Journal of Inorganic Biochemistry 96 (2003) 203

Monomeric TpPrMoVOSR complexes via the chemical reduction of TpPrMoVIOSR.

David J Nielsen, School of Chemistry, University of Melbourne, Australia Christian J Doonan, School of Chemistry, University of Melbourne, Australia Graham N George, Stanford Synchrotron Radiation Laboratory, United States Hugh Harris, Stanford Synchrotron Radiation Laboratory, United States Charles G Young, University of Melbourne, Australia

EPR evidence has suggested the presence of molybdenum(V) intermediates in the catalytic cycle of hydroxylase enzyme systems [1], and references therein], and as such these species are attractive targets for the synthesis of small-molecule model systems. Ongoing work in our group has allowed access to several stable and well characterised monomeric molybdenum(VI) oxo-thio complexes TpPrMoVIOSR (TpPr = hydridotris(3-isopropylpyrazol-1-yl)borate) with co-ligand R = eg. substituted phenolates [2], as shown below.

These Mo(VI) complexes have proved amenable to chemical reduction using cobaltocene (CoCp2) yielding initially the Pr V [CoCp2][Tp Mo OSR] salts [1,2]. Solution and solid state sulfur X-ray absorption spectroscopy (XAS) on selected examples of the chemically reduced species shows pre-edge features attributable to the S 1s → Mo=S π* transition of a [MoOS]+ unit. Further spectroscopic investigations (EPR, IR) are consistent with the presence of a paramagnetic Mo(V) centre bearing a terminal thio ligand. Continuing spectroscopic, structural and reactivity investigations centred on these important species will be presented. References: [1] P. D. Smith, D. A. Slizys, G. N. George and C. G. Young, J. Amer. Chem. Soc., 122(12), 2000, 2946. [2] C. J. Doonan, Unpublished results. 204 Journal of Inorganic Biochemistry 96 (2003)

Nine-Zinc Finger Protein: Infl uence of TFIIIA-Type Linker at N- or C-Terminal on DNA Binding Site

Wataru Nomura, Institute for Chemical Research, Kyoto University, Japan Yukio Sugiura, Institute for Chemical Research, Kyoto University, Japan

In natural zinc fi nger proteins, the short linker sequence, Thr-Gly-Glu-Lys-Pro, is the most typical linkr sequence between each fi nger domain. Zinc fi nger proteins recognize specifi c DNA sequence by locating its α-helix in the major groove of DNA. The utilization of zinc fi nger proteins for the DNA sequence recognition has been investigated from the recognition helix experiments. The central three-zinc fi nger connection of the native nine-zinc fi nger protein transcription factor IIIA(TFIIIA) is composed of unique linker sequences, -NIKICV-, -TQQLP-, -AG-, and -QDL-. As revealed by X-ray crystallographic study of TFIIIA-5S rRNA gene complex, the characteristic DNA binding mode of TFIIIA zinc fi nger protein is brought from the unique linker sequences. By modifi cation of the linker sequences and adjustment of the linker length, several efforts to improve the DNA binding affi nity have been performed. However, it is unknown whether the position of the linker sequences in multi-zinc fi nger protein gives any effect on DNA bindings or not. New artifi cial nine-zinc fi nger proteins, Sp1ZF9TC and Sp1ZF9TN, which use TFIIIA-type linker for their C- and N- terminal three-zinc fi nger connections have been created. To investigate the infl uence of TFIIIA-type linker by their different locations in the proteins, the gel mobility shift assays(GMSA), footprinting assays, and methylation interference analyses were performed.The GMSA revealed similar DNA binding affi nities of the two proteins. The footprinting analyses indicated that the two zinc fi nger proteins recognize the same part of GCII or GCIII DNA. Moreover, the specifi c base contacts were observed in the same sites of the substrate DNA. In the present proteins, Sp1ZF9TC and Sp1ZF9TN, the four zinc fi ngers (the fi ngers 1~4 or 5~9) situated in the opposite site to the TFIIIA-linker position participate in their DNA bindings. Indeed, the position of TFIIIA-linker is important in DNA recognition by multi-zinc fi nger proteins.

Reaction mechanism of nitrile hydratase: analysis by isobutryronitrile

Masafumi Odaka, Bioengineering Laboratory, RIKEN, Japan Masanari Tsujimura, Bioengineering Laboratory, RIKEN, Japan Hiroshi Nakayama, Biomolecular Charactrization Division, RIKEN, Japan Naoshi Dohmae, Biomolecular Charactrization Division, RIKEN, Japan Mikio Hoshino, Synthetic Organic Chemistry Laboratory, RIKEN, Japan Koji Takio, Biomolecular Charactrization Division, RIKEN, Japan Mizuo Maeda, Bioengineering Laboratory, RIKEN, Japan Isao Endo, Faculty of Agriculture, Utsunomiya University, Japan

Nitrile hydratase (NHase) catalyzing the hydration of nitriles to the corresponding amides is industrially important because of the production of acrylamide. NHase is a non-heme iron or non-corrin cobalt enzyme. Recent X-ray crystallographic analyses demonstrated that the structures around the catalytic center were conserved between Fe- and Co-type NHases including two post-translationally modifi ed cysteine ligands, cysteine-sulfi nic (Cys-SO2H) and -sulfenic acids (Cys-SOH). These modifi cations are believed to be responsible for the catalytic reaction. However, the detailed reaction mechanism remains unknown. In general, NHase exhibits wide substrate specifi city. It is of interest that isobutyronitrile (IBN) was known to barely hydrated and to function as a competitive inhibitor of Fe-type NHases of two bacterial strains with Ki values of about 5 µM. These fi ndings sound curious because n-butyronitrile is normally hydrated by these enzymes and because Fe- type NHases of other strains hydrate IBN with relatively slow rate constant. Thus, we have studied the interaction between isobutyronitrile (IBN) and Fe-type NHase in detail. From kinetic studies of the inhibitory effect of IBN on NHase, we found that commercially available IBN contained an unknown compound that strongly inhibited NHase activity. This compound induced signifi cant changes on the UV-Vis absorption spectrum of NHase, suggesting its interaction with the iron center. The compound was isolated by reversed-phase HPLC and identifi ed as 2-cyano-2-propyl hydroperoxide (Cpx) by NMR. Upon the addition of stoichiometric amount of Cpx, NHase was irreversibly inactivated, probably by the oxidation of the Cys-SOH ligand to Cys-SO2H (NHase(double sulfi nate)). Moreover, UV-Vis absorption spectral changes of NHase induced by Cpx suggested that Cpx was associated with NHase(double sulfi nate), to form a stable complex. Cpx is a novel inhibitor for Fe-NHase and gives a clue for understanding its catalytic mechanism. Journal of Inorganic Biochemistry 96 (2003) 205

Expression and spectroscopic characterization of Pseudomonas ovalis [2Fe-2S] ferredoxin involved in the iron-sulfur cluster assembly machinery

Daijiro Ohmori, Department of Chemistry, School of Medicine, Juntendo University, Japan Takeo Imai, Department of Life Science, Rikkyo University, Japan Akio Urushiyama, Department of Chemistry, Rikkyo University, Japan Minoru Akiyama, Department of Chemistry, Rikkyo University, Japan Eiji Watanabe, Department of Chemistry, School of Medicine, Juntendo University, Japan Fumiyuki Yamakura, Department of Chemistry, School of Medicine, Juntendo University, Japan Keikichi Uchida, Department of Biology, School of Medicine, Juntendo University, Japan

We reported that Pseudomonas ovalis contained two types of ferredoxins; 7Fe-ferredoxin and 2Fe-ferredoxin. To investigate detailed features and physiological roles, we cloned the genes encoding 2Fe-ferredoxin(fdxB) from the chromosomal DNA of P. ovalis . The two ORF translations upstream of fdxB has a similarity to the heat shock protein family, hscA and a part of hscB, those are members of the iron-sulfur cluster assembly gene cluster. The fdxB translation has a signifi cant similarity to the primary structures of E. coli 2Fe-ferredoxin, which is involved in the iron-sulfur cluster assembly machinery. The UV-vis absorption spectrum of the recombinant ferredoxin reveals a characteristic feature of [2Fe-2S] ferredoxins. Circular dichroism spectra of the oxidized and reduced protein were similar to those of vertebrate-type ferredoxins than to those of plat-type ferredoxins. The 77K Resonance Raman spectrum of the protein in the Fe-S stretching region displays closely similar band frequencies (287, 317, 327, 332, 350, 394, 421cm-1) to that of bovine adrenodoxin, though it displays signifi cantly different band frequencies from those of spinach ferredoixn-I and ferredoxin-II. EPR spectrum of the reduced protein exhibits an axially symmetric spectrum (g1=2.02, g2=1.94), which is more similar to that of adrenodoxin than that of spinach ferredoxin-I (rhombic symmetry spectrum). These spectroscopic data imply that the [2Fe-2S] cluster environment in the protein is similar to that of a vertebrate-type ferredoxin.

Effi cient N-terminal Peptide Sequencing using Bis(terpyridine)Ruthenium(II) Derivatives

Taka-aki Okamura, Osaka University, Japan Norikazu Ueryama, Osaka University, Japan Taku Iwamura, Osaka University, Japan Mana Ikemori, Osaka University, Japan Maki Kaneko, Osaka University, Japan Ken Masui, Osaka University, Japan Hitoshi Yamamoto, Osaka University, Japan Minoru Yamaguchi, Shimadzu Corporation, Japan Hiroki Kuyama, Shimadzu Corporation, Japan Eiji Ando, Shimadzu Corporation, Japan Susumu Tsunasawa, Shimadzu Corporation, Japan

Proteomics has become one of the most important projects in biological and medical fi elds. For exhaustive identifi cation of proteins in each organism, speedy and powerful analytical techniques are required. The recent development of mass spectroscopy and the improvement of instruments enable high-sensitive detection. We have developed novel reagents having cationic bis(terpyridine)ruthenium(II) moiety for the determination of amino acid sequences of proteins. When [(tpy)Ru(tpy-C6H4NCS)](PF6)2 (tpy = 2,2':6',2'-terpyridine) is used for Edman degradation, the obtained thiohydantoin is detected supersensitively by mass spectroscopy compared to conventional methods. The cationic character facilitates ionization process and the ions are easily identifi ed by the characteristic isotope pattern of the ruthenium compound. The labeling of N-terminus of peptides using the active ester [(tpy)Ru(tpy-C6H4COONSu)](PF6)2 effectively and selectively enhances the intensities of N-terminal product ions in MSn spectra. The stepwise dissociation from C- to N-terminus was performed successfully using MSn technique in iron-trap detector. Entirely sequential N-terminal fragments of oligopeptide (> 10 residues) were clearly detected by PSD (post source decay) MALDI-TOF-MS spectra. 206 Journal of Inorganic Biochemistry 96 (2003)

Synthesis and Properties of Copper(II) Complexes with Bulky Ligand Tris(pyrazolyl)methane

Tetsuya Ono, Department of Chemistry, University of Tsukuba, Japan Yoko Ishikawa, 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

The activation of oxygen process in metalloprotein is very important in vivo. Oxygen binding, activation, and reduction are often carried out by copper containing enzymes. In particular the aerobic oxidation of catechols to o-quinones mediated by copper ion is an important catalytic process in both synthetic and metalloprotein systems. In our laboratory, we have investigated to mimic many metalloprotein models using hydrotris(pyrazolyl)borate anion as N3-type ligands. In this work, hydrotris(3,5-diisopropyl-1-pyrazolyl)borate (L1) anion and new tris(3,5-diisopropyl-1-pyrazolyl)methane (L1') were synthesized and used as N3-type ligands. Catecholato and peroxo complexes were synthesized with these ligands. In order to shed light on the structures and the electronic properties of the intermediate of copper containing monooxygenase, we studied about the structure, property, and the effect on the differences of the charge of ligands about each complex. The geometry around copper(II) ion in [Cu(O2C6Cl4)L1] (1) is considered as square planar environment with two nitrogen atoms of L1 and two oxygen atoms of tetrachlorocatecholate ligand. In 1, L1 is so bulky that the apical N atom of L1 is not ligated to copper(II) ion, making a hydrogen bond with O atom from X-ray analysis. In [Cu(O2C6Cl4)L1'] (2), the Cu(II)-semiquinonato species would be considered from UV-Vis and ESR spectra. Both [(CuL1)2(µ-O2)] (3) and [(CuL1')2(µ-O2)](Y)2 (4) (Y = - - 2 2 PF6 or ClO4 ) show µ-h : h -peroxo binuclear copper(II) structure. The bond distances of Cu-O and O-O, charge transfer energy, and Cu-O and O-O stretching energies show that the Cu-O bond of 4 is stronger than that of 3 and the O-O bond of 3 is stronger than that of 4. There are attribute to the effect on the differences of the charge of ligands.

Roles of NH—O≠- Hydrogen Bonds for Calcium Binding in Aspartate Peptide Fragment

Akira Onoda, Department of Macromolecular Science, Graduate School of Science, Osaka University, Japan Taka-aki Okamura, Department of Macromolecular Science, Graduate School of Science, Osaka University, Japan Hitoshi Yamamoto, Department of Macromolecular Science, Graduate School of Science, Osaka University, Japan Norikazu Ueyama, Department of Macromolecular Science, Graduate School of Science, Osaka University, Japan

NH—O hydrogen bonds to coordinating carboxylate groups are found frequently in the active site of calcium-binding proteins. Such hydrogen bonds are also important in aspartic protease. Aspartic acid protease model peptides, Z-Phe- Asp(COOH)-Thr-Gly-Ser-Ala-NHCy (1), AdCO-Asp(COOH)-Val-Gly-NHBzl (3), and their aspartate anion, (NEt4){Z- Phe-Asp(COO-)-Thr-Gly-Ser-Ala-NHCy} (2) and (NEt4){AdCO-Asp(COO-)-Val-Gly-NHBzl (4), having invariant primary sequence of Asp-X(Thr,Ser)-Gly fragment were synthesized and characterized by 1H NMR, CD and IR spectroscopies. NMR structure analyses indicate that the Asp Oδ atoms of aspartate peptide, 2, are intramolecularly hydrogen-bonded with Gly, Ser, Ala NH and Ser OH supporting the rigid β turn-like conformation in acetonitrile solution. The tripeptide in aspartic acid peptide, 3, forms an inverse γ turn structure, which is converted to β turn-like conformation because of the formation of intramolecular NH—O- hydrogen bonds with Asp Oδ in 4 . Such intramolecular hydrogen bonds lower the pKa value (c.a. 0.3 unit) as the carboxylic acid group. We found that the formation of NH—O- hydrogen bonds involved in a hairpin turn is correlated with the protonation and deprotonation state of Asp side chain in a conserved amino acid fragments. Journal of Inorganic Biochemistry 96 (2003) 207

The Structural Basis of Reductive and Oxidative Denitration Reactions of Organic Nitrochemicals by Flavoenzymes

Allen M Orville, School of Chemistry & Biochemistry, Georgia Institute of Chemistry & Biochemistry, United States Linda Manning, School of Chemistry & Biochemistry, Georgia Institute of Chemistry & Biochemistry, United States Akanksha Nagpal, School of Chemistry & Biochemistry, Georgia Institute of Chemistry & Biochemistry, United States David S Blehert, Department of Bacteriology, University of Wisconsin, United States Michael P Valley, Department of Biochemistry & Biophysics, Texas A&M University, United States Glenn H Chambliss, Department of Bacteriology, University of Wisconsin, United States Paul F Fitzpatrick, Department of Biochemistry & Biophysics, Texas A&M University, United States Brian G Fox, Department of Biochemistry, University of Wisconsin, United States

Nitrochemicals are currently widely used as drugs, biocides, fuels, explosives, and solvents. Consequently, they have been distributed throughout the environment from man-made sources in vast excess over naturally occurring examples. They are often mutagenic and/or toxic, in part because nitro-group metabolism can yield reactive nitrogen intermediates that modify DNA. Nevertheless, studies of microorganisms isolated from environments recently exposed to xenobiotic nitrochemicals have established that many nitrocompounds are catabolized. We present the crystal structure and functional analysis of a) xenobiotic reductase from Pseudomonas putida (XenA), b) xenobiotic reductase from P. fl uorescens I-C (XenB) and c) nitroalkane oxidase from Fusarium oxysporum (NAO). XenA and XenB catalyze the regiospecifi c, NADPH-dependent reductive denitration of nitroglycerin to yield nitrite and 1,2- or 1,3-dinitroglycerin, respectively. They are new members of the a/b-TIM barrel, FMN-dependent oxidoreductase family but share only 34 % sequence identity. In contrast, NAO catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones with production of H2O2 and nitrite. We present the structural analysis of recombinant NAO in two space groups and in a covalent 5-nitrobutyl-1,5-dihydro-FAD adduct trapped during turnover. Thus, these prokaryotic and eukaryotic enzymes use FMN or FAD to catalyze either reductive or oxidative denitration reactions. As a group they transform substrates like: i) organic nitroesters, ii) cyclic nitrochemicals such as 2,4,6-trinitrotoluene, and iii) primary and secondary nitroalkanes present in many fuels, solvents and biocides. The structural basis for catalysis by these fl avoenzymes and insights into their reaction mechanisms will be discussed. Portions of this research were carried out at SSRL, Stanford University, X26C at NSLS, Brookhaven National Laboratory, and SER-CAT at APS, Argonne National Laboratory. Funded in part by the Georgia Tech Research Corporation and the Georgia Institute of Technology offi ce of the vice provost for research to A.M.O., by the NSF (MCB-9733734) to B.G.F. and by the NIH (GM 58698) to P.F.F. 208 Journal of Inorganic Biochemistry 96 (2003)

Oxidation Mechanism of Phenols by Dicopper-Dioxygen (Cu2/O2) Complexesµ Takao Osako, Department of Chemistry, Graduate School of Science, Osaka, Japan Kei Ohkubo, Department of Material and Life Science, Graduate School of Science, Osaka, Japan Masayasu Taki, Department of Chemistry, Graduate School of Science, Osaka, Japan Yoshimitsu Tachi, Department of Chemistry, Graduate School of Science, Osaka, Japan Shunichi Fukuzumi, Department of Material and Life Science, Graduate School of Science, Osaka, Japan Shinobu Itoh, Department of Chemistry, Graduate School of Science, Osaka, Japan

The fi rst systematic studies on the oxidation of neutral phenols (ArOH) by the (µ-η2:η2-peroxo)dicopper(II) complex (A) and the bis(µ-oxo)dicopper(III) complex (B) supported by the 2-(2-pyridyl)ethylamine tridentate and didentate ligands LPy2 and LPy1, respectively, have been carried out in order to get insight into the phenolic O-H bond activation mechanism by metal-oxo species. In both cases (A and B), the C-C coupling dimer was obtained as a solely isolable product in ~50 % yield base on the dicopper-dioxygen (Cu2/O2) complexes, indicating that both A and B act as one-electron oxidants for the phenol oxidation. The rate-dependence in the oxidation of phenols by the Cu2/O2 complexes on the one-electron oxidation potentials of the phenol substrates as well as the kinetic deuterium isotope effects have indicated that the reaction involves a proton-coupled electron transfer (PCET) mechanism. The reactivity of phenols for hydrogen atom transfer (HAT) reactions using cumylperoxyl radical (C) as the hydrogen acceptor has also been investigated to demonstrate that the rate-dependence of the HAT reaction on the one-electron oxidation potentials of the phenols is signifi cantly smaller than that of the reaction with the Cu2O2 complexes. Thus, the result unambiguously confi rmed that the oxidation of phenols by the Cu2O2 complex proceeds via the PCET mechanism. The reactivity difference between A and B has also been discussed by taking account of the existed fast equilibrium between A and B.

Interaction of Monodentate Ligands with a Co(III) Complex containing a N2S3 Coordination Environment Similar to an Active Site of Nitrile Hydratase.

Tomohiro Ozawa, Nagoya Institute of Technology, Japan Takayuki Goto, Nagoya Institute of Technology, Japan Yasuhiro Funahashi, Nagoya Institute of Technology, Japan Koichiro Jitsukawa, Nagoya Institute of Technology, Japan Hideki Masuda, Nagoya Institute of Technology, Japan

Nitrile hydratase is known as an enzyme that catalyzes a hydration of a nitrile compound to the corresponding amide and has a low-spin Co(III) or Fe(III) ion at the active site. Recently, X-ray crystal structural analysis of the Co-type enzyme revealed that three sulfurs from cystein and two amido nitrogens in a main chain coordinated to the Co(III) center as shown in Fig. 1. Two of three sulfurs were interestingly oxygenated to sulfi nate and sulfenate. A mechanism that the activated coordinating water (or hydroxide ion) attacks directly to a nitrile or to a surrounding water molecule has been offered as a candidate, however, is still in dispute because of the unique structure. In relation to this speculated mechanism, the coordination behavior of a small molecule including water to a Co(III) complex is interesting. In this study, a Co(III) complex was synthesized, in which two amide nitrogens, two sulfi nic sulfurs, and a thioether-type sulfur are included as the coordinating atoms. The oxidation of the thiols was performed by exposing its Co(III) complex to the air and confi rmed by the appearance of S-O stretching on the IR spectrum. Coordination of monodentate small molecules at the sixth site as well as the characterization of the Co(III) complex will be discussed. Journal of Inorganic Biochemistry 96 (2003) 209

Metal uptake and confi gurational studies of antiviral macrocycles

Stephen J Paisey, Edinburgh University, United Kingdom Neil McDonald, Dundee University, United Kingdom Paul J Armstrong, Edinburgh University, United Kingdom Peter J Sadler, Edinburgh University, United Kingdom

Cyclam (1,4,8,11-tetraazacyclotetradecane) and its derivatives are potent HIV cell entry inhibitors binding selectively to the human CXCR4 co-receptor.1 The most successful of this class of compounds is xylyl-bicyclam (AMD3100) which reached phase II clinical trials.2 The strength of in vitro binding to the CXCR4 co-receptor correlates well with anti-HIV activity. Metal binding to AMD3100 has a marked effect on its binding strength to CXCR4 as illustrated by the following series: Zn(II)2 > 3 AMD3100 > Ni(II)2 > Cu(II)2 >> Co(III)2 >> Pd(II)2. Metal cyclams can adopt 6 major confi gurations, depending on the chirality of the coordinated nitrogens, and the folding of the macrocycle.4 We have developed multinuclear 2D NMR techniques which allow time dependent changes in the distribution of these confi gurations to be followed in solution.5 Modelling studies suggest that the CXCR4 co-receptor recognises specifi c confi gurations of Zn2-xylyl-bicyclam. Since Fe(III), Zn(II) and Cu(II) are abundant in the blood we have used UV-vis spectroscopy and ICP-AES to investigate the ability of cyclams to sequester Fe(III) from transferrin, and Zn(II) and Cu(II) from albumin both alone and in competition. We thank the E. P. S. R. C and the Wellcome Trust for support. 1) Schols, D., Este, J. A., Henson, G., and De Clercq, E., Antiviral Res. (1997) 35, 147-156. 2) Hendrix, C. W., Flexner, C., MacFarland, R. T., Giandomenico, C., Fuchs, E. J., Redpath, E., Bridger, E., and Henson, G. W., Antimicrob. Agents Chemother. (2000) 44, 1667-1673. 3) Este, J. A., Cabrera, C., De Clercq, E., Struyf, S., Van Damme, J., Bridger, G., Skerlj, R. T., Abrams, M. J., Henson, G., Gutierrez, A., Clotet, B., Schols, D., Mol. Pharmacol. (1999) 55, 67-73 4) Bosnich, B., Poon, C. K., Tobe, M. L., Inorg. Chem.(1965) 4, 1102. 5) Liang, X., Parkinson, J. A., Weishaeupl, M., Gould, R. O., Paisey, S. J., Park, H., Hunter, T. M., Blindauer, C. A.; Parsons, S., Sadler, P. J., J. Am. Chem. Soc. (2002), 124, 9105-9112. 210 Journal of Inorganic Biochemistry 96 (2003)

Synergistic Activation of Soluble Guanylate Cyclase by CO and YC-1: Effects of Substrate

Biswajit Pal, Center for Integrative Bioscience, Okazaki National Research Institutes, Japan Zhengqiang Li, Center for Integrative Bioscience, Okazaki National Research Institute, Japan Shigeo Takenaka, Department of Veterinary Science, Osaka Prefecture University, Japan Shingo Tsuyama, Department of Veterinary Science, Osaka Prefecture University, Japan Teizo Kitagawa, Okazaki National Research Institutes, Japan

Soluble guanylate cyclase (sGC), a physiological receptor of nitric oxide (NO), converts GTP into cyclic GMP (cGMP), which acts as a second messenger in many cell signaling pathways. Catalytic activity of sGC, which binds heme through a conserved histidine (His) residue, increases up to 200 times when NO binds to heme. Formation of a fi ve-coordinate (5c) species in NO bound form and subsequent conformational changes in the protein is responsible for this activation. Carbon monoxide (CO) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzanidazole (YC-1) can synergistically activate sGC to the same level as NO. In native sGC, Fe-His bond cleavage has recently been shown in the presence of CO and YC-1. However, the role of GTP, the substrate of the enzyme, has been overlooked in most of the studies. In fact, we have pointed out the appearance of the 5c CO-bound heme in bovine lung sGC, in the presence of YC-1. However, GTP is necessary for the production of 5c species. This observation directly indicates the possibility of GTP-induced structural changes in sGC. To identify the effect of substrate we carried out resonance Raman study of CO-bound sGC in the presence of YC-1 and -1 a series of substrate analogues. Two Fe-CO stretching (νFe-CO)Raman bands were found in CO-bound sGC; one at 473 cm and the other at 488 cm-1. Upon the addition of YC-1, population corresponding to the 488 cm-1 species increased. Upon further addition of GTP, this conversion was nearly completed and accompanied by appearance of a new isotope sensitive -1 νFe-CO band at 521 cm , which we assigned to the 5c species of CO-sGC. GTP-γ-S produced a similar type of change whereas the effect of cGMP was less pronounced. Most interesting change appeared when ATP was added in the presence of CO and YC-1. Effect of YC-1 was abolished by the addition of ATP and 488 cm-1 band disappeared. On the other hand, intensity of the 488 cm-1 band diminished and the 473 cm-1 component increased upon addition of excess GTP (5mM). These results very clearly indicate the GTP-induced structural changes in sGC in the presence of CO and YC-1. Journal of Inorganic Biochemistry 96 (2003) 211

Spectroscopic Characterization of the Oxo-Transfer Reaction from Bis(µ-oxo)Dicopper(III) Complex to Triphenylphosphine

Svitlana V Pavlova, Academia Sinica, Taiwan, Province of China Kelvin H-C Chen, Academia Sinica, Taiwan, Province of China Sunney I Chan, Institute of Chemistry, Academia Sinica, Taiwan, Province of China

Among the copper-dioxygen complexes so far reported, bis(µ-oxo)dicopper(III) and (µ-η2: η2-peroxo)dicopper(II) have been most extensively studied since these binuclear metal sites have been proposed as a models of the reactive intermediate at the active sites of the copper monooxygenases.

