydroxylaton in cytoplasmic membrane of Methylosinus trichosporium OB3b, the membrane fraction exhibiting pMMO activity and purifi ed pMMO from M. trichosporium OB3b were prepared. pMMO activity in the membrane fraction required NADH and duroquinol as reductant. But purifi ed pMMO required only duroquinol. This result indicates that there is an electron transfer system from NADH to the purifi ed pMMO in cytoplasmic membrane of M. trichosporium OB3b and there is/are the enzyme(s) mediating the electron transfer from NADH to pMMO. The purifi ed pMMO consists of three subunits (43, 27, 24 kDa) estimated by SDS-PAGE. The molecular weight of cross- linked pMMO with DSP was estimated by SDS-PAGE and it was about 200 kDa, indicating that purifi ed pMMO exists as a dimmer. Metal content of purifi ed pMMO was measured and it contains four copper ions per pMMO dimmer. EPR spectrum of purifi ed pMMO indicates that the copper ions are coordinated by four nitrogen atoms. The copper site(s) is/are thought to be active site(s) of pMMO. It was found that NADH-dependent pMMO activity in the membrane fraction was inhibited by rotenone. But quinol- dependent pMMO activity in the membrane fraction and the activity of purifi ed pMMO were not inhibited by rotenone. These results indicate that the enzyme mediating the electron transfer to pMMO is inhibited by rotenone. Journal of Inorganic Biochemistry 96 (2003) 193

Inversion of Helical Chirality in Metal Complexes by Steric Control

Hiroyuki Miyake, Department of Chemistry, Graduate School of Science, Osaka, Japan Hiroshi Tsukube, Department of Chemistry, Graduate School of Science, Osaka, Japan

Helical structure is one of the most important motifs in bio-polymers. Typically, many proteins have α-helical structures, the helicity of which is signifi cantly determined by the nature of amino acid components: poly-L-amino acids usually exhibit right-handed helix, while poly-L-proline forms left-handed one. We report here that several transition metal complexes with amino acid-based tetradentate ligands offer characteristic inversion of helical chirality. The employed ligands are ethylenediamine derivatives in which both nitrogen atoms were modifi ed by chiral amino acid amides. The crystal structure analyses revealed that they form cobalt(II) complexes in a stereoselective fashion. Although amino acid derivatives with same confi guration were introduced, helical chirality could be inversed by optimization of amide substitutes.

Model Compounds of Galactose Oxidase

Garry M Mockler, University of Wollongong, Australia Ray J Butcher, Howard University, United States Roger Kanitz, University of Wollongong, Australia Margaret Sheil, University of Wollongong, Australia Cody Szczepina, University of Wollongong, Australia

Copper(II) complexes, CuL.B where LH2 is a Schiff Base ligand formed by the reaction of salicylaldehyde with amino acids and B is pyridine or imidazole, oxidize sugars to the corresponding aldehydes in basic solutions using acetonitrile/water, methanol, methanol/water or water as the solvent. Two moles of the copper complex react with one mole of the sugar.

Where B is pyridine, Cu2O is precipitated during the redox reaction. Where B is imidazole, Cu(I)LH is precipitated during the reaction. Cu(I)LH re-oxidises to form the starting material in the presence of oxygen and may then be re-used to oxidise more sugar. A possible new method to classify the types of copper in biological systems will be discussed. 194 Journal of Inorganic Biochemistry 96 (2003)

Fine tuning of phosphodiesterasse models activity using allosteric effect of metal ion coordination

Larisa A Mokhir, Anorganisch-Chemisches Institut, Universität Heidelberg, Germany Markus Hoppe, Anorganisch-Chemisches Institut, Universität Heidelberg, Germany Hans Pritzkow, Anorganisch-Chemisches Institut, Universität Heidelberg, Germany Igor O Fritsky, Anorganisch-Chemisches Institut, Universität Heidelberg, Germany Roland Kraemer, Anorganisch-Chemisches Institut, Universität Heidelberg, Germany

In enzyme catalysis allosteric regulation is the control of activity by noncovalent modifi ers (molecules or ions) which bind to the enzyme at a site other than the active site but alter the conformation of the active site. We have recently described polynuclear metal complexes of polypyridyl ligands which may be considered as prototypes of synthetic allosteric catalysts [1]. While two ‘functional’ copper (II) ions cooperate in the cleavage of a phosphodiester, catalytic activity is controlled by the nature of a third ‘structural’ (or allosteric) metal ion Ms. Here we present the expansion of this concept to structurally different ligand systems capable to form polynuclear complexes. [1] I..O.Fritsky, R. Ott, R. Krämer, Angew. Chem., 2000, 112, 3403; Angew. Chem. Int. Ed., 2000, 39, 3255; I..O.Fritsky, R. Ott, H. Pritzkow, R. Krämer, Chem. Eur. J., 2000, 7, 1221.; L. Kovbasyuk; M. Hoppe; H. Pritzkow; R. Kramer. Eur. J. Inorg.

