Journal of Inorganic Biochemistry 86 (2001) 401

Biological control on trace element partitioning into biogenic calcites

Rosali_nd E. M. Rickaby a & Daniel P. Schrag a " Department of Earth and Planetary Sciences, Harvard University, 20, Oxford Street, Cambridge,

MA 02138, USA (e-mail: rickaby@fas.harvard, edu)

The concentration of trace elements (e.g. Sr, Cd, Ba and Zn) in marine biogenic calcites has the potential to act as a tool to probe past ocean conditions and related climate change. The concentration of a trace element such as Sr in the calcite depends on two factors: the oceanic concentration of St, and the partitioning of Sr between seawater and the biogenic calcite. We challenge the traditional view that the partitioning of trace elements occurs according to thermodynamic predictions for inorganic calcite and propose that the chemistry of the biogenic calcite is instead conlxolled by the biological calcification process. As an example, the Sr/Ca of coccolithophore calcite from contrasting areas of the ocean shows significant variations despite the uniform concentration of Sr and Ca within the modem ocean. We investigate the role of Sr during calcification with culture experiments of the coccolithophore Emiliania huxIeyi. The hypothesis is tested that because the calcite liths are constructed in an intracellular vesicle, the biological discrimination between the similar Sr 2. and Ca-'* ions by CaZ+-selective channels and pumps, or the organic template, is the dominant control on the chemistry of the calcite 1. An understanding of environmental controls on the biological discrimination between Ca and trace elements during calcification is vital for using these paleoceanographic tools to record past climates.

1. Rickaby R. E. M., Zondervan I., Riebesell U., and Schrag D. P., submitted to Global Biogeochemical Cycles, 2000.

Dioxygen activation by a nickel thioether complex: characterization of a Nim2(~-O)2 core

Charles G. Riordan. Beaven S. Mandimutsira, Jennifer L. Yamarik Department of Chemistry and Biochemistry, University of Delaware, Brown Laboratory, Newark,

DE 19716, USA (e-mail: riordan@udel.edu)

Oxygenation of the Ni(I) complex [PhTfBU]Ni(CO) 1, yields thermally sensitive [(PhTfnU)Ni]z(~t-O)2 2, Scheme, Based on its collective spectroscopic properties, 2 is formulated as a bis-p-oxo dimer. The complex is deep purple, a consequence of two intense charge transfer bands at 410 nm (e = 12,000 M-~cm -1) and 565 nm (16,000 M-~cm-l). Resonance Raman spectra show the higher wavelength optical band (2ex = 590 nm) is coupled to an lSOz-senstive vibrational mode at 585 cm 1 (Av (1802) -30 cm-l). Analogous vibrational modes have been characterized in Mn, Fe and Cu structures and ascribed to the symmetric stretching mode of the bis-~t-oxo core, Mz(~t-O)2. The low temperature IH NMR spectrum of 2 indicates a diamagnetic species in support of the binuclear structure (electronically-coupled core). Nickel K-edge XAS confirms the Ni(III) oxidation state and the binuclear structure, Ni---Ni, 2.82 A. While this structural motif is precedented, 2 represents the first of its type derived from O2 in nickel chemistry. Preliminary density functional theory calculations and reactivity studies of 2 will be reported.

/ - \ • % . . . 4 , i '~-CO

[PhTtreU]Ni(CO)~ 4

0 2

t ~uene - 7 8 ° C

[(PhTttBU)Ni]2(P-O)2 , 2

402 Journal of lnorganic Biochemistry 86 (2001)

The structure of metal adducts of anthracyclines probed by absorption, circular dichroism and paramagnetic NMR

Silvia Ripoli" Lorenzo Di Bali, a Guido Pintacuda, a'b and Piero Salvadori a

"Centro di Studio de/CNR per le Macromolecole Stereoordinate ed Otticamente Attive, Dipartimento di Chimica e Chimica lndustriale, Via Risorgimento 35, 1-56126 Pisa, haly.( e-mail.silviar@dcci.unipi.i 0 Scuola Normale Superiore, Piazza dei Cavalieri 7, 1-56126 Pisa, Italy