Although the reactivity of copper-dioxygen species toward PPh3 has been known for a couple of years, the mechanistic details have remained elusive1. Toward this end, we have employed various spectroscopic methods to characterize the chemical species formed during the putative oxygen transfer reaction between copper-dioxygen complexes and PPh3. Here we report the parallel EPR, 1H NMR and Cu K-edge X-ray absorption spectroscopic studies of the chemistry between bis ′ ′ (µ-oxo)dicopper(III) complex 1, supported by N,N,N ,N – tetraethylethylenediamine, and PPh3 under various conditions.

We show that under aerobic conditions at low temperature (∼80 °C), complex 1 reacts with PPh3 producing O=PPh3 and a diamagnetic species that has been assigned to an oxo-bridged dicopper(II) compound on the basis of EPR, and Cu K-edge spectroscopic data. As a result of interaction between 1 and PPh3 the intensity of 1s → 3d transition centered at ~8981.1 eV has decreased and preedge feature at 8978.4, corresponding to Cu(II), has appeared. In addition, the low fi eld shift (0.4 eV) for transitions corresponding to the 1s → 4p + LMCT shakedown and 1s → 4p transitions, was also observed. Appearance in the 1H NMR spectra of new resonance signals, with chemical shifts in the normal region (0-10 ppm), support the formation of the µ-oxo- dicopper(II) diamagnetic complex. When the reaction was performed in the absence of excess dioxygen, negligible substrate

(PPh3) oxidation was observed. Instead, a highly symmetrical copper(II) cluster intermediate with characteristic isotropic EPR signal at g = 2.11 was formed. At temperatures above ∼40 °C, oxidative N-dealkylation chemistry occurs. These results will be discussed in terms of parallel reaction channels that are activated under various conditions of temperature and dioxygen. 1 Mahadevan, V.; Henson, M. J.; Solomon, E. I.; Stack, T. D. P. J. Amer. Chem. Soc. 2000, 122, 10249-10250.

Structural and Physical Studies on Thiocyanate complexes of Iron(III) Porphyrins.

Nicole Pearson, University of Natal, South Africa Orde Q Munro, University of Natal, South Africa Giovanni R Hearne, University of the Witwatersrand, South Africa

Novel thiocyanato and bis(thiocyanato) complexes of [FeIIITPP)] have been investigated to delineate the factors which control the coordination geometry (linear vs. bent, N-bound vs. S-bound) and electronic properties of metalloporphyrins.

[Fe(TPP)(NCS)] and [K(18-Crown-6)][Fe(TPP)(NCS2]•2[K(18-Crown-6)][ClO4]•2CH2Cl2 have been synthesized and characterized by X-ray crystallography, IR, NMR, and electronic spectroscopy. The mean Fe-Np distances are 2.066(2) Å and 2.060(13) Å for the fi ve- and six-coordinate complexes, respectively. The Fe-Nax bond length is 1.936(3) Å for the fi ve- coordinate complex and averages to 2.105(20) Å for the six-coordinate complex, consistent with a high-spin d5 electronic confi guration. Various hemeproteins have been reported to possess an electronic structure for Fe(III) in which two electronic states of different spin multiplicity are in a spin equilibrium with one another. This frequently observed low-spin to high-spin transition is linked to the electron transfer/storage functions of these proteins. [57Fe(TPP)(NCS)] has been synthesized for Mössbauer studies on the pressure and temperature dependence of the Fe(III) spin state since the porphyrin/NCS- ligand 5 1 combination places the ground state close to the S= /2 → S = /2 crossover. Our results show that a change in temperature does not cause a change in the spin state of the FeIII ion, but a pressure greater than 6 GPa (achieved in a diamond anvil cell) is suffi cient to cause a change in spin state from high-spin to low-spin in this complex. This is the fi rst ever reported pressure induced spin-state change in an iron porphyrin. Density functional theory calculations (B3LYP method, LACVP** basis set), in conjunction with the crystallographic data, suggest that the observed thiocyanate coordination geometries are dependent on both the electronic confi guration of the metal ion and crystal packing interactions. 212 Journal of Inorganic Biochemistry 96 (2003)

IV V V O and V O2 complexes of schiff base derivatives of ethylenediamine and vitamine B6 João Costa Pessoa, Instituto Superior Técnico, Centro Química Estrutural, Portugal Margarida Castro, Departamento de Bioquímica, University de Coimbra, Portugal Isabel Correia, Instituto Superior Técnico, Centro Química Estrutural, Portugal Carlos Geraldes, Departamento de Bioquímica, University de Coimbra, Portugal Tamás Jakusch, Biocoordination Research Group of Hungarian Academy of Science, Hungary Tamás Kiss, Biocoordination Research Group of Hungarian Academy of Science, Hungary

Investigation of the insulin-mimetic effect of vanadium has focused on neutral mostly bis complexes of bidentate ligands. We started structural, speciation and insulin mimetic studies on complexes of pyridoxine and tetradentate ligands, such as the

Schiff base derivatives of pyridoxal and ethylenediamine pyr2en, and its reduced form 1 51 Rpyr2en by pH-metry, EPR or H and V NMR spectroscopic methods. IV 2+ The reduced derivative Rpyr2en forms much more stable complexes with V O + and VO2 than the Schiff base. In the complexes formed the imine- or the amine-N and the phenolate-O atoms coordinate to the metal ions in a tetradentate way. The pyridine N atoms do not take part in the coordination, but their deprotonation/ protonation occurs in the physiological pH range. IV 2+ + 1 51 In the V O and VO2 -Rpyr2en systems geometric isomers coexist. The H and V NMR measurements indicate two V types of V O2 isomers (I and II) The isomer I proved to be more stable than II. The structures of isomers I, corresponding to V V V O2LH and V O2L were determined by X-ray-diffraction. The results were also corroborated by DFT studies. The complex IV IV IV V O(pyridoximinato)2, V O(pyr2en) and V O(Rpyr2en) were also prepared. Ex vivo toxicity and insulin mimetic studies are also reported. Acknowledgements. The authors thank the Hungarian Academy of Sciences, the NRF (OTKA T31896/2000), the FEDER, the Portuguese FCT and the POCTI Programme (project POCTI/35368/QUI/2000).

Taking Snapshots of Cobalt(III) Cytochrome c Folding: Real-Time NMR Studies

Ekaterina V Pletneva, California Institute of Technology, United States D Bruce Fulton, Iowa State University, United States Jay R Winkler, California Institute of Technology, United States Harry B Gray, California Institute of Technology, United States

Folding of cytochrome c (cyt c) is strongly infl uenced by ligand-substitution reactions at the heme. In folded cyt c at pH 7, His18 and Met80 axially ligate to the heme iron. In GuHCl-unfolded protein, Met80 is replaced by a nitrogenous base of an amino-acid sidechain (pH 7: His26, His33, or His39), while His18 remains bound to the heme. This misligation retards folding because ligand exchange is required for the peptide to adopt its native conformation. We take advantage of the ligand- exchange processes and replace the native iron center with substitution-inert cobalt(III). This modifi cation dramatically slows down folding of cyt c: while folding of Fe(II) cyt is complete in a few hundred milliseconds, folding of Co(III) cyt requires hours. The early steps in cyt c folding have been examined by a variety of spectroscopic techniques, including real- time NMR measurements. Heteronuclear NMR spectroscopy offers a powerful means to monitor folding reaction at the level of individual residues. The 15N-labeled S. cerevisiae Fe(III) cyt was expressed in E. coli, the Fe(III) ion was removed and replaced with Co(III). The amide 1H and 15N chemical shifts of Co(III) cyt are similar to that of Fe(II) cyt, indicating that Co-substitution does not disrupt the native protein fold. Refolding of Co(III) cyt was initiated by manual dilution of concentrated denaturant solution with buffer. For the unfolded protein, amide proton chemical shifts are clustered between 8 and 8.5 ppm. As the protein refolds, chemical shift dispersion becomes apparent. To elucidate the residue-specifi c events in cyt c folding, we performed a series of 2D heteronuclear NMR real-time folding studies. HSQC and 2D HSQC-NOESY spectra were acquired at various time points during refolding. Our studies visualize sequential refolding steps and identify the residues that participate in these steps. Journal of Inorganic Biochemistry 96 (2003) 213

Interactions of Proximate Amino Acid Residues in Polyaza Macrocyclic Systems

Sally E Plush, University of Adelaide, Australia Stephen F Lincoln, University of Adelaide, Australia Kevin P Wainwright, Flinders University, Australia

Interest in the design and development of water soluble polyaza macrocycles with possible applications as enzyme mimics and MRI agents has attracted much attention over recent years. The objective of our studies is to develop a series of water soluble polyaza macrocyclic ligands bearing donor pendant arms derived from amino acids.

The ligands are prepared by substituting [9]aneN3 and [12]aneN4 macrocycles with N-bromoacetyl-amino acid monomer units.1 The amino acids used to date have been L-phenylalanine and L-tryptophane derivatives, these were initially synthesised as methyl esters and later hydrolysed to the acid form. The free acid form of the ligands prepared was found to be water soluble at neutral pH, thus achieving the initial aim of the project. Potentiometric titrations on the L-phenylalanine derived ligands show strong coordination with Cu2+, Zn2+ and Cd2+, exhibiting preferential binding towards Cu2+. Analysis of the stereochemical nature of the ligands by NMR spectroscopy revealed that inversion around the nitrogen centre of the macrocyclic ring occurs to produce two diastereomers. Binding of Zn2+ or Cd2+ the rate of inversion was slowed such that the individual protons on the arms could be distinguished. Thus, incorporation of chiral amino acids as pendant arms induces homochirality in both the substituted [9]aneN3 and [12]aneN4 systems. This is important for chiral recognition studies such as those involving molecular recognition of host guest complexes. The potential for these macrocyclic ligands has been demonstrated and thus warrants further interest into their development. (1) Watson, A. A.; Willis, A. C.; Fairlie, D. P. Inorg. Chem. 1997, 36, 752-753.

On the identity of a novel Fe(IV) intermediate in the catalytic cycle of Taurine Alpha-Ketoglutarate Dioxygenase

John C Price, Pennsylvania State University, United States Eric W Barr, Pennsylvania State University, United States Bhramara Tirupati, Pennsylvania State University, United States Joseph M Bollinger, Pennsylvania State University, United States Carsten Krebs, Pennsylvania State University, United States

Taurine Dioxygenase, (TauD) an alpha-ketoglutarate-dependent dioxygenase, liberates sulfi te from organosulfonates through the oxygen mediated hydroxylation of the alpha carbon. Recently we characterized a novel Fe(IV) intermediate within its catalytic cycle. Using stopped-fl ow absorption, and freeze quench Mössbauer and EPR spectroscopies with modifi ed substrates, we have further elucidated the identity of this intermediate and its position within the TauD catalytic cycle. 214 Journal of Inorganic Biochemistry 96 (2003)

CLaNP – An artifi cial paramagnetic centre to study proteins by NMR

Miguel Prudencio, Leiden Institute of Chemistry, Leiden University, Gorla, The Netherlands Jan Rohovec, Laboratory for Applied Organic Chemistry and Catalysis, The Netherlands Antonietta Impagliazzo, Leiden Institute of Chemistry, Leiden University, Gorla, The Netherlands Joop Peters, Laboratory for Applied Organic Chemistry and Catalysis, The Netherlands Marcellus Ubbink, Leiden Institute of Chemistry, Leiden University, Gorla, The Netherlands

Paramagnetic NMR has been used to obtain structural information about protein molecules (1) and to characterise protein- protein interactions of transient complexes (2). So far, these methods make use of the paramagnetic effects caused by a metal cofactor in a metalloprotein or those resulting from attaching a spin label to the protein. In this work, we have designed an artifi cial paramagnetic centre which can be specifi cally attached to the surface of any protein molecule. This molecule (CLaNP – Caged Lanthanide -NMR Probe) can generate various paramagnetic effects that can be measured by NMR, like pseudocontact shifts, paramagnetic alignment (RDCs without the need for external alignment media) and relaxation. These can be converted into structural information about proteins and protein containing complexes. The probe consists of a lanthanide ion in a cage complex that has two chemical groups for specifi c recognition of the protein. In the current version of the ClaNP, the molecule reacts with thiol groups of exposed Cys residues in a protein. We report the results obtained upon attaching the CLaNP molecule to the surface of a double-Cys mutant of A. faecalis S-6 Zn-pseudoazurin (see fi gure). Distance-dependent pseudocontact shifts were observed in residues all through the protein, up to 45 Å from the Ln ion. It is expected that even more distant effects will be observable in large proteins or complexes. Furthermore, The probe also causes alignment of the protein in strong magnetic fi elds. The results indicate that the CLaNP may be a useful tool to obtain long-range distance and structure information in NMR. (1) Allegrozzi et al., J. Am. Chem. Soc., 122, 4154-4161 (2000) (2) Ubbink et al., Structure, 6, 323-335 (1998) Journal of Inorganic Biochemistry 96 (2003) 215

Dioxygen activation by copper(I) complexes: Electronic effects

on the formation and substrate reactivity of the Cu2O2 core. Lanying D Quant, Johns Hopkins University, United States Jason Shearer, Johns Hopkins University, United States Mike Vance, Stanford University, United States Hong C Liang, Johns Hopkins University, United States Mark J Henson, Stanford University, United States Christiana X Zhang, Johns Hopkins University, United States Edward I Solomon, Stanford University, United States Kenneth D Karlin, Johns Hopkins University, United States

We have tuned the redox potential of a copper(I) complex in order to understand how electronic effects infl uence the II 2+ 2 2 III 2+ facile equilibrium between a [Cu2 (O2)] µ-η :η -(side-on)-peroxo, and a [Cu2 (O)2] bis(µ-oxo) species. The ligands, PYAN, NMe-PYAN, OMe-PYAN, and Cl-PYAN (whereby R-PYAN = N-[2-(4-R-pyridin-2-yl)-ethyl]-N, N’, N’-trimethyl- propane-1,3-diamine) and their corresponding copper(I) complexes have been synthesized, and upon reaction with O2 at low temperature, generate varying amounts of each, side-on peroxo and bis(µ-oxo) isomers. The Cu2O2 species were characterized by UV-Vis, and resonance Raman spectroscopies, and show a trend such that more electron donating ligands favor the formation of the bis(µ-oxo) dicopper(III) isomer over the side-on peroxo dicopper(II) species. Exogenous substrate II 2+ III 2+ reactivity of the [Cu2 (O2)] side-on peroxo and [Cu2 (O)2] bis(µ-oxo) compounds will also be presented.

Detailed mechanistic studies have been done on the oxidative N-dealkylation reaction of dimethylaniline by the Cu2O2 2+ complexes, [Cu2(R-MePY2)2O2] (R-MePY2 = bis-[2-(4-R-pyridin-2-yl)-ethyl]-methylamine), yielding methylaniline and formaldehyde. A variety of mechanistic experiments, including linear free energy correlations, kinetic isotope effects, and inter vs. intramolecular isotope effects signify that the reaction proceeds through an electron transfer mechanism for

R = H, but the reaction can also proceed through a hydrogen atom transfer mechanism for the case of R = OMe, and NMe2, depending on the aniline derivative.

Probing the DNA Selectivity of Ruthenium Metallointercalators using ESI Mass Spectrometry

Stephen F Ralph, University of Wollongong, Australia Jenny L Beck, University of Wollongong, Australia Raj Gupta, University of Wollongong, Australia Thitima Urathamakul, University of Wollongong, Australia Margaret M Sheil, University of Wollongong, Australia Janice R Aldrich-Wright, University of Western Sydney, Australia

Metal compounds that bind non-covalently to DNA have attracted considerable interest. This has been driven partly by a desire to understand these interactions, and partly by studies that have shown these compounds may be useful as nucleic acid probes, synthetic restriction enzymes or DNA repair agents. In this paper we will show how Electrospray Ionisation Mass Spectrometry (ESI-MS) can provide a wealth of information concerning non-covalent complexes formed between double- stranded DNA and ruthenium metallointercalators [1]. For example, the ESI mass spectrum below shows the presence of 3 different non-covalent complexes and a small amount of unreacted DNA in a reaction mixture containing a 1.5:1 ratio of metallointercalator and DNA duplex. By comparing the relative abundances of non-covalent complexes formed in reaction mixtures involving the same ruthenium compound, but different oligonucleotides, information on DNA specifi city was also obtained. Spectra of reaction mixtures containing two different ruthenium compounds and duplex DNA were used to determine the relative binding affi nities of the metal compounds. Competition experiments were also performed using ruthenium compounds and either the intercalator daunomycin, or minor groove binder distamycin, to provide further information on DNA binding modes. [1] J.L. Beck, R. Gupta, T. Urathamakul, N.L. Williamson, M.M. Sheil, J.R. Aldrich-Wright and S.F. Ralph, Chemical Communications, in press (2003). 216 Journal of Inorganic Biochemistry 96 (2003)

The interaction of vanadyl ions with cysteine and cystine containing peptides investigated by model studies

Dieter Rehder, Institute of Inorganic and Applied Chemistry, University of Hamburg, Germany Dongren Wang, Institute of Inorganic and Applied Chemistry, University of Hamburg, Germany Axel Behrens, Institute of Inorganic and Applied Chemistry, University of Hamburg, Germany

Inorganic and organic (coordination) compounds of vanadium have been shown to exert insulin-mimetic activity [1]. A prominent intracellular compound potentially involved in the speciation of an antidiabetic vanadium drug is glutahione in its reduced (GSH, containing cysteine) and oxidised (GSSG, containing cystine) form. Speciation may occur via coordination/ligand exchange and/or redox interaction with complexes containing the vanadyl (VO2+) ion. In order to obtain insight into the role of GSH and GSSG, we have carried out model studies with disulfi des and thiolates. Selected examples are presented in eqns. (1) to (3). They demonstrate that vanadyl ions can reductively cleave the disulfi de bond [eqn. (1)], stabilise the disulfi de by coordination [eqn. (2)], or generate disulfi des from thiolates [eqn. (3)]. In the latter case, VO2+ acts as a catalyst in a three step oxidation of thiolate by oxygen [2]. Vanadyl complexes can thus be expected to have a regulatory function for the GSH/GSSG equilibrium. The relevance of these studies for an assessment of the inhibitory role of vanadate towards phosphatases with cysteine in the active centre is also addressed. 1 D. Rehder, J. Costa Pessoa, C.F.G.C. Geraldes, M.M.C.A. Castro, T. Kabanos, T. Kiss, B. Meier, G. Micera, L. Pettersson, M. Ranger, A. Salifoglou, I. Turel, D. Wang, J. Biol. Inorg. Chem. 7 (2002) 384. 2 D. Wang, A. Behrens, M. Farahbakhsh, J. Gätjens, D. Rehder, Chem. Eur. J., in the press. Journal of Inorganic Biochemistry 96 (2003) 217

Platinum(II) Complexes Containing 1,2- and 1,7-Dicarba-closo-dodecaborane(12): Potential New Agents for Boron Neutron Capture Therapy

Louis M Rendina, The University of Adelaide, Australia Susan L Woodhouse, The University of Adelaide, Australia Jean A Todd, The University of Adelaide, Australia

Boron Neutron Capture Therapy (BNCT) is a bimodal cancer treatment that is currently undergoing Phase I clinical trials in several countries. The key aspect of the therapy is the interaction of slow (thermal) neutrons with 10B-containing drugs that are localised within malignant cells. The resulting nuclear reactions ultimately lead to cell destruction owing to the production of high linear energy transfer (LET) particles that are accompanied by approximately 2.4 MeV of kinetic energy. The effectiveness of the neutron capture reaction is dramatically enhanced if the 10B-containing compounds are placed in close proximity to chromosomal DNA, and the search for new DNA-binding agents for BNCT remains a major research objective. The ability of DNA to act as a template for the organised binding of cisplatin, metallo-intercalators and other types of transition metal complexes allows one to examine whether platinum compounds that are tethered to polyhedral boranes such as the dicarba-closo-dodecaboranes(12) (carboranes) have the capacity to target the macromolecule for potential use in BNCT. A number of key results have been obtained in our laboratory regarding the preparation, DNA-binding, and anti-cancer properties of both mono- and di-nuclear platinum(II) complexes that are tethered to a 1,2- and 1,7-dicarba-closo-dodecaborane(12) moiety, respectively. For example, we have demonstrated that complexes 1, 2 and 3 exhibit avid DNA-binding and anti-cancer characteristics in vitro. This is the fi rst time that transition metal complexes containing boron-containing ligands have been shown to target DNA. The complexes also possess anti-cancer activity against a variety of tumour cell lines including cisplatin-resistant cells. The key results of this work will be presented.