Alpha-keto Acids and 4-Hydroxyphenylpyruvate Dioxygenase

Graham R Moran, University of Wisconsin – Milwaukee, United States Vincent M Purpero, University of Wisconsin – Milwaukee, United States Kayunta Johnson-Winters, University of Wisconsin – Milwaukee, United States Micheal Kavana, University of Wisconsin – Milwaukee, United States

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisate a reaction that involves decarboxylation, substituent migration and aromatic oxygenation. HPPD is a member of the alpha-keto acid dependent oxygenases that require Fe(II) and an alpha-keto acid substrate to oxygenate an organic molecule. We have examined both the binding of ligands and oxygen reactivity of HPPD from Streptomyces avermitilis. The binding of either 4-hydroxyphenylpyruvate, phenylpyruvate or pyruvate to the holo-enzyme produces a weak charge transfer band at ~500 nm that is indicative of bidentate binding of the 1-carboxylate and 2-keto pyruvate oxygen atoms to the active site metal ion. Interestingly, no turnover is observed in the presence of phenylpyruvate or pyruvate. EPR and MCD of these holo-enzyme complexes suggest a geometric switch that increases the reactivity of the enzyme toward molecular oxygen only in the presence of 4-hydroxyphenylpyruvate. The addition of 4-hydroxyphenylpyruvate and molecular oxygen to the holo-enzyme is formally random. The basis of the ordered steady-state kinetic mechanism previously observed by Rundgren (Rundgren, M. (1977) J Biol Chem 252, 5094-9.) is the low oxygen reactivity of holo-HPPD in the absence of 4-hydroxyphenylpyruvate. The rate of reduction of molecular oxygen increases 3,600 fold when holo-HPPD is in complex with this substrate. This complex reacts with molecular oxygen with a second order rate constant of 1.4 ◊ 105 M-1s-1 inducing the formation of a spectrophotometrically observable intermediate that decays at the catalytically relevant rate of 8 s -1. Journal of Inorganic Biochemistry 96 (2003) 195

Heteronuclear NMR and UV-Resonance Raman Studies on the molecular mechanism of Zn2+ sensing with cyanobacterial transcription factor, SmtB

Hayato E Morita, Center for Gene Research, Ehime University, Japan Miki Wakamatsu, Department of Chemistry, Faculty of Science, Ehime University, Japan Shinjiro Okuro, Department of Materials and Biological Sciences, Faculty, Japan Takamitsu Kohzuma, Department of Materials and Biological Sciences, Faculty, Japan Hidenori Hayashi, Department of Chemistry, Faculty of Science, Ehime University, Japan

In cyanobacterium Synechococcus sp. PCC 7942, SmtB, functioning as the sensor to heavy-metal ions (notably Zn-ion) in the dimer form, represses the transcription of smtA gene encoding metallothionein-like protein. The binding of SmtB with Zn-ions reduces the affi nities to recognition DNA sequences located in the operator/promoter region of smtA gene, and this induces the transcription of smtA. To clear the relationships between the structural changes of SmtB following Zn-ion binding and the decreases of affi nities to recognition DNA sequences, we have analyzed the structural changes of SmtB, induced by Zn-ion binding, with hetero-nuclear NMR and UV-resonance Raman spectroscopy. On the basis of the results obtained here, we further compared the affi nities of point-mutated SmtB to modifi ed recognition DNA sequences. From NMR spectroscopic analysis, we assigned the amino acid residues coordinated to the Zn-ions and found that Zn-ion binding induces the structural changes around two leucine residues. Furthermore, we showed that these structural changes induced the structural distortion around two recognition helices in SmtB dimer, and concluded that this the main reason for the decrease in the affi nities of SmtB to recognition DNA sequences, following Zn-ion binding. UV-resonance Raman spectroscopy precisely indicated the orientation of the side chain atoms in histidine residues coordinated to Zn- ions. In consideration of these results, we designed the point-mutated SmtB and compared the affi nities of these mutated proteins to recognition DNA-sequences and certifi ed our models about the relationships between the structure and the function of SmtB. There are two recognition DNA sequences in the operator/promoter region of smtA, and the numbers and the sizes of SmtB-DNA complexes are different. To clear the origin of this difference, we also designed the modifi ed recognition DNA- sequences and compared the numbers and the sizes of SmtB-DNA complexes. 196 Journal of Inorganic Biochemistry 96 (2003)