A structural study of metal ions adducts of a new disaccharide anthracycline (MEN 10755) is undertaken. The trivalent lanthanide ion, Yb(III), was employed as paramagnetic structural probe for JH-NMR analysis. Through a comparative spectroscopic investigation (UV-Vis absorption and CD, IH-NMR), the isomorphism between its adduct with lanthanide ions (La +3, Yb .3, Lu .3) and calcium (one of the most representative biological cations) was verified. Solution behavior and cation binding were also investigated by means of optical titrations. In agreement with other anthracyclines, MEN 10755 was found to dimerize in aqueous solution (estimated g4~m [pH 7.6] = 7 • 103), but not in methanol. A prevalent complex yb+3/MEN 10755 1:I both in buffered aqueous and methanolic solutions (estimated Kcompl = 2100 M ~) was observed. A numerical analysis of the LIR and LIS IH-NMR literature data of a similar adduct (yb+3/daunorubicin) was performed by a newly developed software, PERSEUS (Paramagnetic Enhanced Relaxation and Shifts for Eliciting Ultimate Structures), and the structure of the complex was characterized, locating definitely the binding site on the O-11, O-12 quinone system, The components of the anisotropic part of the magnetic susceptibility tensor were determmed, as well. Finally, a study of the time dependent formation of an yb+3/MEN 10755 complex through 1H-NMR, UV-Vis CD and induced NIR CD was carried out.

Coupling of electron transfer and conformational transitions in cytochrome c immobilised on hydrophobic surfaces

L a u r a R i v a s a, Dan ie l H. M u r g i d a a and Pe te r H i ldeb rand t a

a lns t i tu to de Tecnologia e Bioquimica, Universi ta Nova de Lisboa, Rua da Quinta Grande Apartado 127, P-2780 Oeiras Por tuga l (e-mail." l r ivas@itqb .unl .pO

Cyt0chrome c (Cyt-c) serves as an electron-transferring protein in the respiratory chain of aerobic organism. Prior to the electron transfer to its partner protein, cytochrome c oxidase (CcO), a protein complex is formed that is primary stabilised via electrostatic interactions between the lysine-rich domain around the heine crevice of Cyt-c and an aniomc region on the protein surface of CcO. However, it was shown that also hydrophobic forces play an important role in the interactions of Cyt-c with CcO, either for a proper alignment of the redox sites or for promoting conformational changes that are functionally relevant for the redox process. Thus, it is of particular interest to analyse in more detail the effect of hydrophobic interactions on the heine pocket structure and electron transferring properties of Cyt-c.

In this study we have addressed this issue by employing surface-enhanced resonance Raman (SERR) spectroscopy, which selectively probes the heme sites of those proteins that are bound to coated silver electrodes. Using self-assembled monolayers (SAMs) coatings prepared by n-alkanethiols, binding of Cyt-c occurs predominantly via hydrophobic interactions and is associated with structural changes in the heme pocket of the oxidised protein. Specifically, the native Met-80 ligand is replaced that is directly linked to the hydrophobic patch in the putative binding domain. Potential- dependent SERR studies reveal that lowering the potential leads to the conversion of the bound Cyt-c to the native reduced state, indicating a coupling of electron transfer and conformational transition, i.e., the restoration of the native axial ligation pattern. The dynamics of these processes were studied by time-resolved (TR) SERR spectroscopy in order to analyse the underlying reaction mechanisms in more detail. The TR-SERR spectra provide no indication for contribution from the oxidised form of the native state of Cyt-c, suggesting that its relative contribution must be very small during the redox process. The kinetic analysis reveals that the conformational transition step is slow compare to the electron transfer process, becoming rate limiting with increasing the driving force.