Crystal structures of the nitrosyl and nitrosoethane adducts of ferrous horse heart myoglobin

George B Richter-Addo, University of Oklahoma, United States Daniel M Copeland, University of Oklahoma, United States Ann H West, University of Oklahoma, United States

Nitric oxide and organic nitroso compounds react with many heme proteins to give heme-NO and heme-RNO adducts, respectively. We have determined the crystal structure of horse heart MbNO at a resolution of 1.9 Å. The Fe-N-O angle of 147° in hh MbNO is similar to the 150° angle determined from a MS XAFS study of a frozen solution of hh MbNO (Rich et al. J. Am. Chem. Soc. 1998; 120: 10827-10836), but the Fe-N(O) bond length of 2.03 Å is longer than the 1.75 Å distance determined from the XAFS study, and suggests distal pocket infl uences on FeNO geometry in the crystal. The nitrosyl N atom is located 2.98 Å from the imidazole N-atom of the distal His64 residue suggesting electrostatic stabilization. The crystal structure of ferrous hh Mb(EtNO) was determined at a resolution of 1.7 Å, and reveals N-binding of the nitrosoethane ligand to the iron center. To the best of our knowledge, the crystal structure of hh Mb(EtNO) is the fi rst such determination of a nitrosoalkane adduct of a heme protein. Comparisons of the crystal structure of hh MbNO with the crystal structures of hh Mb(EtNO) and sperm whale MbNO are presented. 218 Journal of Inorganic Biochemistry 96 (2003)

New Generation MCD Spectrometers

Mark J Riley, University of Queensland, Australia Elmars R Krausz, Australian National University, Australia Alex Stanco, Lastek Pty Ltd, Australia

We are in the process of constructing of two specialised, state-of-the art MCD (Magnetic Circular Dichroism) spectrometers to be used in projects involving the structure – spectral correlations in the active sites of metalloenzymes and related compounds of biological interest. Magnetic Circular Dichroism (MCD) is the difference between the absorption of left and right circularly polarised light, in the presence of a magnetic fi eld. It has proved to be extremely powerful in the analysis of the electronic structure of metal- centered active sites in biological systems, especially when used in conjunction with other spectroscopic techniques. MCD has the following advantages: a) It is a very sensitive technique, especially in the near-IR region where it is often diffi cult to observe the absorption bands of metalloenzymes. b) It is selective; the spectrum of a metal centre imbedded in a protein matrix can be obtained. c) It is multidimensional; the MCD spectrum contains information when measured against temperature, magnetic fi eld strength as well as wavelength. d) It can investigate non-Kramers doublet systems that are frequently diffi cult to study with EPR. e) Because an MCD spectrum is a signed quantity, it intrinsically contains more information and has a higher resolution than an absorption spectrum. f) MCD simultaneously provides information about both ground and excited states and thus provides a complimentary link between EPR, and optical spectroscopies. These instruments will draw on recent advances in detectors, modulators and electronics. They will far exceed, by several orders of magnitude, the specifi cations of current commercial instruments; which are often compromised by being based on CD spectrometers. The instruments feature innovative magnetic shielding, large area avalanche diode detectors and will enabling one to carry out high-resolution, high through-put spectroscopy with fast fi eld sweeps. Aspects of instruments are being commercialized by Lastek (Aust) Pty Ltd. The construction of these instruments is concurrent with software development of a suite of open source programs to be used in the fi tting, simulation and interpretation of the MCD spectra. Journal of Inorganic Biochemistry 96 (2003) 219

Dioxygen activation by low valent nickel complexes: characterization of new Ni-dioxygen intermediates

Charles G Riordan, University of Delaware, United States Koyu Fujita, University of Delaware, United States Matthew Kieber-Emmons, University of Delaware, United States Beaven S Mandimutsira, University of Delaware, United States

A series of Ni(I) complexes supported by the [S3] borato ligands, [PhB(CH2SR)3] (abbreviated PhTtR) have been prepared and their reactivity towards dioxygen assayed, Scheme. Ligand design permits for the interception of distinct, thermally- sensitive intermediates. For example, [PhTttBu]Ni(CO) is oxygenated to the purple, bis(mu-oxo) dimer, {[PhTttBu]Ni}2(µ-

O)2. In contrast, the larger, adamantyl-supported complex, [PhTtAd]Ni(CO) reacts with dioxygen to yield the brown, monomeric species, [PhTtAd]Ni(O2). This complex is a useful precursor for the construction of mixed metal species. Each intermediate is characterized uniquely by its combined UV-visible, NMR, resonance Raman and Ni EXAFS spectroscopic features. Density functional theory analysis and reactivity studies of the novel intermediates will be presented.

Inhibitory Interactions between Tripodal Chelating Ligands and Matrix Metalloproteinases

Kenton R Rodgers, North Dakota State University, Department of Chemistry, United States Hongshan He, North Dakota State University, Department of Chemistry, United States Douglas P Linder, North Dakota State University, Department of Chemistry, United States Indrani Chakraborty, North Dakota State University, Department of Chemistry, United States

Matrix metalloproteinases (MMPs) are Zn(II)-containing endopeptidases that play crucial roles in mammalian tissue homeostasis. Dysfunction in MMPs is correlated with a wide variety of disease states including numerous cancers, arthritis, periodontal disease, and opening of the blood-brain barrier. These enzymes have as their substrates various forms of collagen. Their specifi cities for particular types of collagen are relatively low, which makes targeting of a specifi c MMP diffi cult. We have undertaken the synthesis of tripodal chelating inhibitors based on the tren scaffold. The inhibitors are conveniently modifi ed, either symmetrically or asymmetrically, to facilitate targeting of specifi c MMPs. Interaction of these compounds with MMP active sites are being investigated via inhibition studies, characterization of model complexes, and molecular modeling. Results on all three aspects of this study will be presented. 220 Journal of Inorganic Biochemistry 96 (2003)

Comparison of Fe-X-O Bonding in Isoelectronic [FeII(por)CO] and [FeIII(por)NO]+ Complexes

Kenton R Rodgers, North Dakota State University, Department of Chemistry, United States Douglas P Linder, North Dakota State University, Department of Chemistry, United States Graeme R A Wyllie, University of Notre Dame, Department of Chemistry and Biochemistry, United States Mary K Ellison, University of Notre Dame, Department of Chemistry and Biochemistry, United States W Robert Scheidt, University of Notre Dame, Department of Chemistry and Biochemistry, United States Jennifer Banister, North Dakota State University, Department of Chemistry, United States

Diatomic ligand complexes of heme play a wide variety of roles in biology, including the sensing and transport of O2, NO, and CO. The importance of these complexes has focused a great deal of effort on understanding the bonding between the XO ligands and heme iron. Ferric heme-NO complexes are important in heme protein-based NO transport and denitrifi cation.

Recently, it has been recognized that a direct correlation exists between frequencies of the νFe-N and νN-O vibrations in ferric heme-NO complexes, [Fe(por)NO]+. Hence, factors strengthening the FeIII-N bond also strengthen the N-O bond. This is in contrast to the inverse correlations between νFe-X and νX-O frequencies in ferrous heme-CO and heme-NO complexes, which are typically discussed in terms of the widely-accepted π-backbonding model. The direct correlation in ferric heme-NO complexes is not explained straightforwardly in the same terms. This study strives to understand the differences in bonding between these isoelectronic diatomic ligand complexes. Bonding in these complexes will be discussed in the context of results from IR and resonance Raman spectroscopy, x-ray crystallography, and DFT calculations on model ferric heme-NO complexes. Journal of Inorganic Biochemistry 96 (2003) 221

Characterization of a New 8Fe Protein Isolated from Desulfovibrio desulfuricans ATCC 27774

Pedro M Rodrigues, FCMA, CCMAR., Universidade do Algarve, Campus de Gambelas, Portugal Anjos L Macedo, Departamento de Química, Centro de Química Fina e Biotecnologica, Portugal Isabel Moura, Departamento de Química, Centro de Química Fina e Biotecnologica, Portugal José G Moura, Departamento de Química, Centro de Química Fina e Biotecnologica, Portugal

Ferredoxins are small and acidic iron-sulphur proteins that can be used as model compounds in the investigation of electronic, paramagnetic and structural properties of the iron-sulphur clusters in complex enzymes. They are classifi ed according to the type of aggregate [Fe-S];[2Fe-2S] centers originating from ferredoxins of plants and animals, and [3Fe-4S]/[4Fe-4S] typically deriving from bacteria. From sulphate reducers (SR) four distinct types of ferredoxins are found containing [3Fe-4S],[4Fe- 4S],[3Fe-4S] and [4Fe-4S] (7Fe), and 2x[4Fe-4S] (8Fe). The cluster iron atoms are tetraedically coordinate by sulphur atoms. Its binding to the polypeptidic chain is made by a cysteic sulphur atom or sometimes by an oxygen or nitrogen atom. A novel iron-sulfur protein was purifi ed from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judge by polyacrylamide gel electrophoresis. The purifi ed protein is a 13.4kDa dimmer with a polypeptide chain containing 60 amino acids residues, with 8 cysteines that coordinate the two [4Fe-4S] clusters. 1D and 2D 1H NMR studies are reported on the oxidized 2x[4Fe-4S] clusters of the Desulfovibrio desulfuricans ferredoxin ATCC 27774 (Dd27Fd). On the basis of the nuclear Overhauser effect, relaxation measurements and comparison of the present data with the available spectra of the analogous 8Fe ferredoxins namely from Desulfovibrio desulfuricans Norway, Clostridium pasteurianum, Clostridium acidi urici and Peptococcus aerogenes, the clusters ligands were specifi cally assigned to the designated cysteinyl residues. The generated curve using the equation δ=asin2(θ)+bCos(θ)+c originates an a>0 value suggests an essentially p spin delocalization mechanism largely dependent on unpaired spin density on the pz orbital of the sulfur atom.

The factors determining the stability of a minimal cytochrome c

Antonio Rosato, Magnetic Resonance Center, Italy Ilaria Bartalesi, Magnetic Resonance Center, Italy Ivano Bertini, Magnetic Resonance Center, Italy Kaushik Ghosh, Magnetic Resonance Center, Italy Murugendra Vanarotti, Magnetic Resonance Center, Italy Paul R, Vasos, Magnetic Resonance Center, Italy Wei Zhang, Roskilde University, Denmark

Bacillus pasteurii is a Gram+ bacterium, growing optimally at pH 9.2 in the presence of ammonium salts or urea. It expresses a membrane-anchored mono-heme cytochrome c, whose soluble domain (called Bpcytc hereafter) is only 71 amino acid long, making it one of the smallest protein in its family. Oxidized Bpcytc is quite stable towards denaturation, and does not give rise to iron misligation at alkaline pH, nor at high temperature, as it is instead the case for mitochondrial c-type cytochromes. In order to identify the determinants for the different stability of the fold as well as of axial ligation of the iron ion, we have performed an extended characterization of Bpcytc through electronic and NMR spectroscopy, and site-directed mutagenesis. We characterized the structure and backbone dynamics of Bpcytc in both physiologically relevant oxidation states, showing that they vary little in the oxidized vs. reduced state. In addition, a redox-state independent internal water molecule was identifi ed, located at ca. 6 Å from the heme iron ion. The behavior of oxidized Bpcytc in guanidinium highlighted that protein unfolding and variations in the iron axial coordination take place simultaneously. Finally, the NMR determination of backbone amide exchange rates permitted the identifi cation of units opening cooperatively under native conditions, which may be related to folding units. The above results together with the data available in the literature for mitochondrial c-type cytochromes are consistent with the existence of units within the cytochrome c fold, loosely correlated with secondary structure elements, likely driving the folding processes. Such units can constitute a ‘fi ngerprint’ for the cytochrome c fold, which is essentially independent of the primary structure. The stability of the fold and/or of the iron axial ligation are then fi ne tuned by Nature through specifi c interactions, such as hydrogen bonding to internal water molecules or specifi c side chain contacts. 222 Journal of Inorganic Biochemistry 96 (2003)

A DFT investigation of some bioinorganic systems

Nino Russo, Dipartimento di Chimica and Centro di Calcolo ad Alte P, Universita della Calabria, Italy Tiziana Marino, Dipartimento di Chimica and Centro di Calcolo ad Alte P, Universita della Calabria, Italy Marirosa Toscano, Dipartimento di Chimica and Centro di Calcolo ad Alte P, Universita della Calabria, Italy Andrë Grand, CEA Grenoble, France Monica Leopoldini, Dipartimento di Chimica and Centro di Calcolo ad Alte P, Universita della Calabria, Italy

Bioinorganic chemistry is an expanding fi eld of modern chemistry and its progress in the last two decades has interested both experimentalist and theoreticians. Although this grown, many elementary mechanism are not completely known and sometime unknown. Often, the lack of information is due to the diffi culties in their experimental investigation that however, in some cases, can be bypassed employing the wide arsenal of classical and quantum computational tools. We present here the results of our recent studies of a series of biological phenomena in which the metal ligand interactions play a crucial role. In particular we have examined: • The interaction between a series of metal ions with glycine, alanine, cysteine and proline amino acids and with uracil, thymine, cytosine, adenine and guanine nucleic acid bases and nucleotides;

• The Potential energy profi les for CO2 reduction by carbonic anhydrase and for the reduction of nitrate to nitrite by nitrate reductase; • The mechanism for which the phytochelatine are able to complex a series of metal cations All the investigations were performed at high level of theory using the density functional method in its B3LYP formulation and extended basis sets. Work performed in the framework of Memobiomar-MIUR project References: T. Marino, N. Russo and M. Toscano, J. Inorg. Biochem., 79 (2000) 179. N. Russo, M. Toscano, and A. Grand , J. Phys. Chem. B 105 (2001), 4735-4741. N. Russo, M. Toscano and A. Grand, J. Am. Chem. Soc. 123 (2001) 10272-10279 T. Marino, N. Russo and M. Toscano, Inorg. Chem. 40 (2001) 6439-6443 T. Marino, N. Russo and M. Toscano, J. Mass Spectrom 37 (2002) 786-791 N. Russo, E. Sicilia, M. Toscano and A. Grand, Int. J. Quantum Chem. 90 (2002)903 Journal of Inorganic Biochemistry 96 (2003) 223

Far Infrared and Resonance Raman Spectroelectrochemistry of Mo/Fe/S Clusters

Michael D Ryan, Marquette University, United States Lin Li, Marquette University, United States Neal Matyniak, Marquette University, United States

The biochemistry of nitrogenase has been the subject of intense interest by scientists for many decades. Unfortunately, the structure has not brought us closer to understanding how the enzyme works. Since the elucidation of the FeMoco cluster, great efforts have been made to synthesize Mo/Fe/S clusters of high nuclearity, most of which have, at least, 1-2 (quasi)- reversible reduction. The core vibrations of the Mo/Fe/S clusters occur in the far infrared region. In order to investigate the changes that occur when the clusters are reduced and/or oxidized, the far infrared spectroelectrochemistry of linear and cubanes Mo/Fe/S clusters was initiated. Two such clusters are shown in Figure 1. The fi rst cluster was chosen because of the wealth of spectroscopic, voltammetric and visible spectroelectrochemical data that are available, and the second one was representative of the Mo/Fe/S cubane clusters. The far infrared spectroelectrochemistry was carried out in a thin layer cell with CsI windows, which were transparent down to 200 cm-1. Lower energies could be observed using Si-windows, but solvent absorption generally limits the effectiveness of this material. The far infrared spectroelectrochemistry of the linear 2- cluster, [MFe2S4Cl4] (M = Mo, W), was investigated in dichloromethane. The spectrum of the reduced cluster was obtained, and the identity of the vibration bands were confi rmed with 34S substitution for natural abundance S. The differences between the molybdenum and tungsten complexes will be presented, along with resonance Raman data. The far infrared spectrum of the double cubane, 3- [Mo2Fe6S8(PhS)9] , in dichloromethane, is shown in Figure 2 (solvent subtracted). The spectrum was identical to that obtained in a CsI pellet. Reduction of the complex by two electrons yielded a difference spectrum shown in Figure 2. The changes that were observed as a function of potential were used to interpret the effect of reduction of one or both of the cubanes. Additional studies on the far IR spectroelectrochemistry of catechol ligated cubanes will be reported. These results will demonstrate that far infrared spectroelectrochemistry can provide important structural information on Mo/Fe/S clusters. 224 Journal of Inorganic Biochemistry 96 (2003)

Construction of a new expression system of bilirubin oxidase and its mutants to explore the dioxygen reduction mechanism

Takeshi Sakurai, Kanazawa University, Japan Kazuhiro Tanaka, Kanazawa University, Japan Rieko Kitagawa, Kanazawa University, Japan Kunishige Kataoka, Kanazawa University, Japan

Bilirubin oxidase (BO) is a multicopper oxidase used for the clinical test of liver. We have studied the properties and reactions of Myrothecium verrucaria BO and its mutants expressed in Aspergillus oryzae[1-4]. Annoyances of the heterologous expression of BO in A. oryzae could be evaded by constructing the new expression system to use Pichia pastoris GS115 as host. The recombinant BO showed a high enzyme activity similar to that of the authentic BO. The type II Cu EPR signal -3 -1 of the recombinant BO (gII = 2.34, AII = 8.4 x 10 cm ) was different from that of the authentic BO (gII = 2.24, AII = 18.6 x 10-3 cm-1), possibly due to be in the different resting forms. However, after a turnover of the reduction and reoxidation with dioxygen, the recombinant enzyme changed to be in the resting form of that of the authentic BO. By using this multicopying system, Cys457Ser mutant was prepared. Type I Cu site was vacant in this mutant, but the trinuclear center constructed by type II and type III Cuís was fully occupied. The reoxidation process of this mutant is under investigation expecting to isolate a reaction intermediate which cannot be detected in the normal reaction pathway of the authentic (wild type) enzyme (The corresponding intermediate has been isolated from the reaction of T1Cu(II)T2Cu(I)T3Cu(I) laccase with dioxygen ([5]). We are also preparing other mutants, expecting to isolate other intermediate species of dioxygen reduction [6,7]. [1] Biochemistry, 38 (1999) 3034. [2] J. Biochem., 125(1999) 662. [3] Biosci. Biotechnol. Biochem., in press. [4] J. Biochem, in press. [5] J. Biochem. 129 (2001) 949. [6] J. Biol. Chem., 274 (1999) 32718. [7] Inorg. React. Mech., 2 (2000) 79.

Pathways for Decay of the Tryptophan Cation Radical Intermediate in Oxygen Activation by Protein R2 of Escherichia coli Ribonucleotide Reductase

Lana Saleh, The Pennsylvania State University, United States J Martin Bollinger, The Pennsylvania State University, United States

The four-electron reduction of O2 to water at the carboxylate- bridged diiron(II) site of the R2 subunit of ribonucleotide reductase from E. coli is balanced by oxidation of two Fe(II) ions to Fe(III), one-electron oxidation of tyrosine (Y) 122 to the catalytically essential tyrosyl radical, and transfer of an ‘extra’ electron from an exogenous source. In the transfer of the extra electron, a near surface tryptophan residue (W48) is transiently oxidized to W48 cation radical (W48+•) by a kinetically masked two-electron-oxidized adduct between the diiron cluster and oxygen. The W48+• may then be reduced by a facile one-electron reductant (e.g. ascorbate). Under these conditions, the last step in the reaction is the slow oxidation of Y122 by the Fe(IV)Fe(III) cluster X to produce the Y122• of the native protein. The mechanism of decay of the transient W48+• in the absence of a reductant has been examined by stopped-fl ow absorption experiments performed on R2-WT, R2-Y122F, R2-Y356F, and R2-Y122F/Y356F proteins under conditions of varying ionic strength. The results suggest that W48+• decays • -1 via three simultaneous pathways. The fi rst pathway involves the formation of Y122 with kobs = 7 s . The second pathway -1 +• produces an unidentifi ed product with kobs = 1 s . The rate constants of W48 decay by these two pathways are unaffected by ionic strength. The third pathway results in the formation of Y356• with a rate constant that can be expressed by the following -1 equation: kobs = 29 s x ([NaCl]/(0.24 M + [NaCl])). One possible explanation for these observations is that Y356, which is located at the surface of the R2 subunit on the fl exible C-terminus, becomes more ordered and at closer proximity with W48 at higher ionic strengths, allowing for facile electron transfer between it and the W48+•. The salt-dependent ordering of Y356 relative to W48 may have implications for the gating of electron transfer between the R1 and R2 subunits of the enzyme during catalysis. Journal of Inorganic Biochemistry 96 (2003) 225

Functional models of the active sites of nitrosyl [Fe-S] non-heme proteins

Nataliya Sanina, Institute of Problems of Chemical Physics RAS, Russia Sergey Aldoshin, Institute of Problems of Chemical Physics RAS, Russia

The understanding of the chemistry, structure and spectroscopy of the active sites in nitrosyl iron-sulfur non-heme proteins is substantial for studying the reaction mechanisms of endogenous nitrogen monoxide and its intermediates 1,2. It is necessary, for example, for application of new effective adjuvants in chemo- and radiotherapy, and for new cardiovascular drugs in practical medicine. NO interaction with endogenous iron and low-weight molecular thiols in vivo results in the formation of - iron-sulfur nitrosyl complexes of two forms: mononuclear [Fe(NO)2(SR)2] and binuclear [Fe2(SR)2(NO)4]. The mononuclear dinitrosyl iron complexes (DNIC) with sulfur ligands are identifi ed in solutions by a characteristic EPR signal g=2.03. The molecular and crystal structure of the binuclear nitrosyl iron-sulfur complexes is being discussed now. Unfortunately, because of high instability of the complexes in vivo and in vitro, their isolation and crystallization is diffi cult. The overview of the results obtained from X-ray analysis, IR-, Mossbauer and EPR spectroscopy of models of nitrosyl [Fe-2S] and [2Fe-2S] active sites of ‘g~2.03 family’ 3-6 containing two functional groups (NO group as a radio-, chemosensitizing agent or vasodilating agent and RS group as an antibacterial agent, inhibitor of enzymes, antimetabolites) will be presented. 1 A. R.Butler, I. L.Megson , Chem. Rev., 2002,102, 1155. 2 Fontecave, M.; Bonomo, et al., Inorg. Chim. Acta. 2001, 318, 1 3 N.A. Sanina, O.A. Rakova, S.M. Aldoshin et al. Russ. J. Coord.Chem., 2001, 27, 3, C.179-183 4 O.A. Rakova, N.A. Sanina, et al. Doklady Chemistry, 2002, 383/ 3, 75-77 5 O. A. Rakova, N. A. Sanina, S. M. Aldoshin et al., Inorg. Chem. Comm., 2003, 6, 145-148 6 N. A. Sanina, O. A. Rakova, S. M. Aldoshin et al., Mendeleev.Comm., 2003 – in press. The work has been supported by the Russian Foundation for Basic Research (project no.02-03-33344)

Characterization of Heme Environmental Structure of Cytoglobin, A Fourth Globin in Human

Hitomi Sawai, Department of Life Science, Graduate School of Science, Japan Narifumi Kawada, Department of Hepatology, Graduate School of Medicine, Japan Katsutoshi Yoshizato, Developmental Biological Science, Graduate School of Science, Japan Hiroshi Nakajima, School of Materials Science, Japan Advanced Institute of Science and Technology, Japan Shigetoshi Aono, Institute for Molecular Sciece, Okazaki National Research Institutes, Japan Yasuhisa Mizutani, Molecular Photoscience Research Center, Kobe University, Japan Yoshitsugu Shiro, RIKEN Harima Institute/SPring-8, Japan

Cytoglobin (Cgb; 21kDa) is a hemoprotein that is present in a variety of many organs [1, 2, 3]. The amino acid sequence of Cgb shares about 25~45% identity to some globins and key amino acid residues are highly conserved. Therefore, Cgb has been identifi ed as a fourth globin in human, subsequent to myoglobin (Mb), hemoglobin (Hb) and neuroglobin (Ngb), but its function is unknown. We carried out a site-directed mutagenesis, in which six His residues were replaced with Ala, and found that the imidazoles of His81 (E7) and His113 (F8) bind to the heme iron as axial ligands in the hexacoordinate and low spin state. This structural feature is not identical to Hb and Mb, but the same as Ngb as well as hexacoordinate Hbs in animals, protists, cyanobacteria and all plants. In the resonance Raman and IR spectra of the ferrous-CO Cgb, three Fe-C-O stretching modes are obtained, suggesting presence of three confi gurations in the CO coordination to the iron. These conformers would arise from differences in the degree of interaction of the iron-bound CO with its surroundings. In the oxy complex, the υFe-OO stretching mode of Cgb was similar to those of elephant Mb, suggesting that the Fe-O2 band is stabilized through hydrogen bond between the distal oxygen atom and its surrounding, possibly with the His81 imidazole and/or Arg84. Using the pico-second -1 time-resolved resonance Raman spectroscopy, we obtained the νFe-His stretching mode at 229cm . The iron-His113 bond is stronger than other globins. This may be the cause of stable oxy complex of Cgb (τ1/2ªseveral hours) against auto-oxidation.