Molecular Aspects of Denitrifi cation / Nitrate Dissimilation

Isabel Moura, REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidadé Nova de Lisbon, Portugal Ines Cabrito, REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidadé Nova de Lisbon, Portugal Gabriela Almeida, REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidadé Nova de Lisbon, Portugal Carlos Cunha, REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidadé Nova de Lisbon, Portugal Maria J Romao, REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidadé Nova de Lisbon, Portugal Jose JG Moura, REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciencias e Tecnologia, Universidadé Nova de Lisbon, Portugal

Denitrifi cation is one of the most important processes in the Nitrogen Biological cycle. In this process, the nitrate molecule is reduced to molecular dinitrogen in a series of reactions:

2 NO3- → 2 NO2- → 2 NO → N2O → N2 Four enzymes have been identifi ed in this inducible pathway: nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, named after the substrate they transform. Nitrite can also be reduced to ammonia in other group of bacteria that dissimilate nitrate to ammonia, thus providing energy to support cell growth. The nitrite reductase from this organism is a multiheme c enzyme. The structures of these enzymes are known except the one of nitric oxide reductase. We will describe the structure and properties of nitrate reductase (NAP), nitrite reductase (NIR) and nitrous oxide reductase (NOR). NAP from D.desulfuricans ATCC 27774 is a very simple enzyme containing a single subunit with one [4Fe-4S] center and a molybdenum ion bound to two MGD cofactors. The structure of NIR from the same organism was recently solved at 2.3Å. Five heme c and two Ca sites were observed. The spectroscopic properties of this enzyme are discussed in relation with the determined structure. The crystal structure of NOR was solved to a resolution of 2.4 Å.This enzyme contains one binuclear (CuA) and a tetranuclear copper center (CuZ).

The studies performed by us on the N2OR from Ps.nautica revealed the unusual structure of CuZ (catalytic site). The structure reveals that the CuZ center belongs to a new type of cluster, in which four copper ions are ligated by seven histidine residues. Spectroscopic studies will be presented. A model is proposed for the binding of N2O binds to this center. A mechanistic proposal will be presented. Aknowledgements: K.Brown, S.Bursakov, C.Cambillau, P.Cheng, C.Costa, J.Lampreia, M.Tegoni, A.Pereira, M.PrudÍncio, E.Solomon, P.Tavares are acknowledge for many contributions. J.M.Dias et al.Stucture 7,65-79 (1999) K.Brown et al.J.Biol.Chem. 275, 41133-41136 (2000). I.Moura and J.J.G.Moura, Curr. Opin, in Chem.Biol. 5, 168-175 (2001) C.A.Cunha et al. J Biol Chem 2003 Mar 4; [epub ahead of print] Journal of Inorganic Biochemistry 96 (2003) 197

Correlations of Electronic Spectra and Molecular Structures of Blue Copper Proteins

Vera Müller, Universität Heidelberg, Germany Peter Comba, Universität Heidelberg, Germany Rainer Remenyi, Universität Heidelberg, Germany

Blue Copper Proteins are known for their fast and effi cient long range electron transfer in plants and bacteria. Characteristic properties of cupredoxines are an intense blue colour (absorption at ~600 nm, 2000-6000 1/(M*cm), arising from S(Cys)- Cu(II) charge transfer, and high redox potentials (approx. 180-680 mV). These properties depend to a large extent from the structure of the chromophore and the difference between various species must be due to subtle structural changes. Therefore, it is of interest to correlate structural parameters with electronic properties. This might be possible by the computation of electronic properties from structural parameters and vice versa. Accurate spectroscopic data might also be used to validate experimental and computed structural data. Excited state properties were calculated with DFT (Gausssian 98) and AOM (CAMMAG) methods and compared with experimental electronic spectra.