In functional sapects, it is possible that Cgb acts as a useful O2 reservoir in tissues lacking of O2. [1] T. Burmester, B. Ebner, B. Weich, and T. Hankeln (2002) Mol. Biol. Evol. 19, 416-421 [2] N. Kawada, D. B. Kristensen, K. Asahina, K. Nakatani, Y. Minamiyama, S. Seki, and K. Yoshizato (2001) J. Biol. Chem. 276, 25318-25323 [3] H. Sawai, N. Kawada, K. Yoshizato, H. Nakajima, S. Aono, and Y. Shiro (2003) Biochemistry in press 226 Journal of Inorganic Biochemistry 96 (2003)

Crystal structure and mechanism of a purple acid phosphatase from sweet potato: an enzyme with a novel Fe-Mn binuclear metal centre

Gerhard Schenk, University of Queensland, Australia Lyle E Carrington, University of Queensland, Australia Mohsen Valizadeh, University of Queensland, Australia John de Jersey, University of Queensland, Australia Susan E Hamilton, University of Queensland, Australia Luke W Guddat, University of Queensland, Australia

The crystal structure of an isoform of sweet potato purple acid phosphatase (SP-PAP) containing a unique antiferromagnetically coupled FeIII-MnII centre has been determined in the presence of the competitive inhibitor phosphate, and as the free enzyme. The overall fold is similar to that reported for the FeIII-ZnII containing PAP from red kidney bean (RKB-PAP). Furthermore, the seven metal-coordinating amino acids are completely conserved amongst all known PAPs. However, the sweet potato enzyme is clearly distinguished from other PAPs by (i) a more complex pH dependence of its kinetic parameters, (ii) increased catalytic effi ciency for a range of activated and unactivated phosphate esters, (iii) the II absence of a Brönsted correlation (βlg ~ 0), (iv) its requirement for Mn , and (v) the presence of a µ-oxo-bridge instead of a µ-hydroxo-bridge. The identity of the reaction-initiating nucleophile for PAPs is still under debate. The strong nucleophilicity of the µ-oxo-bridge correlates with the increased catalytic rate of SP-PAP, and suggests a role for the solvent bridge as nucleophile. Consistently, no electron density corresponding to a solvent bridge is present in the phosphate-bound form of SP- PAP. In RKB-PAP two histidine residues are proposed to be involved in substrate binding and/or acid catalysis (His295 and 296). Only one of those residues (His296) is conserved in SP-PAP; the other is replaced by an alanine. In addition, a glutamate residue in SP-PAP (Glu367) is positioned appropriately to act as a general acid during catalysis. These observations are also consistent with SP-PAPís higher catalytic effi ciency. Thus, at pH 4.9 the reaction catalysed by SP-PAP follows a mechanism similar to that of other PAPs, with the exception of utilising a more potent nucleophile and a stronger acid to protonate the leaving group. Interestingly, and in contrast to other PAPs, the sweet potato enzyme reaches maximum reactivity at nearly neutral pH. We speculate that at higher pH this enzyme may have an alternative catalytic mechanism, which is similar to that observed in alkaline phosphatases. Tyr760 from the adjacent subunit in SP-PAP is close to MnII, and may play a role as the attacking nucleophile.

tetraazamacrocyclic nickel complexes as models for the active site of methylcoenzyme M reductase

Mark H Schofi eld, Williams College, United States David Y Chung, Williams College, United States

Methylcoenzyme M reductase, which contains a square planar nickel corphin cofactor, F430, catalyzes the fi nal step in methane biosynthesis in methanogenic Archaea. Using chemical reactivity and electrochemical studies as well as spin density (UB3LYP) calculations, several known tetraazamacrocyclic nickel complexes were evaluated for their suitability as chemical mimics for F430. Among the candidates studied, only Nickel(II)-7,15-diphenyl-1,5,9,13-tetraazahexadeca-

1,3,5,7,9,11-hexaenato hexafl uorophosphate (Ni(II)-[16ox]PF6) was found to undergo both reversible oxidation (to Ni(III)) 9 and quasi-reversible reduction (to Ni(I)). Chemical reduction of Ni-[16ox]PF6 by Zn/Hg in DMF affords the Ni(I) (d ) complex confi rmed by EPR spectroscopy. Alkylation of Ni(II)-[16ox]PF6 with MeMgBr in THF affords Me-Ni(II)[16ox] which is dealkylated Hg2+. Journal of Inorganic Biochemistry 96 (2003) 227

Crystal structures of reaction intermediates of BphC

Toshiya Senda, Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Japan Eiji Masai, Department of BioEngineering, Nagaoka University of Technology, Japan Masao Fukuda, Department of BioEngineering, Nagaoka University of Technology, Japan

BphC derived from Pseudomonas sp. strain KKS102 is an extradiol-cleaving catecholic dioxygenase. Extradiol-cleaving catecholic dioxygenases catalyze the addition of two atomic oxygens to the catechol ring of the substrate, resulting in cleavage of the catechol ring. These enzymes typically contain a non-heme iron (Fe(II)) in their active site. In order to elucidate the catalytic mechanism of the extradiol-cleaving catecholic dioxygenase, we have tried to solve the crystal structures of reaction intermediates of BphC. Here we present the crystal structures of the substrate free form of BphC, the BphC-substrate (ES) complex, and the BphC-substrate-NO (ES-NO) complex, all of which were determined under anaerobic conditions. These crystal structures revealed the followings. (a) The substrate directly coordinates to the Fe ion as a monoanionic form. (b) Upon substrate binding, His194 makes a conformational change, forming a strong hydrogen bond with hydroxyl group of the substrate. This hydrogen bond seems to be required to deprotonate the hydroxyl group of the substrate. (c) The NO molecule directly coordinates to the Fe ion. The binding site of the NO molecule, which is highly likely to be the binding site of dioxygen, is the vacant site of the octahedral coordination sphere of the ES complex. The cavity that accommodates the NO molecule is lined by hydrophobic residues. On the basis of these fi ndings, we propose a catalytic mechanism of the extraiol-cleaving catecholic dioxygenase in which His194 seems to play three distinct roles. At the early stage of the catalytic reaction, His194 appears to act as a catalytic base, which likely deprotonates the hydroxyl group of the substrate. Then the protonated His194 seems to stabilize a negative charge on the oxygen molecule located in the hydrophobic oxygen-binding cavity. Finally, protonated His194 seems to function as a proton donor.

Formation mechanism of the Soluble Complexes Containing Human Serum Albumin and Calcium Oxalate*

Yuhua Shen, School of Chemistry and Chemical Engineering, Anhui University, China Anjian Xie, School of Chemistry and Chemical Engineering, Anhui University, China W enjin Yue, School of Chemistry and Chemical Engineering, Anhui University, China

The main composition of urinary stone is protein and calcium oxalate [1]. The role of protein in the formation of urinary stone has been still unclear.[2]. In this paper, Fourier transform infrared spectroscopy was used to study the fi ltrates produced by reactions of different initial mole ratios (1:1,1:10 and 1:100) of human serum albumin (HSA) to calcium oxalate in normal saline with a thin hydration fi lm technique. It was seen that the frequencies of amide I (~1655cm-1), amide II (~1542 cm-1) of HSA shifted and the relative intensity of amide I to amide II increased as initial ratio of HSA and calcium oxalate from 1:1 to 1:100 compared with pure HSA. The secondary structures of HSA were measured using deconvolution and curve fi tting method in the amide I range (1600~1700cm-1)of FTIR spectra. It indicated that calcium oxalate caused the increase of α-helix and β-fold structures and the decrease of corner and random structures of HSA. Apparently the structure of HSA became more orderly. The concentrations of calcium ions in the fi ltrates examined by inductively coupled plasma atomic emission spectrometry were much more than in the saturation aqueous solution of calcium oxalate. The results demonstrated that there were strong interactions between HSA and calcium oxalate by hydrogen bonds and coordination. It suggested that the soluble complexes containing HSA and calcium oxalate formed. The complexes could make calcium oxalate nucleated on the surface of protein as well as the crystal speed of calcium oxalate restrained. References: 1 Smesko A., Singh R.P., .Lanzalaco A.C, et al..,Coll.Surf.30(1988)361 2 Binetle J.P., Binetta M B., Clin. Chim. Acta 296(2000) 59 * Project is supported by NNSFC(20031010 ,29971001),KPME(00180) and NSFAP(0045115) 228 Journal of Inorganic Biochemistry 96 (2003)

O2 Evolution from the High-Valent Oxo-Manganese Porphyrin Dimer. Yuichi Shimazaki, Kyushu University, Japan Taro Nagano, Kyushu University, Japan Hironori Takesue, Kyushu University, Japan Bao H Ye, Kyushu University, Japan Fumito Tani, Kyushu University, Japan Yoshinori Naruta, Kyushu University, Japan

1. The oxygen evolving center (OEC) in photosystem II (PSII), which consists of a tetranuclear Mn active site, can carry out the four electron oxidation of water to dioxygen. As a functional model reaction for OEC, we have already reported that the electrochemical oxidation of an aqueous acetonitrile solution with dimanganese tetraphenylporphyrin dimer evolved oxygen in the potential range >1.2 V vs. Ag/Ag+. However, the mechanism of the oxygen evolution, especially a possible high valent Mn=O intermediate has not been elucidated. In this study, we characterized a high-valent Mn=O species by various spectroscopic methods. Furthermore, we detected O2 evolution from the high-valent Mn=O species. 2. Oxidation of the Mn(III, III) dimer (1) with 4 eq. m-CPBA under basic conditions gave a red-colored species. The new species exhibited a very sharp Soret band at 423 nm. Since this intermediate was diamagnetic species on the basis of the magnetic studies, we assign it as a low spin d2 Mn (V, V) porphyrin dimer. The resonance Raman spectrum of the Mn(V, V) dimer exhibited two isotope-sensitive intense bands at 791 cm-1 and 518 cm-1. By replacing with 18OH-, these bands shifted to 756cm-1 and 491cm- 1. Further, by replacing with 16OD-, the band of 518 cm-1 shifted to 503 cm-1, though the band of 791 cm-1 did not shifted. Therefore, 791 cm-1 and 518 cm-1 bands are assigned to n(Mn=O), and n (Mn-OH), respectively. When an excess amount of V CF3SO3H was added to the high-valent (HO-Mn =O)2 species, O2 evolution was detected quantitatively. We will discuss the mechanism of O2 evolution from water catalyzed by the Mn porphyrin dimer.

A Theoretical study of the Mononuclear Non-hem Iron Complex of Phenylalanine Hydroxylase (PAH)

Yoshihito Shiota, Kyushu University, Japan Kazunari Yoshizawa, Institute for Fundamental Research of Organic Chemistry, Japan

The reaction pathway and the energetics for the hydroxylation of L-phenylalanine to form L-tyrosine are analyzed from B3LYP DFT computations. We propose a reasonable model for an oxygen species responsible for the catalytic reactivities of phenylalanine hydroxylase (PAH). Our model involves the iron-oxo species (FeIV=O), the side chain ligands of His285, His290, and Glu330 and the two water molecules in the binding site as a catalytic active center. The reaction pathway is initiated by a C-H bond cleavage via an H-atom abstraction that leads to a radical intermediate, followed by a C-O bond formation via an oxygen rebound step to lead to L-tyrosine complex. The activation energies for the C-H cleavage step and the C-O bond formation are 11.2 kcal/mol and 8.4 kcal/mol, respectively, at the B3LYP/6-311++G** level of theory. Thus, the rate-determining step in this hydroxylation is the C-H bond dissociation step. Following the DFT calculations of the small cluster model, protein model calculations with the catalytic domain as a surrounding environment are carried out by using a combined quantum mechanical/molecular mechanical (QM/MM) method. The protein model includes total 5432 atoms in the catalytic domain and is a truncated form of the PAH enzyme lacking the N-terminal regulatory and C-terminal tetramerization domains. The QM/MM method can describe the protein environment via non-bonding interactions between the QM (active site and substrate) and MM (catalytic domain) regions. The QM/MM calculations suggest that the iron active site in PAH enzyme is located at the bottom of a cleft. The substrate-binding site and the stable complexes (the reactant complex, the radical intermediate, and the product complex) along the reaction coordinate are determined. Journal of Inorganic Biochemistry 96 (2003) 229

Substrate Recognition and Melocular Mechanism of Fatty Acid Peroxygenase P450 from Bacillus subtilis

Yoshitsugu Shiro, RIKEN Harima Institute/ SPring-8, Japan Isamu Matsunaga, Osaka City University Medical School, Japan Dong S Lee, RIKEN Harima Institute/ SPring-8, Japan

Cytochrome P450BSβ from Bacillus subtilis catalyzes a hydroxylation reaction of fatty acid at the β (67%) and α (33%) positions using hydrogen preoxide as an oxidant. We have determined the crystal structure of the ferric enzyme in the substrate-bound form at a 2.1 angstrom resolution. The substrate palmitic acid is stabilized in the enzyme through two specifi c interactions: electrostatic interaction of its carboxylate with a guanidium group of Arg242, and hydrophobic interaction of the long alkyl chain with some hydrophobic residues in the channel. These interactions are responsible for the site specifi city of the hydroxylation site. The fatty acid carboxylate group interacts with Arg242 in the same fashion as has been reported for the active site of chloroperoxidase, His105 – Glu183, which is an acid-base catalyst in the peroxidation reaction. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by peroxygenase

P450BSβ is proposed in which carboxylate of the bound-substrate fatty acid assits in the cleavage of the peroxide O-O bond.

Metal Ion Complexes of Nucleoside 5’-O-thiomonophosphates and Related Ligands. Quantifi cation of the Extent of Metal Ion-Sulfur Binding

Helmut Sigel, University of Basel, Switzerland

Nucleotide analogues where a phosphate O is replaced by S receive increasing attention. These thio-derivatives are used to study enzymatic reaction mechanisms, to identify phosphate oxygens important in ribozyme catalysis, and they play a role in oligonucleotides used in the antisense strategy, yet only little information exists (see in [1]) on their H+ and M2+ binding properties.

We have studied (pot. pH titr.; aq. sol.; 25 °C; I = 0.1 M, NaNO3) acid-base and metal ion-binding equilibria involving adenosine 5’-O-thiomonophosphate, uridine 5’-O-thiomonophosphate (UMPS2-) and methyl thiophosphate (MeOPS2-) [1,2] but focus here on MeOPS2- and UMPS2- (= PS2-), the properties of which are compared with the parent methyl phosphate 2- 2- 2- - - (MeOPO ) and uridine 5’-monophosphate (UMP ) (= PO ) [2]. The acidity constants of H(PO) and H(PS) differ by ∆ pKa - - = 1.37 as proven for the pKa range 5.0-6.8 for several H(PO) . This result allows to predict the pKa of a H(PS) once that of the parent H(PO)- is known. 2+ 2- The data points for thiophosphates fi t exactly on log K versus pKa straight-line plots constructed for M /R-PO systems (R = non-interacting residue) thus proving that the reduced stability of the M(PS) species with Mg2+, Ca2+, Mn2+ and Ni2+ is due to the lower basicity of the thiophosphates [2]. For the M(PS) complexes of Zn2+, Cd2+ and Pb2+ a considerably enhanced stability compared to that of the parent M(PO) is observed despite the reduced basicity. Indeed, stability constant comparisons reveal that in thiophosphate complexes of Mg2+, Ca2+ and Mn2+ the formation degree of the S-bonded isomer is close to zero, for Zn(PS) it amounts to about 75% and for Cd(PS) it is above 99%. Supp. by the Swiss Nat. Sci. Found. and the Swiss Fed. Off. for Educ. & Sci. (COST D20). [1] (a) C. P. Da Costa, D. Krajewska, A. Okruszek, W. J. Stec, H. Sigel, J. Biol. Inorg. Chem. 7 (2002) 405-415. (b) R. K. O. Sigel, B. Song, H. Sigel, J. Am. Chem. Soc. 119 (1997) 744-755. [2] C. P. Da Costa, A. Okruszek, H. Sigel, submitted. 230 Journal of Inorganic Biochemistry 96 (2003)

A Cold-Adapted Metallo-Oxotransferase, Periplasmic Nitrate Reductase, from an Antarctic Bacterium

Philippa J L Simpson, Centre for Heavy Metals Research, School of Chemistry, The University of Sydney, Australia David S Nichols, Cooperative Research Centre for Antarctica and the South Ocean, Australia Rachel Codd, Centre for Heavy Metals Research, School of Chemistry, The University of Sydney, Australia

Select nitrogen, carbon and sulfur redox reactions fundamental to all life are governed by metallo-oxotransferases that catalyse the 2e- reduction or oxidation of substrates, such as nitrate (Fig. 1), via a net oxygen atom transfer [1]. Intriguingly, instead of the more common Mo-containing form, select metallo-oxotransferases characterised from heat-loving (hyperthermophilic) archaea that live in hydrothermal vents on the ocean bed where temperatures can reach 100 °C contain W at the active site. Whether metallo-oxotransferases from organisms that live under cold conditions (psychrophilic; ‘psychro’ is Greek for ‘cold’) contain Mo, W, or an alternative redox active metal, is not known, since these enzymes have yet to be fully characterised in cold-adapted organisms. We have obtained a bacterium from Antarctic sea-ice, Shewanella gelidimarina [2], and isolated and partially purifi ed the fi rst cold-adapted metallo-oxotransferase, periplasmic nitrate reductase (NAP). The cold-adapted enzyme consists of two subunits, 57 and 33 kDa, with an associated cytochrome-containing subunit which does not co-purify. Cold-adapted NAP possesses increased cold-resistance, retaining 33% of enzyme activity upon decreasing the temperature from 20 °C to 4 °C, compared to a cold-intolerant analogue which retained only 3% of activity under these conditions. Here, we will present further details of the results from our chemical and biochemical studies of this enzyme and shed light upon the molecular mechanisms of cold-adapted metallo-oxotransferases that contribute to the catalytic competency of the enzyme at sub-zero temperatures. Figure 1 The reduction of nitrate by the molybdenum-oxotransferase, periplasmic nitrate reductase (NAP), as described by Dias [3]. (1) Hille, R. Chem. Rev. 1996, 96, 2757-2816. (2) Bowman, J. P.; McCammon, S. A.; Nichols, D. S.; Skerratt, J. H.; Rea, S. M.; Nichols, P. D.; McMeekin, T. A. Int. J. Syst. Bacteriol. 1997, 47, 1040-1047. (3) Dias, J. M.; Than, M. E.; Humm, A.; Huber, R.; Bourenkov, G. P.; Bartunik, H. D.; Bursakov, S.; Calvete, J.; Caldeira, J.; Carneiro, C.; Moura, J. J. G.; Moura, I.; Romao, M. J. Structure 1999, 7, 65-79. Journal of Inorganic Biochemistry 96 (2003) 231

Electronic structure of the oxygen activating intermediates of Ribonucleotide Reductase: the peroxo intermediate and a model complex of the high-valent intermediate, X

Andrew J Skulan, Stanford University, United States Thomas C Brunold, Stanford University, United States Melissa A Hanson, Stanford University, United States Jeffrey Baldwin, Pennsylvania State University, United States Hua-fen Hsu, University of Minnesota, United States J Martin Bollinger, Pennsylvania State University, United States Lawrence Que, Jr, University of Minnesota, United States Edward I Solomon, Stanford University, United States

Identifying the contributions of the electronic and geometric structures of the oxygen-activating intermediates of binuclear non-heme iron enzymes to their reactivity is vital to understanding their reaction mechanisms. Ribonucleotide reductase (RR) is believed to pass through peroxo and high-valent intermediates, though their structures are the focus of much discussion. The putative peroxo-level intermediate of wild-type RR is kinetically masked, while the fi rst observed iron-oxygen intermediate observed in the W48F/D84E mutant of RR from E. coli is a peroxo-level complex. Combining LT absorption, resonance Raman and resonance Raman profi ling shows that only a µ-1,2 structure is consistent with experimental data. DFT studies on computational models (90-95 atoms) of the active site correlate the wild-type intermediate to that of W48F/ D84E RR and predict that the formation of a µ-1,2-peroxo intermediate is more thermodynamically favored in the double mutant than in wild-type, consistent with kinetic data on RR. This result may have parallels in other enzymes as methane monooxygenase (MMO) shares the same coordinating ligand set as the W48F/D84E RR mutant and has similar spectroscopic signatures for its peroxo intermediate, P. The key reactive intermediate X of RR possesses bridging oxo ligands and an FeIIIFeIV oxidation state. The structurally III IV 3+ characterized Fe Fe bis-µ-oxo complex, [Fe2O2(5-Et3-TPA)2] , shows radical formation, oxygenase and desaturase activity, paralleling the functions of the non-heme iron enzyme family. LT and VTVH MCD, absorption, EPR and resonance Raman spectroscopies supplemented by density functional calculations have lead to identifi cation of the origin of this molecule’s unusual spectroscopic and magnetic signatures. These studies provide a detailed electronic structure description of the Fe2O2 diamond core and its contribution to reactivity. 232 Journal of Inorganic Biochemistry 96 (2003)

Kinetic and Theoretical Studies of cyanide binding to the extracted FeMo-cofactor of Nitrogenase

Barry E Smith, John Innes Centre, Norwich, United Kingdom Zhen Cui, John Innes Centre, Norwich, United Kingdom Adrian J Dunford, Chemistry School of Natural Sciences, University of New, United Kingdom Marcus C Durrant, John Innes Centre, Norwich, United Kingdom Richard A Henderson, Chemistry School of Natural Sciences, University of New, United Kingdom

The active site of molybdenum nitrogenase is the iron-molybdenum cofactor (FeMoco) , an MoFe7S9X.homocitrate cluster (Einsle, O. et al. Science 2002, 297, 1696) that can be extracted intact from the enzyme. There have been many studies of substrate and inhibitor binding to the extracted cofactor (Burgess, B. Chem.Rev. 1990;90, 1377) but none have addressed the kinetics of binding. Here we describe the kinetics of the reactions between CN- and various derivatives of extracted FeMoco {FeMoco-L; where L is bound to Mo, and is NMF, ButNC or imidazole (ImH)} using a stopped-fl ow, sequential-mix method. The course of the reaction is followed indirectly by monitoring the change in the rate of the reaction of the cofactor with PhS-. The kinetic results indicate that the initial site of CN- binding to FeMoco-L is controlled by a combination of the electron-richness of the cluster core and the lability of the Mo-L bond. The reactions between FeMoco-L and CN- occur with a variety of rates and rate laws and ultimately involve displacement of L and binding of CN- to Mo. For FeMoco-NMF the reaction with CN- is complete within the dead-time of the apparatus whilst with FeMoco-CNBut the reaction is much slower and exhibits fi rst order dependences on both [FeMoco-CNBut] and [CN-].The reaction of FeMoco-ImH with CN- occurs at a rate which exhibits a fi rst order dependence on [FeMoco-ImH] but is independent of [CN-]. We conclude that CN- binds directly to the Mo site for FeMoco-NMF and FeMoco-ImH, but with FeMoco-CNBut CN- binds initially at an Fe site subsequently moving to Mo. Complementary DFT calculations are consistent with this interpretation, indicating that the Mo-L bond is stronger for L = ImH than for CNBut and that electron-release from the imidazole group makes the cluster core in FeMoco-ImH more electron-rich than FeMoco-CNBut . DFT calculations also indicate that the binding of CN- to Mo is stronger than to any Fe atom.

Novel Separation Process for Ultra pure and High Specifi c Activity Cu-64

Suzanne V Smith, Australian Nuclear Science and Technology Organisation, Australia David J Waters, Australian Nuclear Science and Technology Organisation, Australia Nadine M Di Bartolo, Australian Nuclear Science and Technology Organisation, Australia Rosalie Hockings, Australian Nuclear Science and Technology Organisation, Australia

Copper-64 (T1/2) has been identifi ed as an excellent tool for monitoring copper in medical, industrial and environmental processes. In nuclear medicine it has been identifi ed as one of the emerging positron emission tomography isotopes. However its routine production in an ultra pure state and at high specifi c activities remains a challenge. While Cu-64 may be readily produced in a reactor or a cyclotron, the limiting factor in its commercial availability has been the result of either poor specifi c activity ( 1000 Ci/gram at EOB), poor yields, contaminating isotopes, or the cost of target material. Cu-64 and Cu-67 were reported to be co-produced during routine commercial production of Ga-67.1 Preliminary work by us, demonstrated for the fi rst time the separation of Cu-64 from the Ga-67 production process.2 After a comprehensive study on the binding affi nity of contaminating ions (e.g Co-57, Ni-57 and Zn-65) with AG1-X8 in organic/acid mixtures and the re-design of the target plate, we have developed a new process for similtaneous separation of high purity Cu-64 and purifi cation of enriched Zn-68 target for re-use. The production yields for Cu-64 are typically 1.8 mCi/Ahr and high specifi c activities of up to 30,000 Ci/g are achievable. Most signifi cant is the extremely high chemical purity of the Cu-64 that allows the monitoring of Cu-67 (cp:. It is felt that use of this new method should substantially increase the potential use of Cu-64 on a worldwide basis. 1 TE Boothe Nucl Instru & Method in Phys Res B56 1266 1991 2 SV Smith DJ Waters N Di Bartolo Radiochimica Acta 75 65-68 1996 3 I Voskokoinik H Brook SV Smith et al Febs Letters 435 178 1998 4 SA Gale SV Smith RP Lima et al Aquatic Toxicology 62 (2) 135 2003 5 SV Smith N Di Bartolo J Harrowfi eld AM Sargeson PCT/WP 00/40585 Journal of Inorganic Biochemistry 96 (2003) 233

Resonance Raman study of folding intermediates of cytochrome c

Giulietta Smulevich, Department of Chemistry,University of Florence, Italy

Spectroscopic studies can elucidate the folding pathway of proteins and characterize intermediate states. In particular, misfolded intermediates of cytochrome c (cyt c) have been studied by optical and magnetic resonance techniques, exploiting the spectral features of the heme to probe for protein structural changes. Resonance Raman (RR) spectroscopy has particular advantages, since it is extremely sensitive to changes in the heme geometry and environment. In this contribution, RR results for different types of unfolded cyt c states and related models will be presented. Acid unfolding of horse ferric cyt c at low ionic strength leads to a heme coordination change from the native 6- coordinated low-spin (6LS) Met80-Fe-His18 species to a 6-coordinated high-spin (6HS) H2O-Fe-His18 species. A different 6LS species is also observed at low pH, identifi ed as a misligated His-Fe-His form. Further pH lowering induces the formation of an aquo 5-coordinated, high-spin (5HS) species. A comparison with the heme-containing 1-56 N-fragment of cyt c shows that pH-dependent changes occur also for this protein. The ferric 1-56 N-fragment is characterized by His-Fe-His ligation at pH 7. In fact, the RR spectra are identical with those of urea-unfolded cyt c, which has His-Fe-His coordination. At pH 4.8, an aquo 6HS species is observed, whereas a 5HS species is formed at pH 3.0. It is, therefore, apparent that the 1-56 N-fragment is a good model for the misfolded His-Fe-His cyt c intermediate. The molten globule, or A-state, of cyt c is stabilized by salts in an acidic environment. The salt-induced collapse of acid-denatured cyt c leads to a number of equilibria between high spin and low spin heme states, and between two types of 6LS states. The two latter states are characterized by native-like Met-Fe-His coordination and a misfolded His-Fe-His coordination. The equilibrium between these states is dependent on the anion concentration and/or size.

Probing the Mechanism of Radical Abstraction of the Small Subunit of E. coli Ribonucleotide Reductase

Jessica L Sneeden, University of Washington, United States Lawrence A Loeb, University of Washington, United States

Ribonucleotide reductase from Escherichia coli consists of two dissociable subunits, R1 and R2. The R2 (NrdB) subunit contains a stable radical at tyrosine 122, buried within a hydrophobic pocket of the folded protein. Although inaccessible to solvent, the radical is subject to abstraction by radical scavengers, notably the DNA synthesis inhibitor hydroxyurea. Hydroxyurea has a number of clinical applications; it has been used as an anticancer and antiviral agent, and in the treatment of sickle cell anemia. How hydroxyurea is able to access the radical is unknown; it has been suggested that small molecules are able to access Tyr122 directly via channels from the surface of the protein to its hydrophobic core. To test this hypothesis, we have mutated fi ve contiguous residues (I74, S75, N76, L77, K78) that comprise one side of a channel where Tyr122 is visible from the protein surface. Our data represent the fi rst demonstration of of NrdB ORF mutants in E. coli highly resistant to hydroxyurea, and provide evidence for direct access to the radical by hydroxyurea. Using the method of random oligonucleotide mutagenesis, we have replaced these amino acids with partially random sequence, creating a large library of NrdB mutants. We have selected this library for survival on increasing concentrations of hydroxyurea and identifi ed active mutants that are greater than 104-fold resistant to hydroxyurea over wild-type NrdB. 234 Journal of Inorganic Biochemistry 96 (2003)

New Structural and Functional Models for Quercetin 2,3-dioxygenase

Gabor Speier, Department of Organic Chemistry, University of Veszprém, Hungary Jozsef Kaizer, Research Group for Petrochemistry, Hungarian Academy of Sciences, Hungary Michel Giorgi, Chimie, Biologie et Radicaux Libres, UMR CNRS 6517, Un, France Marius Reglier, Chimie, Biologie et Radicaux Libres, UMR CNRS 6517, Un, France

The X-ray structure of quercetin 2,3-dioxygenase isolated from Aspergillus japonicus revailed that the Cu(II) ion at the active site is surrounded by three histidine and a water molecule or by additional carboxylate coordination of glutamate

[1]. We have found that the rate of oxygenolysis of [Cu(fl a)(idpa)]ClO4 (1) (fl a = favonolate, idpa = 3,3-iminobis(N,N- dimethylpropylamine) is enhanced by carboxylate addition (acetate or benzoate) [2]. We prepared and structurally characterized further [Cu(fl a)(bpg)] (2) [bpgH = N,N-di(2-picolyl)glycine], which is a more relevant mimic for the enzyme. The oxygenolysis of 2 proceeds with the incorporation of two O-atoms of

O2 and release of CO. The corresponding (O-benzoylsalicylato)copper(II) complex [Cu(O-bs)(bpg)] was formed and characterized. On the basis of kinetic studies of the oxygenation of 2 a plausible mechanism is proposed. [1] F. Fusetti, K.H. Schröter, R.A. Steiner, P.I. van Noort, T. Pijning, H.J. Rozeboom, K.H. Kalk, M.R. Egmond, B.W. Dijkstra, Structure, 2002, 10, 259. [2] …. Balogh-Hergovich, J. Kaizer, G. Speier, J. Mol. Catal. A., submitted. ’ 4 2

Redox Interplay of Molybdenum- and Tungsten-thio Centres: A Possible Catalytic Role for Pterin-dithiolene

Stephen A Sproules, School of Chemistry, University of Melbourne, Australia Jason P Hill, University of Melbourne, Australia Jonathon M White, School of Chemistry, University of Melbourne, Australia Graham N George, Stanford Synchroton Radiation Laboratory, SLAC, Stanfor, United States Charles G Young, University of Melbourne, Australia

The pterin-dithiolene co-ligand is present in all molybdo- and tungstoenzymes, with the exception of the molybdenum nitrogenases.[1] Apart from anchoring the active site to the protein, it is believed to ‘fi ne tune’ the reduction potential of the metal centre facilitating the catalysis of metabolites. However, the catalytic mechanism of these enzymes is yet to be fully established. Stiefel and co-workers have noted that dithiolenes can be converted into trithiolenes with the inclusion of an adjacent terminal thio ligand.[2,3] Due to the facile nature of this reaction, such a conversion occurring in the enzyme cannot be discounted. This paper outlines the effect of one-electron oxidation and reduction processes with Tp*MOS(pyS) and Tp*MOS(S2PPh2) (M = Mo, W) complexes. Oxidation and reduction leads to M(V) (M = Mo, W) species, through direct or induced internal redox reactions. The intriguing redox interplay observed is mechanistically analogous to the proposed trithiolene formation.[4,5] The produced complexes are isolated and characterised by spectroscopy, including XAS and X- ray crystallographic studies. [1] R. Hille, TRENDS in Biochemical Sciences, 2002, 27, 360. [2] R. S. Pilato, K. A. Eriksen, M. A. Greaney, E. I. Stiefel, S. Goswami, L. Kilpatrick, T. G. Spiro, E. C. Taylor, A. L. Rheingold, J. Am. Chem. Soc., 1991, 113, 9372. [3] E. I. Stiefel, Pure & Appl. Chem. 1998, 70, 889. [4] J. P. Hill, L. J. Laughlin, R. W. Gable, C. G. Young, Inorg. Chem. 1996, 35, 3447. [5] A. A. Eagle, S. Thomas, J. P. Hill, S. A. Sproules, J. M. White, E. R. T. Tiekink, G. N. George, C. G. Young, Inorg. Chem., submitted for publication. Journal of Inorganic Biochemistry 96 (2003) 235

Crystal structure of the R2 subunit of Ribonucleotide Reductase from Chlamydia trachomatis

Pâl Stenmark, Department of Biochemistry and Biophysics, Stockholm University, Sweden Martin Högbom, Department of Biochemistry and Biophysics, Stockholm University, Sweden Grant McClarty, Department of Medical Microbiology, University of Manitoba, Canada Pär Nordlund, Department of Biochemistry and Biophysics, Stockholm University, Sweden

Ribonucleotide reductases (RNRs) catalyze a reaction essential for all living cells, namely the production of precursors for DNA synthesis. Inhibition of RNR would stop DNA synthesis and cell proliferation; it is therefore a potential target for anti -bacterial, -viral and -cancer drugs. The role of protein R2 is the generation of a stable tyrosyl radical via the reductive cleavage of dioxygen at a di-iron carboxylate site. The tyrosine residue carrying the radical in R2 is conserved among all sequenced R2 proteins with the exception of the Chlamydia family, which has a phenylalanine in this position (F127). We have solved the crystal structure of Chlamydia trachomatis R2 at 1.7Å resolution. Phenylalanine 127 is located at the position were the radical harboring tyrosine in other R2 proteins is located. The radical species of this protein thus has to be stored and/or generated in a different way compared to the other R2's. It can be hypothesized that the radical is stored either directly at the iron site or on one of the other tyrosines in the vicinity of the di-iron site. We have also solved the structure of the F127Y mutant of Chlamydia trachomatis R2, this structure shows signifi cant changes at the di-iron site compared to the wild type protein. We have shown that the radical site in the Chlamydia trachomatis R2 is very different from all other R2 proteins of known structure. Different possibilities for how the Chlamydia family of R2 proteins can function without the ‘normal’ radical site will be discussed.

Copper and silver homeostasis by Escherichia coli assessed with a biosensor

Jivko Stoyanov, Institute of Clinical Pharmacology, Switzerland David Magnani, Institute of Clinical Pharmacology, Switzerland Marc Solioz, Institute of Clinical Pharmacology, Switzerland

Copper and silver are potentially toxic metals and cells must be able to control their cytoplasmic concentrations. Copper is essential for life and functions as a cofactor in key redox enzymes such as cytochrome c oxidase or superoxide dismutase. In contrast, there is no known biological role for silver and it has not been recognized as a metal that is under homeostatic control. Copper homeostasis has been extensively studied in bacteria. In Escherichia coli, the chief transporter to excrete excess cytoplasmic copper has been shown to be the CopA ATPase, a member of the new family of heavy metal ATPases. Strains deleted in the copA gene are exquisitely sensitive to copper. To assess changes in cytoplasmic metal ion concentrations, we constructed reporter plasmid based on the lux gene cluster of Vibrio fi scheri. This system has been widely used to construct biosensors. We constructed a heavy metal ion reporter system by placing the lux operon under the control of the copA promoter (PcopA) of E. coli. This promoter has been shown to control the expression of the CopA copper ATPase via the action of the CueR repressor, which responds to copper, silver, and other heavy metals. To record changes in cytoplasmic copper levels, an E. coli wild-type strain and a ∆copA strain were transformed with the lux reporter plasmid. By measuring the luminescence, we could thus show variations in cytoplasmic copper levels at different ambient metal ion concentrations in wild-type and mutant cells. In wild-type cells, maximal luminescence was observed at 60 µM copper or 0.8 µM silver in the media. In the strain deleted in CopA, maximal luminescence was induced by only 4µM copper and 0.1 µM silver, respectively. At the same time, the maximal luminescence responses were enhanced 5 to 7-fold. This suggests that the CopA copper ATPase participates in the control of both, copper and silver levels and that cytoplasmic silver concentrations can also be controlled by CopA. 236 Journal of Inorganic Biochemistry 96 (2003)

Using a Principle of Heterogeneous Catalysis to Achieve Enzyme-like Molecular Catalysis

Gerhard F Swiegers, CSIRO Molecular Science, Australia Jun Chen, IPRI, University of Wollongong, Australia Junhua Huang, CSIRO Molecular Science, Australia Chee O Too, IPRI, University of Wollongong, Australia Gordon G Wallace, IPRI, University of Wollongong, Australia

The primary role of a catalyst is to bring reactant molecules or atoms into close proximity to each other so that they can react. Different classes of catalyst achieve this in different ways. Synergistic molecular catalysts, such as enzymes, typically achieve this through the use of a few proteinaceous catalytic groups which are spatially arranged to make attached reactants or intermediates ideally proximate for reaction. Heterogeneous catalysts depend on high proportions of the atoms at their surfaces bearing reactants or intermediates at certain temperatures, pressures, or concentrations. This creates a statistically relevant probability that many of the reactants or intermediates will be adventitiously proximate to each other and able to react. In this work we report a new hybrid approach to bio-inspired or bio-mimetic synergistic molecular catalysis in which discrete, molecular catalytic groups are concentrated within a porous support such that a statistically signifi cant proportion of them are adventitiously proximate to each other and able to catalyse the reaction. Coating of a platinum electrode with conducting polypyrrole doped with ferrocene sulfonate induces a 0.3 V anodic shift in the most positive potential for hydrogen gas evolution in 1 M sulfuric acid and a seven-fold amplifi cation in hydrogen generation when poised for 12 h at ∼0.44 V (vs. Ag/AgCl). The amplifi cation per unit electrochemical area is 3.5-fold. Similar improvements are observed over the full voltage range to ∼3.0 V in two-electrode water electrolysis experiments. Comparative and fundamental studies indicate that this effect is due to the ferrocene sulfonate which acts, at high local concentrations, as a synergistic catalytic group in the reaction. The resulting system is possibly the fi rst truly practical example of an ‘artifi cial enzyme’ catalyst.

Synthesis and characterization of dicopper-dioxygen complex supported by novel unsymmetrycal pentapyridine ligand

Yoshimitsu Tachi, Department of Chemistry, Graduate School of Science, Osaka, Japan Kazuki Aita, Graduate School of Science, Japan Shinichi Teramae, Department of Material and Life Science, Graduate School of Science, Osaka, Japan Fumito Tani, Institute for Fundamental Research of Organic Chemistry, Japan Yoshinori Naruta, Institute for Fundamental Research of Organic Chemistry, Japan Shunichi Fukuzumi, Department of Material and Life Science, Graduate School of Science, Osaka, Japan Shinobu Itoh, Department of Chemistry, Graduate School of Science, Osaka, Japan

Reactive intermediates derived from the reaction of copper(I) complexes with O2 play important roles in many biological and industrial oxidation processes. Among the series of copper-active oxygen species, dicopper-dioxygen (Cu2/O2) complexes such as (trans-µ-1,2-peroxo)dicopper(II), (µ-η2:η2-peroxo)dicopper(II), and bis(µ-oxo)dicopper(III) complexes have been most extensively studied in model systems. Most of the dicopper-dioxygen complexes so far been reported involve symmetrical ligand environments, but few examples have been reported on Cu2/O2 complexes supported by unsymmetrical ligands. Since most of the dinuclear metal sites in metalloenzymes have an unsymmetrical coordination environment, it is highly desirable to investigate the dioxygen reactivity of unsymmetrically coordinated dicopper(I) complexes in artifi cial system. In this study, a novel unsymmetrical dinucleating ligand L has been designed and synthesized in order to investigate ligand effects on the copper(I)-dioxygen reactivity. The ligand has an unique unsymmetrical structure combined with tetradentate and tridentate coordination sites. The cyclic voltammetric(CV) study as well as the X-ray structure of the dicopper(I) complex clearly indicated that the ligand provided different coordination environments for the two copper ions.

Reaction of the dicopper(I) complex with O2 at a low temperature provided a peroxo intermediate with unique spectroscopic features, implying the formation of a new active oxygen-dicopper species. Journal of Inorganic Biochemistry 96 (2003) 237

Palladium(II) and Platinum(II) Complexes with Pendent Phenol Rings as Receptors for Biomolecules

Masako Takani, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan Tatsuo Yajima, Faculty of Engineering, Kansai University, Japan Osamu Yamauchi, Faculty of Engineering, Kansai University, Japan

Enzymes bind with substrates at the active site cavity through various weak interactions. Cyclodextrins, calixarenes, etc., which incorporate molecules into the cavity, have been extensively studied as hosts for a variety of compounds. We have been studying intermolecular interactions in the systems involving nucleotides and platinum(II) complexes with an aromatic nitrogen ligand and an amino acid etc. and have shown that the planar Pt(II)-aromatic ring moiety effectively binds with an uncoordinated aromatic ring. In order to attain selective binding of molecules in an aromatic environment, we now investigated adduct formation by metal complexes containing pendent aromatic rings. The ternary Pt(II) complexes containing phen or bpy and N,N’-bis(o- or m-hydroxybenzyl)ethylenediamine (o- or m- hben) such as Pt(phen)(o-hben) and binary Pd(II) complexes such as Pd(o-hben)2 were prepared, and the adducts with AMP and GMP were isolated. 1H NMR spectra indicated that the complexes bind with the nucleotides to cause upfi eld shifts of the ring proton signals due to stacking, which were larger for the ternary complexes than for the binary complexes. Large shifts were observed for [Pt(phen)(m-hben)]-GMP as compared with [Pt(phen)(en)]-GMP, which suggests that pendent aromatic rings close to the coordination plane contribute to adduct formation. Interactions with nucleotides and other molecules will be presented.

Emission Ratiometric Probes for Zinc Ion

Masayasu Taki, Department of Chemistry, Northwestern University, United States Janet L Wolford, Department of Biochemistry, Molecular Biology, and Cell, United States Thomas V O’Halloran, Department of Chemistry and Department of Biochemistry, United States

The important role of the zinc ion (Zn2+) has been recognized not only in a wide range of enzymatic processes but also in biological systems such as DNA and RNA synthesis, gene expression, metabolism, apoptosis, and signaling. While the total concentration of Zn2+ in cells is thought to be ~0.3 mM, its intracellular concentration is tightly controlled by zinc uptake and export genes, such as Zur and ZntR metalloregulatory proteins in E. coli, respectively.(1) However, little is known about the intracellular behavior of Zn2+, especially in comparison to other physiologically important cations such as Ca2+. In order to investigate the functions of such spectroscopically silent metal ions in biological systems, new fl uorescent sensor molecules that respond to a specifi c metal ion in the excitation or emission spectrum have been useful tools. We will herein describe the synthesis and characterizations of a novel emission ratiometric probe for Zn2+, Zinbo-5. The family employs a benzoxazole fl uorophore core which exhibits a large shift in the emission spectrum as well as in the excitation spectrum upon the complexation with Zn2+. Both the apo form and the Zn2+-bound form are excitable at the same wavelength in the ultraviolet range (340 nm to 360 nm). Zinbo-5 exhibits an emission band at 407 nm, which shifts to 443 nm upon addition of Zn2+. The dissociation 2+ constant for Zn under physiological conditions (50 mM HEPES, pH 7.20, 0.1 M KNO3) was determined as cp:. Biological applications using Zinbo-5 will also be discussed. (1). C. E. Outten, T. V. O’Halloran, Science 2001, 292, 2488-2492. 238 Journal of Inorganic Biochemistry 96 (2003)

Hydrolysis of 4-Nitrophenyl Acetate by a Novel (N2S)Zinc-Hydroxide Complex: A Kinetic and Thermodynamic Study of a Reactive Peptide Deformylase Model

Robert C diTargiani, Johns Hopkins University, United States Sechin Chang, Johns Hopkins University, United States Michael H Salter, Jr, University of North Carolina-Wilmington, United States Robert D Hancock, University of North Carolina-Wilmington, United States David P Goldberg, Johns Hopkins University, United States

Metallohydrolases use a terminal water/hydroxide moiety to catalyze hydrolysis at biological pH. While there are many synthetic model complexes of these systems that contain all-nitrogen coordination spheres, there are few examples of mixed II nitrogen/sulfur(alkylthiolate) complexes. We have recently developed the chemistry of a new family of (N2S)M L complexes with the pyridine-amine-thiolate ligand, PATH.1 This presentation will focus on the formation and reactivity of (PATH)ZnOH,2 which represents a reactive model of the zinc form of peptide deformylase (PDF). The zinc-hydroxide complex is generated 3 by slow dissolution of (PATH)ZnCH3 in water and protonolysis of the zinc- carbon bond to give (PATH)ZnOH and methane. (PATH)ZnOH has been shown to promote the hydrolysis of 4-nitrophenyl acetate, an excellent test substrate for both models and enzymes. Product analysis of the hydrolysis reaction by 1H NMR spectroscopy shows that this reaction proceeds cleanly to give 4-nitrophenolate and acetate as the only products. A complete kinetic study, including a pH-rate profi le, has led to the determination of the pKa and the pH-independent second-order rate constant for (PATH)ZnOH. In addition, activation parameters for the hydrolysis reaction have been obtained by an Arrhenius and

Eyring analysis. The pKa for the metal-bound hydroxide has been confi rmed by an independent potentiometric study. These data support a mechanism of simple nucleophilic attack by the metal-bound hydroxide. The basicity and nucleophilicity of this (N2S)ZnOH complex will be discussed in comparison to other zinc model complexes and enzymes. 1 Chang, S., Karambelkar, V. V., diTargiani, R. C., Goldberg, D. P. Inorg. Chem.,194-195 (2001). 2 diTargiani, R. C., Chang, S., Salter, M. H. Jr., Hancock, R. D.; Goldberg, D. P. Inorg. Chem. submitted. 3 Chang, S., Sommer, R. D., Rheingold, A. L., Goldberg, D. P. Chem. Commun., 2396-2397 (2001). The NIH (GM62309) is acknowledged for the generous support of this work.

Stereoselective Oxidation of Co(II) Complexes in the Milieu of Bovine Serum Albumin

Toshiaki Taura, Laboratory of Molecular Recognition and Coordination Chemistry, University of Aichi Prefecture, Japan

Serum albumin is the main transport protein in the blood. It has been reported that serum albumin specifi cally interacts with a variety of anionic or hydrophobic substrates: fatty acid anions, warfarin, bilirubin and so on. By contrast, this protein potentially has chiral recognition capabilities for substrates with the asymmetric centers. Recently we have found that the circular dichroism (CD) of a cobalt(III) or chromium(III) complex anion (tris(oxalato)cobaltate(III) or tris(oxalato)chromate(III)) is induced in the visible region by the chiral interaction with bovine serum albumin. On the basis of the CD intensity the enantiomeric excess of the metal complex in the optical resolution by bovine albumin was estimated. In this work, using this CD technique, we investigate how metal complexes, especially cobalt(II) complexes including ethylenediaminetetraacetatocobaltate(II), are oxidized in the milieu of bovine serum albumin stereoselectively. Journal of Inorganic Biochemistry 96 (2003) 239

NMR studies probing the directionality of DNA interstrand cross-linking by the trinuclear platinum complex BBR3464

Donald S Thomas, School of Biomedical and Chemical Sciences, University of Western Australia, Australia Murray S Davies, School of Biomedical and Chemical Sciences, University of Western Australia, Australia Junyong Zhang, School of Biomedical and Chemical Sciences, University of Western Australia, Australia Susan J Berners-Price, School of Biomedical and Chemical Sciences, University of Western Australia, Australia Nicholas Farrell, Department of Chemistry, Virginia Commonwealth University, United States

Previous DNA binding studies using molecular biology techniques have revealed that the dinuclear Pt complex [{trans- 2+ PtCl(NH3)2}2(µ-NH2(CH2)6NH2)] (1,1/t,t (n=6); (1)) forms predominantly interstrand cross-links in the 5′-5′ direction. On 4+ the other hand, the clinical candidate, [{trans-PtCl(NH3)2}2-µ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}] (1,0,1/t,t,t, (n=6,6); BBR3464 (2)) produces a smaller percentage of interstrand cross-links and footprinting studies indicate the formation of cross-links in both the 5′-5′ and 3′-3′ directions.1 It is possible that pre-association of the central linker of 2 in the minor groove of DNA affects the directional nature as well as the extent of the interstrand cross-links We have used [1H,15N] NMR methods to follow the stepwise formation of the cross-links and to compare the binding of the dinuclear (1) and trinuclear (2) compounds to the 14-base pair duplex 5′-d(ATACATGGTACATA)-3′•5′-d(TATGTACCATGTAT)-3′. The sequence offers several possible bifunctional binding sites including 1,4-interstrand cross-links in both 5′-5′ and 3′-3′ directions. Studies with 1 showed the product is primarily the 5′-5′ 1,4-interstrand cross-link between G(8) and G(18) bases and exists in two conformational forms.2 Here we present a similar NMR study of the binding of 2 to the same 14-mer duplex. Molecular modelling studies have been used to probe the initial pre-association of the drug with the DNA. The authors thank the Australian Research Council, U.S. National Institute of Health, U.S. National Science Foundation and the American Cancer Society for fi nancial support. 1 Kasparkova, J., Zehnulova, J., Farrell, N., Brabec, V. J. Biol. Chem. 2002, 277, 48076-48086. 2 Berners-Price, S. J., Davies, M. S., Cox, J. W., Thomas, D. S., Farrell, N. Chem. Eur. J. 2003, 9, 713-725.