Chromium(III) Complexes Used as Nutritional Supplements: Structures and Reactivities

Irma Mulyani, School of Chemistry, University of Sydney, Australia Aviva Levina, School of Chemistry, University of Sydney, Australia Peter A Lay, University of Sydney, Australia

Chromium(III) is considered by most nutritionists as an essential trace element for humans, enhancing the activity of insulin in glucose and fat metabolism [1]. It is suggested that more than 90% of population in developed countries do not obtain enough Cr(III) with food, which led to a widespread use of Cr(III)-containing nutritional supplements, particularly Cr(III) picolinate (I) [1]. Due to the concerns about safety of Cr(III) picolinate, a new supplement, Cr(III) propionate (II) has been recently proposed [1]. The mechanism of biological action of Cr(III) complexes remains unclear, and no well-defi ned Cr(III)- containing biomolecules has been characterised as yet [2]. In this work, structures and reactivities of I, II, and other Cr(III) complexes have been studied under biologically relevant conditions. Decompositions of the complexes in neutral aqueous solution and in artifi cial gastric juice have been studied by UV-visible spectroscopy, electrospray mass spectroscopy, and X-ray absorption spectroscopy. The developed methods will be applied to the studies of the speciation of Cr(III) complexes in commercial nutritional supplements. For the fi rst time, the ability of enzymatic systems (such as glucose + glucose oxidase +O2) to oxidise Cr(III) to Cr(VI) in aqueous solutions at pH ~7 have been established. The Cr(III) (II) + glucose + glucose oxidase system causes extensive oxidative damage of DNA in vitro. This fi nding raises concerns about the safety of the use of II as a nutritional supplement. Acknowledgment. Financial support of this work was provided by ARC and ASRP grants. References [1] Vincent, J. B. Polyhedron 2001, 20, 1-26 [2] Levina, A.; Cood, R.; Dillon, C. T; Lay, P. A. Progr. Inorg.Chem. 2003, 51, 145-250. 198 Journal of Inorganic Biochemistry 96 (2003)

Changes of Tryptophan Residues in Human Hemoglobin upon Ligand Binding: Ultraviolet Resonance Raman Study of Mutants at α14, β15 or β37 Tryptophan

Masako Nagai, Kanazawa University Faculty of Medicine, Japan Yayoi Jin, Kanazawa University Faculty of Medicine, Japan Hiroshi Sakurai, Kanazawa University Faculty of Medicine, Japan Yukifumi Nagai, Kanazawa University Faculty of Medicine, Japan Shigenori Nagatomo, Center for Integrative Bioscience, Okazaki National Research Institutes, Japan Teizo Kitagawa, Okazaki National Research Institutes, Japan

The intensity of tryptophan (Trp) Raman bands in the UV resonance Raman (RR) spectrum of human hemoglobin (Hb A) changes markedly upon ligand binding [1, 2]. There are three Trp residues (α14, β15 and β37) in Hb A for each αβ dimer. These Trp residues are located in functionally and structurally important regions, i.e. α14Trp and β15Trp in the α1β1 subunit interface, and β37Trp in the α1β2 subunit interface. To evaluate which Trp residue is mainly responsible for the UVRR spectral changes upon the deoxy (T) to oxy (R) structure transition, we prepared three mutant hemoglobins, α14Trp- >Leu, β15Trp->Leu, and β37Trp->His, by the site-directed mutagenesis and examined the 235-nm excited UVRR spectra. Oxy-rHb(βW37H) dissociated into dimer and exhibited a high oxygen affi nity and decreased cooperativity, but it stayed as a tetramer in the presence of inositol haxaphosphate (IHP) and restored signifi cant cooperativity (Hillís n = 2.0). Mutants at α14 and β15 showed slightly decreased cooperativity (Hillís n = 2.0~2.2). The RR spectrum of each Trp residue in the deoxy-form was extracted as the difference spectra between Hb A and mutants. Three Trp residues gave distinct differences in their peak frequencies of RR bands (labeled W) of W3, W7 doublet, W16, W17 and W18, i.e. at 1556, 1361/1339, 1010, 879 and 755 cm-1 for α14Trp, at 1560, 1359/1339, 1016, 884 and 761 cm-1 for β15Trp, and at 1547, 1365/1346, 1011, 877 and 755 cm-1 for β37Trp. Intensity change of all the Trp RR bands upon ligand binding in rHb (αW14L) was similar to that observed in Hb A, implying that α14Trp was not responsible for the change of Trp band upon the T->R transition. On the other hand, two β subunit mutants, rHb(βW15L) and rHb(βW37H) in the presence of IHP, exhibited a substantial decrease in intensity change compared with that in Hb A and the decrease in the latter was twice as large as that in the former. These results indicate that intensity change of Trp RR band upon the T->R transition in Hb A is mainly due to the environmental change of β37Trp residue. [1] Rodgers, K. R., et al. (1992) J. Am. Chem. Soc., 114, 3697-3709. [2] Nagai, M., et al.(1995) J. Biol. Chem. 270, 1636-1642. Journal of Inorganic Biochemistry 96 (2003) 199