Models Reactions of Haloperoxidases. Halogenation of - - Organic Substrates by RuO4 /X /H 2 O2 Tsz Yan Tong, City University of Hong Kong, Hong Kong

Haloperoxidases are enzymes that catalyze the oxidation of a halide (e.g. chloride, bromide or iodide) by hydrogen peroxide, which can result in halogenations of organic substrate. For example, chloroperoxidases - (CPO) can catalyse the oxidation of Cl to Cl2 by H2O2; the Cl2 produced can then be used to chlorinate organic substrates.

- + H2O2 + Cl + H → H2O + HOCl

+ - HOCl + H + Cl → Cl2 + H2O

- + AH + H2O2 +Cl + H → ACl + 2H2O - - We report here some model reactions of haloperoxidases using the system: RuO4 /Br /H2O2. 1-phenyl propyne and phenol - - are brominated by RuO4 /Br /H2O2 according to the following equations: 240 Journal of Inorganic Biochemistry 96 (2003)

The redox stability and processes of manganese based metallo-enzyme mimetics

George E Tranter, Imperial College London, United Kingdom Delphine D Le Pevelen, Imperial College London, United Kingdom Elaine C Taylor, Imperial College London, United Kingdom Markus Sikkel, Imperial College London, United Kingdom James Booth, Imperial College London, United Kingdom

The redox mechanisms and stability of some manganese based enzyme mimetics, with potential as anti-oxidant therapeutics, have been investigated using Spectro-Electro-Array Spectroscopy (SEAS). The in vitro SEAS technique is able to reproduce the redox cycling and catalytic anti-oxidant activity of the manganese containing complexes whilst providing structural and electrochemical information of the intermediates formed. It was found that some manganese complexes were unstable in their Mn(II) state, the complexes rapidly dissociating with subsequent decomposition of the ligand. This would be consistent with the loss of catalytic activity and complex degradation in the presence of hydrogen peroxide (i.e. during catalase-like activity) and instability on reduction by glutathione (e.g. as may be found in red blood cells, liver and kidney). The implications for the design of effi cient enzyme mimetics for therapeutic anti-oxidant use are discussed.

Structural Characterization of the Particulate Methane Monooxygenase by Electron Paramagnetic Resonance and X-ray absorption Spectroscopy

Chiu-Fou Tseng, Institute of Chemistry, Academia Sinica, Taiwan, Province of China Kelvin H-C Chen, Institute of Chemistry, Academia Sinica, Taiwan, Province of China Shyue-Chu Ke, Department of Physics, National Dong-Hua University, Hu, Taiwan, Province of China Steve S-F Yu, Institute of Chemistry, Academia Sinica, Taiwan, Province of China Sunney I Chan, Institute of Chemistry, Academia Sinica, Taiwan, Province of China

The pMMO isolated from Methylococcus capsulatus (Bath) consists of a three-subunit hydroxylase (45, 27 and 23 kDa) and a NADH oxidoreductase (38 kDa). The hydroxylase is a copper protein, with 15 copper ions arranged in fi ve trinuclear copper clusters. The functional form of the enzyme is the fully or partially reduced copper hydroxylase. The pMMO exhibits unusual substrate specifi city: only small normal alkanes are hydroxylated and similar alkenes epoxidated. From the EPR spectrum of the as-isolated copper enriched particulate methane monooxygenase, we observed a type II copper signal with g//=2.24, A//=185 G, and g⊥=2.058 associated with hyperfi ne splitting of about 150 G centered at g//=2.29. From 15N labeling experiments, the superhyperfi ne splittings observed may be assigned to a catalytic center coupled with three nitrogens. In order to explore the structure of the active site as well as the nature of the reaction intermediate(s) formed at the active site during turnover of the enzyme, we have subjected the pMMO to different levels of reductants such as dithionite, as well as oxidants, including ferricyanide or oxygen under different oxygen tension, and examined the copper ions at various stages of oxidation of the copper clusters. Both the catalytic and electron transfer clusters (C- and E-clusters, respectively) were examined by EPR spectroscopy and X-ray absorption spectroscopy to distinguish between various multi-oxidation states of the copper clusters. From the disappearance and the presence of the type II copper signals and superhyperfi ne splittings with incremental addition of reductive as well as oxidative reagents, we have been able to distinguish between the two catalytic sites from the titration experiments. Upon fully oxidizing the pMMO by ferricyanide, we observe a g≅2.096 isotropic EPR signal, which we have identifi ed and characterized by computer simulation analysis to be associated with a fully oxidized tricopper cluster signal. With the above results, we have made an important step forward in our efforts toward defi ning the functional role of some of the copper ions in the catalytic pathway of the pMMO. Journal of Inorganic Biochemistry 96 (2003) 241

Ruthenium(III)-DMSO complexes of the antiherpes drug acyclovir

Iztok Turel, University of Ljubljana, Slovenia Milena Pecanac, University of Ljubljana, Slovenia Amalija Golobic, University of Ljubljana, Slovenia Enzo Alessio, University of Trieste, Italy Barbara Serli, University of Trieste, Italy Alberta Bergamo, Fondazione Callerio, Trieste, Italy

The antitumor properties of simple ruthenium(III) coordination compounds have been discovered over 30 years ago. Later, several tumor inhibiting ruthenium complexes have been isolated and characterized; DMSO-ruthenium-N donor ligand ternary complexes are amongst the most promising. The main advantages of ruthenium-DMSO complexes are the selectivity for solid tumor metastases and the lack of signifi cant host toxicity. Acyclovir (9-(2-hydroxyethoxymethyl)guanine, acv) is a well known antiviral drug used in clinical practice and was used as a ligand in this study. Treatment of precursor X[trans-RuCl4(DMSO-S)2] 4+ + . (X=NH , Na , or protonated DMSO) with acv in methanol yielded brown-red crystals of mer-[RuCl3(acv)(DMSO-S)(CH3OH)]

0.5 CH3OH (1); when the same reaction was performed in water or various non-dried solvents (such as ethanol, propanol, . acetone/DMSO mixtures, nitromethane) crystals of mer-[RuCl3(acv)(DMSO-S)(H2O)] H2O (2) were isolated instead. In both complexes 1 and 2 the geometry about the Ru(III) ion is as expected distorted octahedral, with three chloride ions in a mer- confi guration and DMSO (coordinated through S) trans to acv (coordinated through N(7)). An oxygen atom from methanol (in 1) or water (in 2) is additionally coordinated to ruthenium. Inspection of the crystal structures of both compounds suggests that the replacement of chloride with methanol (in 1) or water (in 2) is induced by the formation of intramolecular hydrogen bonds. The oxygen atom O(6) from acv is hydrogen bonded to either coordinated methanol (1) or water (2). Both compounds have been additionally characterized by various physico-chemical methods and their biological activity was also studied. 242 Journal of Inorganic Biochemistry 96 (2003)

Resonance Raman studies of the heme chaperone protein, CcmE

Takeshi Uchida, Center for Integrative Bioscience, Okazaki National Research Institutes, Japan Julie M Stevens, Department of Biochemistry, University of Oxford, United Kingdom Oliver Daltrop, Department of Biochemistry, University of Oxford, United Kingdom Chris W Higham, Department of Biochemistry, University of Oxford, United Kingdom Stuart J Ferguson, Department of Biochemistry, University of Oxford, United Kingdom Teizo Kitagawa, Okazaki National Research Institutes, Japan c-Type cytochromes are widespread proteins and involved in a number of metabolic pathways. The heme of cytochrome c is attached to the polypeptide chain via thioether linkages between two heme vinyl groups and two cysteine residues in the conserved CXXCH motif. In many Gram-negative bacteria such as Escherichia coli, heme attachment to cytochromes c occurs via a system that involves at least 12 gene products, including the Ccm (cytochrome c maturation) proteins A-H1. CcmE is a key player of this system and is classifi ed as a heme chaperone. Heme is delivered to CcmE from CcmC and makes a covalent bond with a conserved His, and is then transferred to apocytochrome c. However, the molecular mechanism of heme binding and release remains to be described. We reveal some insights into the structure of CcmE using resonance Raman in this study. Resonance Raman spectra of the high-frequency region of reconstituted CcmE (b-type CcmE) suggests that the protein has a mixture of 5- and 6-coordinate hemes in both the ferric and dithionite-reduced forms. In the presence of CO, reduced b-type CcmE binds CO to form a six-coordinate structure. Fe-C and C-O stretching modes of CO-bound b-type-CcmE were observed at 496 and 1959 cm-1, respectively, which fall on a line for proteins with proximal His ligation in the correlation plot between Fe-C and C-O stretching frequencies. This suggests that CcmE has His as a proximal ligand in the CO-bound form. These frequencies also refl ect the polarity of the distal heme pocket. The obtained frequencies of CcmE indicate the absence of interaction between iron-bound CO and distal amino acid residues as observed in myoglobin. The maturation mechanism of cytochrome c will also be discussed. 1Thony-Meyer, L. (1997) Microbiol. Mol. Biol. Rev. 61, 337-376.

Spectroscopic and Electrochemical Properties of the Blue Copper Protein Pseudoazurin H6K Mutant

Yoshiko Uchida, Ibaraki University, Japan Rehab F Abdelhamid, Ibaraki University, Japan Takamitsu Kohzuma, Ibaraki University, Japan

The blue copper protein pseudoazurin functions as an electron carrier to the respective copper ion containing nitrite reductase in the respirtory chain of denitrifying bacteria. Pseudoazurin is a typical blue copper protein, which has a single copper atom at the active site and an intense absorption band due to S(Cys) to Cu(II) charge transfrer in the visible region. It has been reported that pseudoazurin gives reversible electrochemical behavior at a (4-pyridyl) disulufi de-modifi ed gold electrode, and the redox potential of pseudoazurin have been estimated to be 260mV vs. NHE at pH 7.0. In the previous electrochemical studies of pseudoazurin, the higher redox potential shift due to the protonation of uncoordinated histidine residue, His6 (pKa = 6.6) has been considered under the acidic pH conditions (280 mV at pH 6.0). Such long distance electrostatic effect on the redox potential regarding with the electronic structure of the active center of protein molecules has been attracted for the optimization of many enzymatic reactions. Lysine residue having positively charged sidechain group (pKa = 10.5) at neutral pH was introduced instead of the His6 residue for the purpose of the estimation of electrostatic effect on the redox potential. Cyclic voltammogram of the mutant H6K indicated the well defi ned reversible electrochemical behavior, and the redox potential was estimated to be 237mV vs NHE at pH7.0. The preliminary pH dependency of the redox potential indicates the similar pKa to the wild type pseudoazurin. It is most likely the pH dependency of the redox potential by the acid-base equilibria of amino acid resides except His6. Journal of Inorganic Biochemistry 96 (2003) 243

Construction of artifi cial metalloenzymes:Insertion of Chromium(III) Schiff Base Complexes into Apo-Myoglobin

Takafumi Ueno, Research Center for Materials Science, Nagoya University, Japan Tomomi Koshiyama, Graduate School of Science, Nagoya University, Japan Masataka Ohashi, The Graduate University for Advanced Studies, Japan Masaharu Kono, Graduate School of Engineering, Nagoya University, Japan Takashi Yamane, Graduate School of Engineering, Nagoya University, Japan Yoshihito Watanabe, Graduate School of Science, Nagoya University, Japan

Construction of artifi cial metalloenzymes is one of the exiting subjects in the fi eld of bioinorganic chemistry. We have developed noncovalent insertion of metal complexes into protein scaffolds to conjugate apo-myoglobin (apo- Mb) as a protein matrix with chromium(III) schiff base complexes which are

oxidation catalysts in organic solvents. The crystal structure of Cr(3,3í-Me2 -Salophen)(1)/apo-A71G Mb indicates that the ligation of imidazole of His93 to the Cr atom and hydrophobic interactions of the Schiff base ligand with the protein matrix make the complex stable (Figure 1). Furthermore, Cr(5,5í-

tBu2 -Salophen)/apo-H64D/A71G Mb can catalyze the enantioselective sulfoxidation of thioanisole in aqueous solution. These results suggest that our strategy to utilize synthetic cofactors and apo-Mb mutants provides a novel approach for the bionano-material design. 1 M. Ohashi, T. Koshiyama, T. Ueno, et al. Angew. Chem. Int. Ed., 42, 1005 (2003).

Two novel trans-tetrachloro(dimethyl sulfoxide)azoleruthenate(III) complexes, their antiproliferative activity and the coordination mode of triazole in

H 2trz[RuCl4(dmso)(Htrz)] Peter Unfried, Institute of Inorganic Chemistry of the University of Vienna, Austria Erwin Reisner, Institute of Inorganic Chemistry of the University of Vienna, Austria Madeleine Fremuth, Institute of Inorganic Chemistry of the University of Vienna, Austria Michael A Jakupec, Institute of Inorganic Chemistry of the University of Vienna, Austria Roman Lichtenecker, Institute of Inorganic Chemistry of the University of Vienna, Austria Vladimir B Arion, Institute of Inorganic Chemistry of the University of Vienna, Austria Bernhard K Keppler, Institute of Inorganic Chemistry of the University of Vienna, Austria

Halo-ruthenium(dimethyl sulfoxide) complexes exhibit anticancer or antimetastatic activity. Quite recently, the complex

Him[trans-RuCl4(dmso)im] entered phase I clinical trials as an antimetastatic drug. This discovery stimulated further the interest towards this class of compounds. Herein we report on the synthesis, X-ray diffraction study, magnetic

and spectroscopic properties of mixed heterocycle-dmso ruthenium(III) complexes Hind[trans-RuCl4(dmso)ind] (1),

H2trz[RuCl4(dmso)Htrz] (2) and Ph3PCH2Ph[trans-RuCl4(dmso)Htrz] (3). The solid state structures of 1 and 3 revealed an S-bonded dmso ligand trans- to nitrogen donor of indazole and 1H-1,2,4-triazole respectively. The distribution of electron density over the triazole ring does not allow to locate the double bonds. All ring bonds are essentially the same. However, analysis of the crystal structure of 3 showed the presence of interanion contacts, which we are prone to describe as hydrogen bridges involving H-N1-N2 part of the triazole rings. Taking this into account coordination of triazole via N4 is proposed. In addition, complexes 1 and 2 were tested in vitro in three human cell lines and their antiproliferative activity is compared to that of NAMI-A. 244 Journal of Inorganic Biochemistry 96 (2003)

Cytochrome c without Tryptophan: The W59A Variant

J Antonio Villegas, University of British Columbia, Canada Federico I Rosell, University of British Columbia, Canada A Grant Mauk, University of British Columbia, Canada

Mitochondrial cytochromes c possess a single, phylogenetically conserved Trp residue at position 59 that forms an H-bond with heme propionate-7 that is part of an internal hydrogen-bonding network that includes R38, H39, Y48 and an internally- bound water molecule. While previous reports indicate that replacement of W59 with residues bearing small side chains produces variants incapable of supporting yeast respiration (1), diffi culties in obtaining such variants in suffi cient quantities for physical characterization have prevented more detailed studies (2). We have now expressed the W59A and W59A/N52I/ Y67F variants effi ciently in E. coli and initiated functional and spectroscopic characterization of them. Our initial fi ndings are three-fold: (i) At pH 7, the W59A variant exhibits no absorption maximum at 695 nm while the triple variant does, consistent with the view that the W59A substitution prevents normal axial ligation of the native conformation while the stabilizing substitutions at residues 52 and 67 restore normal axial ligation (alkaline pKa = 8.4). (ii) Thermal stability (pH 7) of the W59A variant determined by UV-CD spectrometry is greatly compromised (Tm = 36.9 °C) relative to that of the wild-type protein

(Tm = 59.4 °C) while that of the triple variant is intermediate (Tm = 40.9 °C). (iii) Reaction of wild-type cytochrome with

1000-fold excess of H2O2 (pH 7) results in rapid bleaching of the Soret maximum while the spectrum of the triple variant is less reactive under the same conditions. We conclude that the stabilizing infl uence of the substitutions at residues 52 and 67 partially overcomes the destabilizing infl uence of the W59A substitution that the triple variant provides a suitable variant with which the structural and functional consequences of placing a small residue at position 59 may be evaluated. Supported by CIHR Grant MOP-7182 (to A.G.M.). (1) Hampsey et al., JBC 261, 3259 (1986); (2) Black et al., Biochem. J. 359, 715 (2001).

Electrochemical insights into inhibition of multi-copper oxidases

Kylie A Vincent, University of Oxford, Inorganic Chemistry Laboratory, United Kingdom Shahid M Kadodia, University of Oxford, Inorganic Chemistry Laboratory, United Kingdom Sarah J Gurr, University of Oxford, Plant Sciences, South Parks Road, United Kingdom Fraser A Armstrong, University of Oxford, Inorganic Chemistry Laboratory, United Kingdom

Some small anionic ligands have long been known to act as inhibitors of multi-copper oxidases and there is evidence from EPR that these bind to type 2 Cu(II) centres. In the present study, protein fi lm voltammetry provides a different perspective on anion inhibition and distinguishes the contrasting ways in which anions inhibit catalytic electron transport. Laccase isolated from the white rot fungus Coriolus hirsutus adsorbed onto a pyrolytic graphite ‘edge’ electrode in oxygen-saturated citrate buffer (pH 4 – 5) gives a well-defi ned catalytic wave corresponding to the four-electron reduction 1 of O2 to H2O. This property has been noted by others. Here we are concerned with how different inhibitors alter the catalytic waveform and the ways in which this may be interpreted. For certain anionic inhibitors such as cyanide, azide or fl uoride, the effect is to cause strong attenuation of the catalytic current. A signifi cantly different result is obtained with chloride. As the chloride concentration is raised, the catalytic O2 reduction wave shifts to more negative potentials but with little attenuation of current. Chloride-inhibited laccase thus becomes a poor oxidant for those phenolic substrates having a higher potential. In conventional solution assays laccase activity is measured by oxidation of a substrate-dye molecule and therefore depends on the reduction potential of this redox partner relative to that of the enzyme. In an electrochemical experiment the inhibition of laccase is readily overcome by application of a higher electrochemical driving force. This study addresses kinetic, thermodynamic and mechanistic aspects of enzyme inhibition. In more specifi c terms, understanding the nature of inhibitor effects on fungal laccases has relevance for applications in biosensors and biofuel cells. 1 Thuesen, M. H., Farver, O., Reinhammar, B., and Ulstrup, J. (1998) Acta Chemica Scandinavica 52, 555-562 Journal of Inorganic Biochemistry 96 (2003) 245

Sequence-selective metallation of double-helical oligodeoxyribonucleotides with Pt(II), Mn(II) and Zn(II).

Jo Vinje, Department of Chemistry, University of Bergen, Norway John A Parkinson, Department of Pure and Applied Chemistry, University of Strathclyde, United Kingdom Peter J Sadler, School of Chemistry, University of Edinburgh, United Kingdom Einar Sletten, Departement of Chemistry, University of Bergen, Norway

Reactions of [PtCl(dien)]+ (dien = diethylenetriamine), Mn2+ and Zn2+ ions with three different double-helical oligodeoxyribonucleotides, containing the central sequence GGXY (XY=AT, TA or CC) have been followed by NMR spectroscopy. 2D [1H, 15N] HSQC/HMQC NMR spectroscopy using 15N-labelled Pt(dien) shows that the rate of formation of 3'-G-N7 and 5'-G-N7 platinated adducts is highly sequence dependent. The relative rates of platination of 5'-G vs. 3'-G are largest for -GGCC-, for which only a small fraction of the 3'-G adduct is formed; for -GGTA-, the rate of 5'-G platination is about 8 times that of 3'-G, and for -GGAT- the ratio is 1.2. These values are in qualitative agreement with those obtained for G-N7/Mn2+ selectivity as determined by paramagnetic line broadening of the adjacent G-H8, and G-N7/Zn2+ selectivity as determined by G-H8 chemical shift changes [1, 2], and are also in agreement with ab initio calculations of the highest occupied molecular orbitals (HOMO) of double stranded DNA [3]. The reaction mixtures containing platinated 3'-G and 5'-G fractions were separated by HPLC, annealed to duplexes and analyzed by 2D [1H, 1H] NOESY NMR spectroscopy. Unexpectedly, the 5'-G-H8 resonances of both 5'-G and 3'-G platinated duplexes showed large downfi eld shifts in the range 0.3 ppm to 0.6 ppm while the 3'-G-H8 resonances in both cases exhibited no, or only slight, upfi eld shifts. This observation is important since a downfi eld shift of ~ 0.5 ppm often is used as a diagnostic of N7-platination [4]. References: (1) N. A. Fröystein, J. T. Davis, B. R. Reid, E. Sletten, Acta Chem. Scand. 1993, 47, 649-657. (2) E. Moldrheim, B. Andersen, N. A. Fröystein, E. Sletten, Inorg. Chim. Acta 1998, 273, 41-46 (3) I. Saito, T. Nakamura, K. Nakatani, J. Am. Chem. Soc. 2000, 122, 3001-3006. (4) D. Lemaire, M-H. Fouchet, J. Kozelka, J. Inorg. Biochem. 1994, 53, 261-271. 246 Journal of Inorganic Biochemistry 96 (2003)

Towards the Determination of the Mechanism of Antitumour Activity of Molybdocene Dichloride

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

The biological chemistry of the metallocene dihalides, Cp2MCl2 (M = Ti, Mo, V, Nb), has attracted signifi cant interest since the antitumour activity of these organometallic complexes was fi rst reported in 1979 [1]. Titanocene dichloride is the most studied of the metallocenes, and is currently in phase II clinical trials. Titanium has been shown to accumulate in nucleic acid rich regions of the tumour cell, and Cp2TiCl2 forms adducts with both nucleic acid components and bulk DNA. While the other antitumour metallocenes have been less studied, current evidence strongly suggests that the different metallocene complexes interact differently with cellular components, and therefore probably have different antitumour mechanisms of action [2]. Although molybdocene dichloride coordinates to nucleotides [3], molybdocene-oligonucleotide adducts are not formed under physiological conditions [4], suggesting that this metallocene is likely to have an alternative cellular target to DNA. Extensive studies on the interaction and competitive coordination of molybdocene dichloride with nucleic acid components, glutathione and amino acids have been performed. Molybdocene dichloride preferentially coordinates to thiols over amino and carboxylate groups or nucleotides, indicating that formation of molybdocene-thiol complexes is related to their biological activity. The stable and highly water soluble derivatives

Cp2Mo(Cys)2 (1) and Cp2Mo(GSH)2 (2) have been isolated and fully characterised. In parallel, studies have been performed to determine the uptake of molybdocene dichloride into V79 Chinese hamster lung cells. These studies, along with visualisation of the cellular distribution of the metal through the use of micro-SRIXE techniques, should allow an improved understanding of the mechanism of action of molybdocene dichloride. [1] Köpf-Maier, P.; Köpf, H. Angew. Chem. Int. Ed. 1979, 18, 477-478. [2] Harding, M. M.; Mokdsi, G. Curr. Med. Chem. 2000, 7, 1289-1303. [3] Kuo, L.Y.; Kanatzidis, M.G.; Sabat, M.; Tipton, A.L.; Marks, T.J. J. Am. Chem. Soc. 1991, 113, 9027-9045. [4] Harding, M.M.; Mokdsi, G.; Mackay, J.P.; Prodigalidad, M.; Lucas, S.W. Inorg. Chem. 1998, 37, 2432-2437.

Structural Analysis of a De Novo Designed Metalloprotein

Herschel V Wade, University of Pennsylvania, United States Steven Stayrook, University of Pennsylvania, United States William F DeGrado, University of Pennsylvania, United States

The de novo design of metalloproteins provides an attractive approach to test the essential features required for function. To this end, DF2 has been designed to serve as a minimal model of the diiron-carboxylate proteins. The members of this family contain carboxylate-bridged diiron centers that share many common structural features, but also contain subtle differences that give rise to different activities, making the system attractive to study how protein environments can infl uence enzyme activities. Here we present crystallographic analyses of the diferric-oxo form of DF2. This complex is important as it is the resting state of all of the diiron-carboxylate proteins. Structural analysis of this complex will guide protein engineering efforts towards catalysis. The structures of DF2 complexes with divalent metal ions is also presented. A comparison of the diferric- oxo and divalent forms of DF2 will provide insight into what kinds of structural rearrangement are need to accomodate metal ion oxidation state changes. These types of rearrangement have been implicated in the mechanisms of the natural diiron proteins. Journal of Inorganic Biochemistry 96 (2003) 247

A Bismuth(III) Complex of 1, 4, 7, 10-Tetrakis(2-pyridylmethyl)-1, 4, 7, 10- tetraazacyclododecane with Potent Cytotoxic Activity

Xiaoyong Wang, State Key Laboratory of Coordination Chemistry, Nanjing, China Jun Lin, State Key Laboratory of Coordination Chemistry, Nanjing, China Jingwen Chen, State Key Laboratory of Coordination Chemistry, Nanjing, China

The antitumor potential of bismuth compounds has not been well explored as other metal compounds although they have long been associated with medicine [1],[2]. Proteins such as transferrin appear to be the biological target for Bi(III) compounds [3], but the evidence for DNA binding is lacking. In this work a Bi(III) complex of 1, 4, 7, 10-tetrakis(2-pyridylmethyl)-1, 4, 7, 10-tetraazacyclododecane (BiTPC) was synthesised and characterised by 1H NMR and the ES-MS. The binding ability of BiTPC to DNA was investigated via its interaction with guanosine-5’-monophosphate (5’-GMP) and calf thymus DNA (CT-DNA). As can be seen from the following Figure, the absorption bands of BiTPC at 334 nm exhibited a hypochromism of about 25.7 % when DNA concentration increased from 0 to 5.13x10-6 M, and that at 235~265 nm showed a similar tendency. The mechanism of reaction of BiTPC towards DNA is discussed based on the UV and CD spectral data. The in vitro data showed that BiTPC is highly cytotoxic against melanoma B16-BL6 cells with an inhibition rate of 81.8% -7 -8 at a concentration of 2.5x10 M. The IC50 of BiTPC is 4.1x10 M which is 100 times more potent than the currently used antitumour drug cisplatin. Figure. The UV absorption spectra of BiTPC with the increasing concentration of CT-DNA. Acknowledgement This work is supported by the National Natural Science Foundation of China (No.s 29925102, 20231010). References [1] U. Dittes, E. Vogel and B. K. Keppler, Coor. Chem. Rev. 163 (1997) 345-364. [2] E. R. T. Tiekink, Crit. Rev. Oncol. Hemat. 42 (2002) 217-224. [3] P. J. Sadler, H. Li, H. Sun, Coor. Chem. Rev. 185-186 (1999) 689-709. 248 Journal of Inorganic Biochemistry 96 (2003)

Correlation of molecular structure, EPR and moessbauer spectral type and reduction potential in low-spin ferriheme models of the cytochromes

F Ann Walker, University of Arizona, United States Liliya Yatsunyk, University of Arizona, United States Michael D Carducci, University of Arizona, United States Thomas Teschner, University of Luebeck, Germany Volker Schuenemann, University of Luebeck, Germany Alfred X Trautwein, University of Luebeck, Germany

2 3 Low-spin Fe(III) porphyrinates can have two possible electron confi gurations, either (dxy) (dxz,dyz) or the less common 4 1 (dxz,dyz) (dxy) . The former is the usual electron confi guration of the b-type cytochromes found in the membrane-bound mitochondrial complexes II and III, all of which have bis-histidine coordination. All of these cytochromes b exhibit single-feature EPR spectra known as ‘large gmax’ or ‘HALS’ signals, that have previously been shown to be characteristic of ferriheme centers having axial ligands in perpendicular planes. However, at the present resolution of the structure o of cytochrome bc1 it does not appear that the histidine imidazole plane dihedral angles are 90 for either heme bH or bL. Furthermore, it has also been shown that low-spin Fe(II), the redox partner in cytochrome electron transfer, strongly disfavors axial ligands binding in perpendicular planes, at least in cases where macrocycle ruffl ing accompanies ligand binding. In an attempt to better understand the limits in axial ligand plane dihedral angle and ligand orientation with respect to the porphyrinate ring that give rise to normal rhombic vs. ‘large gmax’ EPR spectra, we have prepared and investigated the molecular structures, EPR and Moessbauer spectra and redox properties of a number of axial ligand complexes of Fe(III) and Fe(II) octaalkyltetra -phenylporphyrinates. This class of model hemes provides a rich diversity in structural features that leads to new insights into the relationship between structure and spectroscopic and redox properties. The results narrow the limits in ligand dihedral angles for observation of the two types of EPR and Moessbauer signals and provide new understandings of the relationship between EPR data, axial ligand dihedral angles, and reduction potentials that increase our understanding of the membrane-bound cytochromes b. Journal of Inorganic Biochemistry 96 (2003) 249

Polyammonium macrocycles as receptors for biologically relevant oxoanions

Andrew C Warden, Monash University, Australia Mark Warren, Monash University, Australia Leone Spiccia, Monash University, Australia

Phosphates and sulfates are among the most signifi cant oxoanions known, playing a wide variety of roles in almost all biological systems, forming an essential part of life processes. Biological systems have evolved a plethora of proteins and enzymes with which to handle and utilize these entities. In recent years, rapid advances in crystallographic and computational techniques have lead to the elucidation of the crystal structures of many oxoanion-binding enzymes (see Figure), furthering our understanding of how nature has elected to use these species. The knowledge gained in this pursuit has stimulated interest in the synthesis of efficient receptors for phosphates and sulfates. One such class comprises the polyammonium macrocycles, as they usually bear positive charges at physiological pH and hence have a strong affi nity for a wide variety of anions. After a brief overview of some naturally occurring phosphate and sulfate binding sites, this presentation will address the versatility of the macrocycle 1,4,7,10,13,16-hexaazacyclooctadecane ([18]aneN6) in interacting with phosphates and sulfates and how these interactions vary with the degree of protonation of the macrocycle. Although thorough studies have been performed on these and similar systems in solution only a handful of crystal structures containing [18]aneN6/anion complexes have been described. We will also examine the anion-anion and anion-water interactions, the effects of hydration, ion pairing, hydrogen bonding modes of the anions and the novel supramolecular features found in the structures, including a new isomer of the W7 water heptamer, and how they relate to biological systems. 1 Supramolecular Chemistry of Anions; A. Bianci, K. Bowman-James, E. Garcia-España, Eds.: Wiley-VCH,: New York, 1997 and references therein 2 C. Bazzicalupi, A. Bencini, A. Bianchi, M. Cecchi, B. Escuder, V. Fusi, E. Garcia-España, C. Giorgi, S. V. Luis, G. Maccagni, V. Marcelino, P. Paoletti and B. Valtancoli, J. Am. Chem. Soc., 1999, 121, 6807-6815. 250 Journal of Inorganic Biochemistry 96 (2003)

Circular dichroism and magnetic circular dichroism studies of the reduced binuclear non-heme iron sites of D84E and D84E/W48F ribonucleotide reductase mutants. Comparison to reduced methane monooxygenase and contributions to O2 reactivity

Pin-pin Wei, Department of Chemistry, Stanford University, United States Andrew J Skulan, Department of Chemistry, Stanford University, United States Natasa Mitic, Department of Chemistry, Stanford University, United States Yi-Shan Yang, Department of Radiology, Stanford University, United States Edward I Solomon, Department of Chemistry, Stanford University, United States Joseph M Bollinger, Jr, Department of Biochemistry and Molecular Biology, Pennsylvania State University, United States Lana Saleh, Department of Biochemistry and Molecular Biology, Pennsylvania State University, United States

Circular dichroism (CD) and magnetic circular dichroism (MCD) techniques have been used to probe the active site of two mutants of ribonucleotide reductase (R2). The aspartate to glutamate mutation at the binuclear iron site modifi es the R2 ligand set to match the binuclear center of the hydroxylase component of methane monooxygenase (MMOH).1 The reduced crystal structure of D84E R2 suggests that the ligand orientations at the active site parallels those in MMOH.2 However, CD/MCD and variable-temperature variable-fi eld (VTVH) MCD data combined with spin-Hamiltonian analysis of reduced D84E R2 indicate a structure with a different coordination environment than MMOH and no µ-(η1,η1) carboxylate bridge. To further understand the variation in geometry of the active site which lead to profound differences in reactivity, DFT calculations have been carried out to explore the effect of varying the ligand set, positions of free waters, and additional protein constraints on the geometry and energy of the binuclear site of both wild type R2 and mutant R2s to determine how these relate to the reduced MMO active site structure. 1 Bollinger, J. M., Jr.; Krebs, C.; Vicol, A.; Chen, S.; Ley, B. A.; Edmondson, D. E.; Huynh, B. H. J. Am. Chem. Soc. 1998, 120, 1094-1095 2 Voegtli, W. C.; Khidekel, N.; Baldwin, J.; Ley, B. A.; Bollinger, J. M., Jr.; Rosenzweig, A. C. J. Am. Chem. Soc. 2000, 122, 3255-3261 Funded by the NSF Biophysics Program, Grant MCB 9816051 (E.I.S.) and by the NSF Pre-Doctoral Fellowship to P.Wei

A functional model for the on-off coordination of nitrogen donors in iron redox enzymes

Stefan Woerl, Inorganic Chemistry Department, University of Heidelberg, Germany Hans Pritzkow, Inorganic Chemistry Department, University of Heidelberg, Germany Roland Kraemer, Inorganic Chemistry Department, University of Heidelberg, Germany

In biological redox catalysis, on-off coordination of a histidine imidazole group in the course of the catalytic cycle has been reported for at least two iron enzymes, a diiron peroxidase and soybean lipoxygenase.1,2 We have prepared a low-molecular- weight iron complex of 2,2’,2’’-nitrilotribenzoate, in which on-off coordination of the nitrogen donor is relevant to redox catalysis. The complex catalyses the oxidation of hydroquinone by H2O2 at pH 7 while related 1:1 iron complexes of polycarboxylate ligands (nitrilotriacetate, aspartic acid, citrate) are inactive. The triaryl nitrogen atom forms a coordinative bond with iron(II) but not with iron(III). This on-off transition is accompanied by a change of coordination number and polyhedra and appears to be crucial for catalytic activity. 1 S. Jin, D. M. Kurtz, Z.-J. Liu, J. Rose, B.-C. Wang, J. Am. Chem. Soc. 2002, 124, 9845-9855. 2 C. Pham, J. Jankun, E. Skrzypczak-Jankun, R. A. Flowers, M. O. Funk, Jr., Biochemistry 1998, 37(51), 17952-7. Journal of Inorganic Biochemistry 96 (2003) 251

2 The Stereospecifi c Hydroxylation of [2- H2]butane and Chiral Deuterated Butanes by the Particulate Methane Monooxygenase from Methylococcus Capsulatus (Bath)

Lo-Ying Wu, Department of Chemistry, National Tsin-Hua Unitersity, Taiwan, Province of China Steve S-F Yu, Institude of Chemistry, Academia Sinica, Taiwan, Province of China Sunney I Chan, Institude of Chemistry, Academia Sinica, Taiwan, Province of China

Experiments on cryptically chiral ethanes (JACS,1996) have indicated that the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) catalyzes the hydroxylation of ethane with total retention of confi guration at the carbon center attacked. This result would seem to rule out a radical mechanism for the hydroxylation chemistry mediated by this enzyme. The interpretation of subsequent experiments on n-butane and n-pentane has been complicated by hydroxylation at secondary ‘C-H’ bonds. It has been suggested that these results merely refl ect presentation of both the pro-R and pro-S ‘C- H’ bonds to the hot ‘oxygen atom’ species generated at the active site, and that the oxo-transfer chemistry, in fact, proceeds concertedly with retention of confi guration. In the present work, we have augmented these earlier studies with experiments on 2 [2- H2]-butane and designed d,l form chiral deuterated butanes. GC analysis of the stereochemical distribution of the products by the Mosher method, together with GC-MS in conjunction with the isotopic chiral labeling, have been used to follow the hydroxylation chemistry and to obtain independent and direct determinations of the extent of confi gurational inversion 2 2 during the hydroxylation process. Essentially equal amounts of (2R-[3- H2]butan-2-ol and (2R-[2- H]butan-2-ol are produced 2 upon hydroxylation of [2- H2]butane. The chemistry is stereospecifi c with full retention of confi guration at the secondary carbon oxidized. In the case of the various chiral deuterated butanes, the distribution of products mirrors the stereochemical confi gurations of the chiral butane substrates, after allowance is made for the expected deuterium isotope effect on the kinetics of the ‘oxygen atom’ insertion step. The extent of confi gurational inversion has been shown to be negligible for all the chiral butanes examined. Thus, the hydroxylation of butane takes place with full retention of confi guration in butane as well as in the case of ethane. These results are interpreted in terms of an oxo-transfer mechanism based on side-on singlet ‘oxene’ insertion across the ‘C-H’ bond similar to that previously noted for singlet carbene insertion (JACS,1992).

The C-terminal dormain of the membrane copper pump Ctr1 in yeast binds Cu as a cuprous-thiolate polynuclear cluster

Zhiguang Xiao, School of Chemistry, University of Melbourne, Victoria, Australia Graham N George, Stanford Synchrotron Radiation Laboratory, Stanford University, United States Anthony G Wedd, School of Chemistry, University of Melbourne, Victoria, Australia

Copper is an essential but highly toxic element for all living cells. Thus copper uptake and traffi cking must be regulated strictly.1 In yeast, copper uptake is mediated by the plasma membrane copper pump Ctr1 and delivered inside the cells to various copper-requiring compartments via different carrier proteins (chaperones). Ctr1 features three separate structural domains which are proposed to mediate different aspects of Cu transport. The cysteine-rich C-terminal domain, Ctr1c, is hydrophilic, is located in the cytosol and may interact directly with and deliver Cu to various copper chaperones. We have expressed and purifi ed Ctr1c.2 Copper binding and exchanging studies indicated that Ctr1c exhibited high affi nity for Cu(I) and can bind up to 4 Cu(I) ions which can be exchanged rapidly with one of the proposed partners, Atx1 in vitro. Site-directed mutagenesis suggested that all six cysteines in Ctr1c were involved in the Cu binding and the EXAFS data indicated that the copper ions were bound as a cuprous-thiolate polynuclear cluster. Detailed results will be presented. 1 Puig S.and Thiele D.J. Current Opinion in Chemical Biology 2002, 6, 171-180. 2 Xiao Z. and Wedd, A.G.. J. Chem. Soc., Chem. Commun. 2002, 588-589. 252 Journal of Inorganic Biochemistry 96 (2003)

Study on the Relationship Between the Gallstone Formation and the Properties of Patient Bile*

Anjian Xie, School of Chemistry and Chemical Engineering, Anhui University, China Y uhua Shen, School of Chemistry and Chemical Engineering, Anhui University, China Fanfzhi Huang, School of Chemistry and Chemical Engineering, Anhui University, China

Many people suffer from the disease of gallstones[1], but there are still no effective pharmaceuticals to control and cure this disease. Obviously the main components of gallstones exist in the patient bile. It is very important to study the relationship between gallstone formation and the properties of patient bile. In this paper, the patient bile was centrifugated at the speed region of 3000-18000 r/min. FTIR spectrum, UV-Vis spectroscopy, analysis of particle size and determination of zeta potential were used to investigated the patient bile and its centrifugates compared with the normal pig biles because the properties of both bile were quite similar[2]. It was seen that the patient bile was a heterogenetic system and its stability was less than that of normal pig bile. The Content of bilirubin in patient bile was much higher than that of the pig bile. The concentration of bilirubin and some substances with carboxylic and hydroxy groups, such as protein, choleric acid, cholesterol etc., decreased and more precipitates were produced in the patient bile with the increase of the centrifugated speed. FTIR spectra displayed that the compositions of the precipitates were mainly bilirubin, calcium bilirubinate, cholesterol and protein,which were much closer to that of the core of the patient gallstone. It is suggested that the higher content of the bilirubin and the product of the undissolvable calcium salt and some cholesterol and protein in the patient bile has a crucial infl uence on the stability of the patient bile, playing important roles in the nucleation and initial growth of the gallstone. Reference [1] Liu G, Xing D, Yang H M, et al. Spectroscopy and Spectral Analysis,2002,22(4):519 [2] Juste C, Catala I , Riottot M, Hepatology 1997,26:711 * Project is supported by NNSFC(20031010,29971001),KPME(00180) and NSFAP(0045115)

Nitrous oxide reductase from Hyphomicrobium denitrifi cans A3151

Kazuya Yamaguchi, Osaka University, Japan Hideyuki Ogawa, Osaka University, Japan Akiro Kawamura, Osaka University, Japan Shinnichiro Suzuki, Osaka University, Japan

Nitrite oxide reductase located in the periplasm of denitrifying bacteria

catalyzes two-electron reduction of N2O to N2. The Cu-containing nitrous

oxide reductase from Hyphomicrobium denitrifi cans (HdN2OR) has been

isolated, purifi ed, and spectroscopically characterized. Two forms of HdN2OR have been isolated under aerobic condition. Both form are homodimers of ca. 65 kDa subunits, and contain 4.1- 4.5 mol of copper per subunit. The pH profi le of activity for both form is identical, and the specifi c activities at optimum pH 8.8 are 45 ± 5 and 29 ± 3 U/mg for form A and B, respectively. The visible

spectra of HdN2OR exhibited absorption maxima at 480 nm , 540 nm , and 650 nm with a shoulder at 780 nm (form A), and a broad maximum at 650 nm (form B). On one-electron reduction of form A with sodium dithionite, the absorption peaks at 480, 540, and 780 nm disappeared.

The spectral change is reversible on one-electron oxidation with K3Fe(CN)6 of form B. X-band EPR spectra of HdN2OR at

77 K show seven-line hyperfi ne signal in g// region, g = 2.18, with 4.5 mT, characteristic of mixed valence couple Cu(1.5+)-

Cu(1.5+) of CuA site (form A), and a broad featureless signal, g = 2.06 (form B). Comparison of spectral data with other

N2ORs suggests that the form A of HdN2OR contains a mixed valence dinuclear CuA site in the Cu(1.5+)-Cu(1.5+) state and a ∗ mixed valence tetranuclear CuZ site in high redox potential Zmv state, while the form B of HdN2OR have a reduced dinuclear ∗ CuA site in the Cu(I)-Cu(I) state and a Zmv of CuZ site. Journal of Inorganic Biochemistry 96 (2003) 253

CO2-Fixation Mediated upon Zinc-Hydroxo Complex Containing Amino Group. Syuhei Yamaguchi, Department of Applied Chemistry, Nagoya Institute, Osaka University, Japan Teppei Takahashi, Department of Applied Chemistry, Nagoya Institute, Osaka University, Japan Yasuhiro Funahashi, Department of Applied Chemistry, Nagoya Institute, Osaka University, Japan Koichiro Jitsukawa, Department of Applied Chemistry, Nagoya Institute, Osaka University, Japan Hideki Masuda, Department of Applied Chemistry, Nagoya Institute, Osaka University, Japan

Biotin is a critical cofactor in biotin-dependent carboxylases, and operates the fi xation of carbon dioxide. Although the structure of biotin carboxylase has been characterized for its apo form, there is almost no information about detailed mechanism on the binding mode of biotin and the fi xation of carbon dioxide. In biotin-dependent carboxylases, Mg-H2O (aquo) species bound to urea oxygen is proposed as an active species[1]. In order to clarfy the participation of such metal species, we prepared a new zinc complex containing tripodal ligand, tris(6-amino-2-pyridylmethyl)amine (TAPA) (Scheme

1). Reaction of [Zn(tapa)(OH)]ClO4 (1) (Scheme 1-a) with CO2 gas gave a CO2-fi xation zinc complex 2 (Scheme 1-c). The structures of these complexes (1 and 2) were confi rmed by the X-ray crystallography and 1H- and 13C-NMR spectra, which indicated that the hydrogen bonding interaction between amino NH and hydroxide oxygen atom stabilizes Zn-OH species for complex 1. Our results suggested that, in biotin-dependent carboxylases, biotin-Mg-hydroxo or -aquo species, judging from examination using Zn-OH one (Scheme 1-a), plays very important roles in generation of carboxybiotin from biotin and carbon dioxide. [1] E. R. Sanchez, et al., J. Am. Chem. Soc., 124, 1933 (2002) 254 Journal of Inorganic Biochemistry 96 (2003)

Redox function and thermostability of various

P. aeruginosa cytochrome c551 mutants Yasuhiko Yamamoto, Department of Chemistry, University of Tsukuba, Japan Norifumi Terui, Department of Chemistry, University of Tsukuba, Japan Hajime Mita, Department of Chemistry, University of Tsukuba, Japan Yoshihiro Sambongi, Faculty of Applied Biological Science, Hiroshima University, Japan Hikaru Hemmi, National Food Research Institute, Japan Susumu Uchiyama, Graduate School of Engineering, Osaka University, Japan Yuji Kobayashi, Graduate School of Pharmaceutical Science, Osaka University, Japan Yasuo Igarashi, Department of Biotechnology, University of Tokyo, Japan Naoki Tachiiri, University of Tsukuba, Japan Shin-ichI Joseph Takayama, University of Tsukuba, Japan

Mesophile P. aeruginosa cytochrome c551 (Pa) and thermophile H. thermophilus cytochrome c552 (Ht) exhibit high sequence identity (56%), and their main-chain folding is almost identical. But Pa is considerably less stable than Ht. A series of Pa mutants, in which amino acid substitutions had been structurally selected with reference to the corresponding residues in Ht, exhibited increased stability compared with the wild-type Pa. Oxidized forms of the single mutant (F34Y) and quintuple mutant (F7A/V13M/F34Y/E43Y/V78I) in the presence of 1.5 M guanidine hydrochloride exhibited the denaturation temperatures of 66.4°C and 80.2°C, respectively, as opposed to the value of 50.4°C for the wild-type Pa under the identical conditions. Electrochemical and NMR spectral properties for the proteins that differ in the overall protein stability have been characterized in detail. We found that the redox potential (E°′) of the protein is correlated well with the protein stability and that the differences in the E°′ among the wild-type Pa and mutants are attributed predominantly to the enthalpic contribution to the E°′. Temperature-dependent appearance of the NMR spectra of both oxidized and reduced forms of the proteins facilitated a direct comparison of the thermostability of the heme active site among the proteins. The higher stability of the reduced form than that of the oxidized one, for a given protein, was confi rmed by the NMR study. Since only a small redox-dependent polypeptide conformation change has been reported for electron transfer proteins, the difference in the thermodynamic stability between the two different redox forms of the proteins should be attributed to structural properties of the heme active site, which are largely affected by the oxidation state of heme iron. The results strongly suggested that the stability of the heme coordination structure in the proteins regulates the thermodynamic stability of both the redox forms of the proteins, which in turn affects the E°′. Journal of Inorganic Biochemistry 96 (2003) 255

1H-NMR study of the binding of ternary platinum(II) complexes, Pt(NN)(AA) (AA=L-lysine or L-leucine; NN=1,10-phenanthroline, 5,6-dimethyl-1,10- phenanthroline, or 2,2’-bipyridine), with oligonucleotides

Kousuke Yamanaka, Department of Applied Chemistry, Chuo University, Japan Kazuhiko Motegi, Department of Applied Chemistry, Chuo University, Japan Yoh Matsuki, Department of Applied Chemistry, Chuo University, Japan Shin Morita, Department of Applied Chemistry, Chuo University, Japan Makoto Chikira, Department of Applied Chemistry, Chuo University, Japan

The orientation of mono(1,10-phenanthroline)copper(II) and the ternary complexes with various amino acids on DNA has been investigated by EPR spectra of the complexes on the oriented DNA fi bers (1). To refi ne the binding structure and to assess the base sequence specifi city, we have measured 1H-NMR spectra of several ternary platinum(II) complexes, Pt(phen)(AA)

(phen = 1,10-phenanthroline, AA = Lys or Leu), Pt(Me2phen)(Lys) (Me2phen = 5,6-dimethyl-1,10-phenanthroline) and

Pt(bpy)(Lys) (bpy = 2,2’-bipyridine) bound to self-complementary oligonucleotides, DO1 (d(CGCGAATTCGCG) 2) and

DO2 (d(CGCGTATACGCG) 2). The characteristic concentration dependence of the chemical shift and 2D-NOESY signals showed that Pt(phen)(Lys), Pt(Me2phen)(Lys), and Pt(phen)(Leu) intercalated between G2C11 and C3G10 base pairs of DO1 and between G4C9 and T5A8 base pairs of DO2. In contrast to the phenanthroline complexes, Pt(bpy)(Lys) bound to the AT region in the minor groove of DO1, inducing a small rise in the melting point. It was confi rmed that the methyl groups of

Me2phen enhanced the intercalative binding of the complex. The change in the chemical shift of the protons of amino acid side-chain was small and no intermolecular NOE cross peaks with the protons in the oligonucleotides could be observed for all the complexes employed, suggesting that the conformation of the side-chains are not fi xed on the DNA. (1) M. Chikira, et al., J. Inorg. Biochem. 89, 163-173, 2002.