A tetracopper(II) complex as a functional model of multi-copper

proteins mediates an effi cient four-electron transfer to O2 in an aerobic oxidation of catechols

Jun Nakazawa, Doshisha University, Department of Molecular Science and Technology, Japan Tomohisa Kawata, Doshisha University, Department of Molecular Science and Technology, Japan Masahito Kodera, Doshisha University, Department of Molecular Science and Technology, Japan Koji Kano, Doshisha University, Department of Molecular Science and Technology, Japan

A new long-chain octadantate ligand having four ethylenediamine units (L) and copper(II) complex of L, [Cu4(OH)4(L)](ClO4)4 (1) have been synthesized to gain some insight into the mechanism for the aerobic oxidation by multi-copper proteins.

Compound 1 has been structurally characterized and used as a catalyst for the aerobic oxidation of DBCH2. The crystal structure shows that 1 is a dissymmetric dimer of di-µ-OH dimer, and its Cu2(µ-OH)2 core is similar to those of tetracopper complexes 2 and 3. Compound 1 shows higher catalytic activity for the aerobic oxidation of DBCH2 than 2 and 3. In the reaction catalyzed by 1, H2O2 is not generated at all. Dioxygen molecule is consumed to water molecule via the effi cient four-electron redox. This is the fi rst example for the four-electron reduction of dioxygen molecule in the aerobic oxidation of catechols catalyzed by copper complexes. Moreover, UV-vis spectra show that 1 reacts with H2O2 to form a O2-complex upon.

From these results, it is suggested that in the aerobic oxidation of DBCH2 catalyzed by 1, dioxygen molecule is reduced to water via the O2-complex, similarly to multi-copper proteins. 200 Journal of Inorganic Biochemistry 96 (2003)

An Active Site Model of Prostagrandin H Synthase: Formation of an Aryloxyl Radical above an Iron(IV) Porphyrin and its Oxygenation of 1,4-Diene

Yoshinori Naruta, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan Eiki Matsui, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan Fumito Tani, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan Yuichi Shimazaki, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan

Prostaglandin H synthases (PGHSs) are heme-containing enzymes that catalyze the key step in the conversion of arachidonic acid to prostaglandin H2 (PGH2). Each of them have two enzyme active sites (cyclooxygenase and peroxidase) and these locations are distinct but structurally and functionally interconnected by sharing the common heme cofactor. The catalytic cycle for PGHS involves a tyrosyl radical formation by an intramolecular electron transfer of the nearby tyrosine residue to an oxoiron(IV) porphyrin -cation radical and the initial abstraction of 13-pro-S hydrogen from arachidonic acid in cyclooxygenase. In spite of the discoveries of the catalytic roles of the tyrosine residue in cyclooxygenase activity, no structural and functional model complexes have been constructed, because of the diffi culty in fi xing an ArOH group close to a porphyrin. In order to mimic the functions of the active sites of PGHS, we designed a ferric ‘twin- coronet’ porphyrin [FeTCP(OH)4], which contains naphthol groups in hydrophobic and chiral cavities on both faces of the porphyrin ring. X-ray crystal structure of CoTCP(OMe)8 (Fig 1), where all the eight naphthol groups are methylated, showed the naphthol group is located with a suitable distance from the porphyrin ring

(ca. 6.4 Å). The oxidation of FeTCP(OH)4 with m-CPBA (2.5 equiv.) at ~40 °C gave the corresponding naphthoxyl radical and the iron(IV) porphyrin. The resultant naphthoxyl radical was characterized by various spectroscopic methods, such as UV-vis (365, 478 nm), ESR (g = 2.0017) and resonance Raman spectroscopies. The reaction of the resultant intermediate with an 1,4-pentadienyl derivative in the presence of O2 gave the corresponding trans, cis-2,4- pentadienol in high stereoselectivity. Thus, we attained the fi rst successful modeling reaction of PGHs. Journal of Inorganic Biochemistry 96 (2003) 201

Preparation and charactarization of dinuclear iron(III) and vanadium(IV) complexes with bridging salbn or salpentn ligands

Sei Negoro, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Ryoji Okumura, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Hideyuki Asada, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Manabu Fujiwara, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan Takayuki Matsushita, Department of Materials Chemistry, Faculty of Science and Technology, Ryukoku University, Japan