Indole Radical Formation in a Pd(II) Complex Involving a Coordinated Indole Ring

Osamu Yamauchi, Faculty of Engineering, Kansai University, Japan Takeshi Motoyama, Faculty of Engineering, Kansai University, Japan Yuichi Shimazaki, Institute for Fundamental Research of Organic Chemistry, Japan Tatsuo Yajima, Faculty of Engineering, Kansai University, Japan Yasuo Nakabayashi, Faculty of Engineering, Kansai University, Japan

Metal-coordinated phenoxyl radical formation is well established and characterized for galactose oxidase, and a number of model systems have been reported. The tryptophan indole radical, on the other hand, has been reported for cytochrome c peroxidase and mutated ribonucleotide reductase protein R2, but little is known about formation and properties of the indole radical. We previously obtained a radical coupling product from two indole rings in the reaction of a Cu(I) complex having pendant indole moieties with dioxygen, but no indole radical species was detected.1 We here report two Pd(II) complexes, one of which gives the indole radical upon oxidation. A 2N1O-donor tripodal ligand, N-2-pyridylmethyl-N-2-hydroxy-4,6-di(tert-butyl)benzyl-2-aminoethyl-3-indole (Htbu- iepp) reacted with PdCl2 to give [Pd(tbu-iepp)Cl] as orange (1) and yellow (2) crystals depending on the reaction conditions. While the phenolate oxygen is bound to Pd(II) in 1, the indole ring instead of the phenolate moiety coordinates to Pd(II) through C(2) in 2, giving a planar 1C2N1Cl coordination structure. The cyclic voltammogram of 2 exhibited two irreversible oxidation peaks at 0.83 and 1.22 V (vs. Ag/AgCl). Upon oxidation with Ce(IV) in DMF complex 2 gave an ESR signal at g = 2.00 and an absorption peak at 550 nm (half-life, 20 sec.), which are characteristic of an indole π-cation radical. No such spectra were observed for 1, suggesting the possible effect of indole coordination through the carbon atom on radical formation in 2. 1 Y. Shimazaki, T. Nogami, F. Tani, A. Odani, and O. Yamauchi, Angew. Chem. Int. Ed., 40 , 3859-3862(2001). 256 Journal of Inorganic Biochemistry 96 (2003)

A New and Specifi c Detection Method for HPO42- and ATP in Aqueous Solution

Caixia Yin, Institute of Molecular Science, Shanxi University, China Fei Gao, Institute of Molecular Science, Shanxi University, China Pin Yang, Institute of Molecular Science, Shanxi University, China

The detection for phosphate and ATP in water is a challenging task. Now we have found a simple and sensitive detection 2- method for both HPO4 and ATP in aqueous solution. This new ensemble is prepared by mixing ytterbium chloride and pyrocatechol violet in a 2:1 molar ratio in aqueous solution of 10mM HEPES buffer at pH7.0. Upon the addition of [YbCl3], the maximum absorption peak gradually shifted from 444nm (yellow) to 623nm (blue). With the addition of phosphate or ATP solution into the system, the ensemble resulted in a change back of color from blue to yellow and caused a variation 2- in UV/Vis absorption spectra. The ensemble exhibits excellent selectivity for HPO4 and ATP over other common anions - 2- - 3- including Cl , SO4 , CH3COO and HCO . References and notes [1] Stephen J. Lippard and Jeremy M.Berg, Principles of Bioinorganic Chemistry, University Science Books, Mill Valley, California, 1994, p.133. [2] P. D. Beer and P. A. Gale, Angew. Chem. Int. Ed. 2001, 40, 486-516. [3] M. S. Han and D. H. Kim, Angew. Chem. Int. Ed., 2002, 41, 3809-3811. [4] D. A. Skoog and J. J. Leary, Principles of Instrumental Analysis, Saunders College Publishing, Harcourt Brace College Publishers, Orlando, Florrida, 1992. [5] F. A. Cotton and G.Wilkinson, Advanced inorganic Chemistry, 5th ed., John Wiley & sons, Inc, 1988. p. 959-960. Journal of Inorganic Biochemistry 96 (2003) 257

Denitrosyl Reaction of Linkage Isomers of Dinitrosyl-Molybdenum Complexes with 2-Pyrimidinethiolate Delivatives

Toshiaki Yonemura, Kochi University, Japan Tomoharu Ama, Kochi University, Japan Hiroshi Kawaguchi, Kochi University, Japan

In recent years, NO has become clarifi ed as an important biological molecule acting as a neurotransmitter, an immune system to kill tumor cells, and intracellular parasites. We have already reported the preparation and characterization of some dinitrosyl-molybdenum complexes with thiolate ligands.[1] Now, we investigate the properties and denitrosyl reaction of linkage isomers of dinitrosyl- molybdenum [Mo(didentate-N,S)2(NO)2]-type complexes, together with some X-ray crystal structures.

[MoL2(NO)2] {L = 4-amino-2-pyrimidinethiolate (apymt) 1, 4-methyl-2- pyrimidinethiolate (mpymt) 2, 4,6-diamino-2-pyrimidinethiolate (dapymt) 3, and 4,6-dimethyl-2-pyrimidinethiolate (dmpymt) 4} were newly prepared. The geometrical structures of the isomers 1 and 2 were assigned to the cis(NO,NO)- trans(S,S) confi guration on the basis of their UV-Vis, IR, and 13C NMR spectral data. For this confi guration, three pairs of linkage isomers, adjacent-adjacent, adjacent-remote, remote-remote, are possible. In the 1H NMR spectrum of 1, the broad peaks of two amino protons were observed at 7.65 and 7.61 ppm. As such difference of the chemical shifts refers to the existence of the weak intramolecular hydrogen bonding between N-H and S, 1 takes an adjacent-adjacent structure. While, 2 takes a remote-remote structure without the formation of hydrogen bonding. 3 also takes cis(NO,NO)-trans(S,S) confi guration and has also weak intramolecular hydrogen bonding. On the other hand, it is clear that 4 has cis(NO,NO)-trans(N,S) confi guration, as a result of the X-ray diffraction method

(Fig. 1). As dmpymt complex has a large steric hindrance due to the CH3} groups of dmpymt ligands, 4 hardly takes the cis(NO,NO)-trans(S,S) confi guration. The reaction of 1-4 with PPh3 was examined. The decrease in the absorbance of the MLCT band and the disappearance of one of two NO stretching bands observed in the starting complexes indicates the elimination of one of the coordinated NO ligands. It was proven that unexpected compound [{(ON)MoL2}2(µ-OH)2], which has two seven-coordinated Mo ions, and Ph3PO were formed. The details of these structures and properties are also reported. [1] T. Yonemura, et al., Inorg. Chem. Commun., 2001, 4, 661. 258 Journal of Inorganic Biochemistry 96 (2003)

Relationships between Tetramer Formation, Heme Incorporation and Catalysis of a Heme-regulated Phosphodiesterase from Escherichia coli: A Study of Deletion Mutants

Tokiko Yoshimura, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Japan Ikuko Sagami, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Japan Yukie Sasakura, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Japan Toru Shimizu, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Japan

A heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is tetrameric. Each monomer is composed of an N-terminal sensor domain (amino acids: 1-201) containing two PAS domains (PAS-A: 21-84 and PAS-B: 144-201) and a C-terminal catalytic domain (336-807). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. We examined the relationships between tetramer formation, heme binding and PDE activity using deletion mutants. Deletion of the PAS-B domain resulted in loss of heme binding affi nity to PAS-A. However, the mutant protein was still tetrameric and signifi cantly active compared to its wild-type counterpart, suggesting that the PAS-B domain plays an important role in modulating the appropriate structure of the PAS-A domain to maintain binding to the heme and that heme binding is not essential for catalysis. An N-terminal truncated mutant (amino acids: 148-807) was active, confi rming that the heme-bound PAS domain is not essential for catalysis. C-terminal truncated mutants (amino acids: 1-490 or 1-705) were monomeric, indicating that specifi c amino acids located within the deleted regions are responsible for tetramer formation. In view of the fi nding that the isolated PAS-A domain is dimeric, these amino acids in the C-terminal region appear to play a major role in enzyme architecture. One C-terminal truncated mutant (1-490) did not display any PDE activity, whereas another mutant (1-605) was active, indicating that the catalytic site is located between positions 491 and 605. In addition, tetramer formation appears to be important in Ec DOS catalysis.

A Non-Radical Mechanism for Methane Hydroxylation at the Diiron Active Site of Soluble Methane Monooxygenase

Kazunari Yoshizawa, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan Takashi Yumura, Institute for Fundamental Research of Organic Chemistry, Kyushu University, Japan

In this presentation we propose a non-radical mechanism for the conversion of methane to methanol by soluble methane monooxygenase (sMMO), the active site of which involves a diiron active center. We assume the active site of the MMOHQ intermediate that has a direct reactivity to substrate methane to be a bis(µ-oxo)diiron(IV) complex where one of the iron atoms is coordinatively unsaturated (fi ve-coordinate). Is such a diiron complex reasonably formed in the catalytic reaction of sMMO? The answer to this important question is positive from the viewpoint of energetics in density functional theory (DFT) calculations. Thus, our model has a vacant coordination site for substrate methane. If MMOHQ involves a coordinatively unsaturated iron atom at the active center, methane is effectively converted into methanol in the broken-symmetry singlet state in a non-radical mechanism; in the fi rst step a C-H bond of methane is dissociated via a four-centered transition state (TS1) resulting in an important intermediate that involves a hydroxo ligand and a methyl ligand, and in the second step the binding of the methyl ligand and the hydroxo ligand via a three-centered transition state (TS2) results in the formation of a methanol complex. This mechanism is essentially identical to that of the methane-methanol conversion by the bare FeO+ complex and relevant transition-metal-oxo complexes in the gas phase. Neither radical species nor ionic species are involved in this mechanism. We look in detail at kinetic isotope effects (KIEs) for the H-atom abstraction from methane on the basis of transition state theory with Wigner tunneling corrections. Journal of Inorganic Biochemistry 96 (2003) 259

Cavity-Directed Syntheses within Self-Assembled Coordination Cages

Michito Yoshizawa, Department of Applied Chemistry, School of Engineering, Japan Makoto Fujita, Japan

Selective encapsulation and isolation of molecules by self-assembled discrete space are one of the most attractive features of inorganic cage-like molecules as well as biological superstructures. If two or more substrates are effi ciently encapsulated in the self-assembled cage, intermolecular chemical transformation can be remarkably accelerated and suitably controlled owing to the dramatically increased concentration and the strictly regulated orientation of substrates in the cavity. Recently we reported that structurally well-defi ned coordination cages, which self-assemble from six metals and four tridentate ligands, selectively encapsulate large organic molecules at the fi xed position of the nano-sized cavities. In the present paper, we will introduce their utilization for highly selective chemical reactions such as (i) the polycondensation of trialkoxysilanes and (ii) the intermolecular [2+2] photodimerization of large olefi ns within the cavity. These results are referred to as ‘cavity-directed synthesis’, where confi ned space strictly controls the encapsulation by steric nature, and the subsequent chemical transformation can be strictly controlled as desired. Ref. (i) M. Yoshizawa, T. Kusukawa, M. Fujita, S. Sakamoto, K. Yamaguchi, J. Am. Chem. Soc. 2001, 123, 10454. (ii) M. Yoshizawa, Y. Takeyama, T. Kusukawa, M. Fujita, Angew. Chem. Int. Ed. 2002, 41, 1347 & M. Yoshizawa, Y. Takeyama, T. Okano, M. Fujita, J. Am. Chem. Soc. 2003, 125, in press. 260 Journal of Inorganic Biochemistry 96 (2003)

Synthesis of Ruthenium-Platinum Di- and Tri- nuclear Complexes

Sarah Yousouf, University of Western Sydney, Australia Craig R Brodie, University of Western Sydney, Australia Janice R Aldirch-Wright, University of Western Sydney

The effi ciency of platinum complexes as anticancer agents has been established in a number of animal tumour models and in human cancer.1 The effectiveness of ruthenium(II) complexes as chemotherapeutic agents is restricted due their relatively small size, low cytotoxicity and modest DNA binding affi nity limits. This work aims to combine effective chemotherapeutic action of platinum complexes with the useful luminescent properties of ruthenium complexes. Work so far indicates that: • The binding affi nity that this complex exhibits will be greater than the mononuclear equivalents.2,3 • The addition of fully coordinated platinum will increase the binding selectivity for the minor groove.4

• Ruthenium complexes incorporating 4,4’-dimethyl-2,2’-bypyridine (Me2bpy) have been demonstrated to bind to DNA at adenine bulge sites with an affi nity considerably greater than that for standard duplex DNA.5

We report the synthesis and characterisation of the di- and tri- nuclear complexes [(Me2bpy)2Ru(5-phen-(NH2-(CH2)6- 4+ NH2)-Pt(NH3)2-(NH2-(CH2)6-NH2))] (Ru-Pt) and [(Me2bpy)2Ru(5-phen-(-(NH2-(CH2)6-NH2)-Pt(NH3)2-(NH2-(CH2)6-NH2) 6+ -)-5-phen)Ru(Me2bpy)2] (Ru-Pt-Ru) (where phen = 1,10-phenanthroline) (see Figure). The problem of low affi nity at high ionic strengths2,3 previously shown for metal complexes with a single metal centre should be overcome. As a result biological activity should be substantial and the complex will be suited to in vivo conditions. We report the synthesis and characterisation of these metal complexes as well as any initial studies of their interactions with DNA. (1) Natile, G.; Coluccia, M. Coordination Chemistry Reviews 2001, 216-217, 383-410. (2) O’Reilly, F. M.; Kelly, J. M. J. Phys. Chem. B 2000, 104, 7206-7213. (3) O’Reilly, F.; Kelly, J.; Mesmaeker, A. K.-D. Chem. Commun. 1996, 1013-1014. (4) Moniodis, J. J.; Thomas, D. S.; Berners-Price, S. J.; Hegmans, A.; Farrell, N. In Conference of the Inorganic Chemistry Division Royal Australian Chemical Institute: The University of Melbourne, 2003, p 99. (5) Patterson, B. T.; Collins, J. G.; Foley, F. M.; Keene, F. R. J. Chem. Soc., Dalton Trans. 2002, 23, 4343-4350. Journal of Inorganic Biochemistry 96 (2003) 261

Studies on planaramineplatinum(II) with trans-geometry

Jun Q Yu, University of Sydney, Australia Fazlul Huq, University of Sydney, Australia Philip Beale, Sydney Cancer Centre CSAHS, NSW, Australia

Although cisplatin is a widely used anticancer drug, it has a number of side-effects and also cancer cells develop resistance to the continued use of cisplatin. In an attempt to reduce toxicity and widen the spectrum of activity thousands of cisplatin analogues have been prepared and tested by varying the nature of the labile and non-labile ligands. However, it is found that all cisplatin analogues essentially have similar spectrum of activity and develop similar resistance. The reason why transplatin is toxic rather than being anticancer active is believed to be associated with its higher reactivity than cisplatin. It is therefore thought that the introduction of sterically hindered ligands may reduce the reactivity of the trans-complexes suffi ciently so as to result into tumour-active compounds. One such class of compound is transplanaramineplatinum(II) complexes. The aim of this project is to prepare new trans-planaramine platinum(II) complexes of the type trans-Pt(NH3)LCl2 where L stands for 2-hydroxypyridine, 3-hydroxypyridine, imidazole, and imidazo(1,2-a)pyridine and determine the activity of the designed complexes against a number of cancer cell-lines and also to quantify the nature of nteraction with DNA. Four new transplanaramineplatinum(II) complexes have been synthesizd using modifi ed Dhara and Kelland methods. Interaction with DNA has been studied using gel-electrophoresis, HPLC, AAS, UV-visible spectrophotometry and restriction enzyme digestions. Activity against a number of cisplatin-responsive and cisplatin-resistant cancer cell-lines has been determined using MTT assay.It has been found that all of the designed complexes have activity somewhat less than that of cisplatin. However, the compounds have resistance factors much lower than that of cisplatin as applied to cisplatin-resistant cell lines. For all the compounds, cell accumulation is found to be similar or greater than that of cisplatin. This poster will describe the results of the studies.

Over-expression of High Quality pMMO in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor

Steve S-F Yu, Institute of Chemistry, Academia Sinica, Taiwan, Province of China Kelvin H C Chen, Institute of Chemistry, Academia Sinica, Taiwan, Province of China Sunney I Chan, Institute of Chemistry, Academia Sinica, Taiwan, Province of China

Particulate methane monooxygenase (pMMO) was over-expressed in the methanotroph Methylococcus capsulatus (Bath) at high copper-biomass ratios in NMS buffer. It is well known that this methanotroph has diffi culty surviving at copper concentrations in the range of 10 to 20 mM because of the instability of the system under these conditions. On the other hand, the copper ions become toxic to the cells at concentrations higher than 50 mM. In order to obtain pMMO-enriched membranes with high activity and in high yields, we have devised a method to process the cell growth in a fermentor adapted with hollow-fi ber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in the NMS medium over the time course of the culturing of the cells. This technical improvement in the method of culturing bacterial cells has allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes as well as the specifi c activity of the enzyme. The optimal copper concentration in the growth medium is shown to be 30-35 mM. Under these conditions, the pMMO is over-expressed, accounting for 90% of total cytoplasmic membrane proteins, and with a specifi c activity of 88.9 nmoles propylene/min mg protein with NADH as the reductant. The copper stoichiometry is 12-13 per pMMO. Analysis for other metal contents has provided no evidence for zinc, and only traces amounts of iron present in the pMMO-enriched membranes. Finally, it is apparent on the basis of this study that Methylococcus capsulatus (Bath) expresses the membrane-associated methane monooxygenase with multi-functionality. One of the functions is suspected to associate with NADH for reduction of the copper catalytic centers and electron transfer domains. The other function is to convert the ubiquinone to ubiquinol for the use of down stream pathways of the respiratory chain in these bacteria. 262 Journal of Inorganic Biochemistry 96 (2003)

Electron Transfer in Tyrosyl Radical Formation in the R2 Subunit of Mouse Ribonucleotide Reductase

Danny Yun, The Pennsylvania State University, United States Lana Saleh, The Pennsylvania State University, United States Boi Hanh Huynh, Emory University, United States J Martin Bollinger, The Pennsylvania State University, United States

Our previous studies on the mechanism of tyrosyl radical (Y177•) and µ-(oxo)diiron(III) cluster formation in the R2 subunit of ribonucleotide reductase from mouse established that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177• in the reaction sequence and is probably the Y177 oxidant. In the E. coli R2 cofactor assembly reaction, cluster X formation proceeds by the transfer of an electron from the near-surface residue tryptophan 48 (W48) to the initial O2 adduct to the buried diiron cluster. Substitution of W48 or its cognate in mouse R2 (W103) has been shown to disable electron transfer, but published data would suggest that the mechanisms of the electron-transfer-defi cient variants of E. coli and mouse R2 differ markedly. To understand the basis for these differences, we have re-examined the mechanism of Y177• formation in two electron-transfer-defi cient variants of mouse R2, R2-W103Y and R2-D266A. Preliminary results suggest, in contrast to previous proposals, that either addition of O2 to the diiron(II) cluster becomes rate-limiting or that electron transfer is rate-limiting and O2 addition is highly reversible in these variant proteins. Ongoing kinetics experiments will allow these possibilities to be distinguished.

An examination of the aquation kinetics and equilibria of dinuclear platinum anticancer agents

Junyong Zhang, Chemistry, School of Biomedical and Chemical Sciences, University of Western Australia, Australia Donald S Thomas, Chemistry, School of Biomedical and Chemical Sciences, University of Western Australia, Australia Murray S Davies, School of Pharmacy and Molecular Sciences, James Cook University of Townsville, Australia Susan J Berners-Price, Chemistry, School of Biomedical and Chemical Sciences, University of Western Australia, Australia Nicholas Farrell, Department of Chemistry, Virginia Commonwealth University, United States

2+ The dinuclear platinum complexes 1,1/c,c (n=6) and 1,1/t,t (n=6), ([{cis/trans-PtCl(NH3)2}2(µ-NH2(CH2)6NH2)] ) belong to a family of anticancer agents including the trinuclear platinum complex BBR3464 (1,0,1/t,t,t n=6,6) which is currently undergoing clinical trials. To understand the DNA binding interactions of these platinum complexes it is crucial to fi rst understand their simple aquation chemistry. [1H, 15N] HSQC NMR spectroscopy has been used to investigate the hydrolysis profi le of these compounds under conditions of different ionic strength and temperature and in the presence of phosphate or acetate. Rate and equilibrium constants for the aquation reactions have been determined, together with the acid dissociation constants for the aquated species. For 1,1/ c,c (n=6) the equilibrium lies further towards the aquated species than occurs for the related dinuclear complex 1,1/t,t (n=6) under the same conditions.1 The pKa value of the aquated compound is signifi cantly higher for 1,1/c,c (n=6) (pKa =5.99) than for 1,1/t,t (n=6) (5.62) or BBR3464 (5.63). The fi nal product profi le obtained in the reaction of 1,1/c,c (n=6) with phosphate and acetate yield substantially more phosphato- and aceto- bound species as compared to 1,1/t,t (n=6). The authors thank the Australian Research Council, U.S. National Institute of Health, U.S. National Science Foundation and the American Cancer Society for fi nancial support. 1 Davies, M. S., Cox, J. W., Berners-Price, S. J., Barklage, W., Qu, Y and Farrell, N., Inorg. Chem., 39, 1710-1715 (2000). Journal of Inorganic Biochemistry 96 (2003) 263

New Mechanism for Metal Capture by a Bacterial Protein

Haizhong Zhu, University of Edinburgh, United Kingdom Dmitriy Alexeev, University of Edinburgh, United Kingdom Dominic J Campopiano, University of Edinburgh, United Kingdom Peter J Sadler, University of Edinburgh, United Kingdom

Iron uptake by bacteria involves a highly-conserved 34 kDa periplasmic iron transporter Fbp – ferric-ion binding protein. We have recently discovered1 that the characteristic dityrosyl motif of Fbp from the human pathogen Neisseria gonorrhoeae can assemble/bind metal clusters in the open metal-binding cleft of the protein. Closure of the hinged metal-binding cleft and the presence of the synergistic phosphate anion are usually considered to be obligatory for strong metal binding by Fbp.2 We observe that the phosphate ion in the crystal structure of apo-Fbp (Figure) can be displaced from 2- the phosphate binding site by mononuclear [Hf(NTA)2] , yielding a range of oxo Hf(IV) clusters containing from 3 to 5 metal ions. Structural comparison between the crystal structures of apo-Fbp and the Hf-bound form of Fbp shows no hinge closure upon metal binding, suggesting a novel metal acquisition mechanism. We report the crystal structure of the apo-form of Fbp together with data on reloading the protein with Fe(III), and discuss the possibility that bacterial metal uptake is mediated by novel Fbp intermediates. References 1. D. Alexeev, H. Zhu, M. Guo, W. Zhong, D.J.B. Hunter, W. Yang, D.J. Campopiano, P.J. Sadler Nat. Struct. Biol. 2003, 10, 297-302. 2. M. Guo, I. Harvey, W. Yang, L. Coghill, D.J. Campopiano, J.A. Parkinson, R.T.A. MacGillivray, W.R. Harris and P.J. Sadler J. Biol. Chem. 2003, 278, 2490-2502. We thank The Wellcome Trust (Edinburgh Protein Interaction Centre), The Darwin Trust (Fellowship for DA) and CVCP (ORS award for HZ) for support. We also thank the University of Edinburgh Development Trust for assist HZ with attendance at ICBIC11. 264 Journal of Inorganic Biochemistry 96 (2003)