Dinuclear iron centers bridged by oxo or hydroxo groups have been found to exist in hemerythrin, ribonucleotide reductase and methane monooxygenase. Moreover, the magnetic properties of oxo-bridged diiron(III) complexes are of interest for understanding the extent of exchange-coupling interactions between iron ions. Moreover, vanadium ions have been known to be involved in metalloenzymes such as haloperoxidases and nitrogenases. In this paper, we describe the preparation and characterization of dinuclear iron(III) and vanadium(IV) complexes III IV IV bridged by two X-salbn or X-salpentn ligands, [Fe (µ-X-salbn)]2(µ-O) (Fig.1), [V O(µ-X-salbn)]2 and [V O(µ-X- salpentn)]2 (Fig.2). The dinuclear iron(III) complexes were obtained by the reaction of the Schiff base ligands X-salbnH2 (N,N'-di-substituted-salicylidene-1,4-diaminobutane, X = H, 3-Me, 3-MeO, 5-Me, 5-MeO, 5-Br, 5-Cl, 5,6-Benzo), with III Fe Cl3•6H2O in the presence of Et3N in ethanol. The dinuclear vanadium(IV) complexes were obtained by the reaction IV of X-salbnH2 or X-salpentnH2 (N,Ní-di-substituted-salicylidene-1,5-diaminopentane) with V O(acac)2 in ethanol. All the dinuclear complexes have been identifi ed and characterized by elemental analysis and physicochemical methods. The X-ray crystallographic determination revealed that the Fe-O-Fe bond angles of the µ-oxo dinuclear iron(III) complexes are nearly 180°. The magnetic data for these µ-oxo complexes indicated that antiferromagnetic coupling constants, J between iron ions were estimated to be about -100 cm-1. 202 Journal of Inorganic Biochemistry 96 (2003)

Synthesis and Characterization of Copper(II) Complexes with Nicotinate in Different Coordination Style

Fengmei Nie, Department of Chemistry, Capital Normal University, Beijing, China Yuheng Deng, Department of Chemistry, Capital Normal University, Beijing, China Yanmei Li, Bio-organic Phosphorus Chemistry Laboratory, School of Life Science & Engineering, China Yufen Zhao, Bio-organic Phosphorus Chemistry Laboratory, School of Life Science & Engineering, China

Nicotinate acid plays important roles in the metabolism of all living cells, much interest has been directed towards their metal complexes. Nicotinate is a ligand with two types of binding sites. It can coordinate with transition metal ion via one or two O atoms of carboxylate group or N atom of pyridine or both of them. In the course of our systematic study of the coordination mode of nicotinate with Cu(II) in mixed-ligand system, we isolated two complexes [Cu(ntb)(Nic)]ClO4∑H2O (1) and

[Cu2(dibim)2(Nic)2](ClO4)2•2CH3OH(2), where ntb is tris(2-benzimidazolylmethyl)amine, dibim is bis(benzimidazolyl-2- methyl)amine, Nic is nicotinate.

The two mixed-ligand Cu(II) complexes [Cu(ntb)(Nic)]ClO4•H2O (1) and [Cu2(dibim)2(Nic)2](ClO4)2•2CH3OH(2) were synthesized from the reaction of Cu(ClO4)2•6H2O and sodium nicotinate with ntb and dibim. They were structurally characterized by single-crystal X-ray analysis. In complex 1, the Cu(II) atom is in a distorted trigonal bipyramidal environment with three benzimidazole N atoms of ntb defi ning the equatorial plane and one amine N atom of ntb and one O atom of carboxylate of nicotinate occupying the axial positions. In complex 2, the central Cu(II) atom is in a distorted square pyramid confi guration with three benzimidazole N atoms of dibim and one N atom of the pyridine of the fi rst nicotinate defi ning the base and one O atom from the carboxylate group of the second nicotinate occupying the apex of the pyramid. The nicotinate anions bridge the adjacent Cu(II) through both the pyridine N atom and one carboxylate group O atom to form a dinuclear copper complex with two nicotinates in an anti parallel bridging way. The striking feature of the two complexes is that nicotinate coordinates to Cu(II) in different way. Reference [1] Waizumi, K.; Takuno, M.; Fukushima, N. and et al, J. Coord. Chem., 1998, 44, 269-279. [2] Palicova, M.; Segl’a, P.; Miklos, D. and et al, Polyhedron, 2000, 19, 2689-2695. Acknowledgments This work is supported by Beijing Natural Science Foundation (2032006), P. R. China.