Journal of Inorganic Biochemistry 86 (2001) 403

Solid-phase synthesis of peptide-tethered platinum(II) complexes

Marc Robillard, Sophie van Alphen, Elise van der Hout, A. Rob P. M. Valentijn, Nico Meeuwenoord, Gijs van der Marel, Jacques van Boom, Jan Reedijk Leiden Institute of Chemistry, Leiden University P.O. Box 9502, 2300 RA Leiden, The Netherlands, (e-mail: Robillar@chem. leidenuniv, nl)

Recently, we reported for the first time a solid-phase synthesis of a trimeric arginine-containing peptide-dichloroplatinum(II) complex [1]. An ~mmobilized arginine-glycine dipeptide tethered to an ethylenediamine moiety was platinated, and the resulting peptide-dichloroplatinum complex (1) was deprotected and released from the solid support. The finding that the reactive dichloroplatinum moiety is stable under the deprotection and cleavage conditions opens the way to a rapid synthesis and simple purification of a wide variety of platinum complexes via a combinatorial synthesis approach, as well as the preparation of oligopeptide-platinum

HN

H2N'~NH2 * AcO

conjugates designed to target specific cells or DNA regions. To investigate this potential, we explored the introduction of diversity in the platinum chelating moiety, the appended peptide and the platinum coordination mode. This work and methods to evaluate the platination on the solid support will be discussed.

~.. M. S. Robillard, A. R. P. M. Valentijn, N. J. Meeuwenoord, G. A. van der Marel, J. H. van Boom, J. Reedijk, Angew. Chem. Int. Ed. 2000, 39, 3096-3099. This research has been sponsored by the Netherlands Foundation for Technical Sciences (STW, project 349-4427). The authors are indebted to the EU for a grant as Host Institute in the EU Program Human Capital and Mobility. Sponsorship by COST is kindly acknowledged. The authors wish to thank Johnson & Matthey (Reading, UK) for their generous loan of K2PtCI4.

Influence of familial ALS-associated mutations on the thermal stability of distinctly metallated species of human superoxide dismutase

Jo rge A. R o d r i g u e z a, J o a n S e l v e r s t o n e Va len t i ne a, D a r y l K. E g g e r s a, J a m e s A. R o e b, R o b e r t H.

B r o w n , Jr c, and L a w r e n c e J. H a y w a r d d

" Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California 90095, USA (e-mail. jar@chem.ucla.edu) Department of Chemistry and Biochemistry, Loyola Marymount University, Los Angeles, California 90045, USA Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA

,1 Department of Neurology, University of Massachusetts Medical School, Worcester, Massachusetts' 01655, USA

The thermal stability of as-isolated wild type (WT) and of specific FALS associated mutant copper-zinc superoxide dismutases (SODls) was measured by differential scanning calorimetry (DSC). Reconstitution experiments upon the metal free (apo) WT enzyme were carried out to assign the transitions observed in the DSC profile of the SOD 1 variants. Cu 2÷, bound to the copper site of as-isolated WT and mutant A4V enzymes, was reduced to Cu +~ with dithionite to confirm the assignment of a transition that arose from proteins containing copper. WT and a subset of 8 mutant SODls (A4V, L38V, G41 S, G72S, D90A, G93A, and E133A) bound substantial amounts of copper and zinc (0.16-0.74 and 0.84- 1.77 equivalents per dimer, respectively) when compared to five mutants (H46R, G85R, D 124V, D 125H and S 134N) that were markedly deficient in copper and zinc (0-0.15 and 0-0.36 equivalents per dimer, respectively). Despite these differences in metal binding capabilities, we found that all mutant apo-proteins are less stable (M°C to 12°C) than the WT apo enzyme. Results show that either zinc or copper binding increase thermal stability to a comparable extent (M4°C to 16°C and -22°C to 24°C, respectively) in the WT and the former set of mutants. Transitions whose origins remain unassigned were observed in the DSC profile of D124V, G85R and D125H. We speculate that unfolding of either apo monomers, mis-folded apo dimers, or apo forms with a reduced disulfide bond give rise to the transitions in the DSC profile of D124V and G85R. In contrast to all the other mutants studied, zinc and copper binding yielded a greater stabilization of H48Q, 19.3°C and 26.3°C, respectively. Although the Tms for apo-H48Q and WT apoSOD are similar, apo-H48Q exhibited a broader transition that may include a less stable species. In general, all ALS-associated SOD1 mutant apo proteins were less stable than the WT apo form.

Supported by the NIH and ALS Association.

404 Journal of Inorganic Biochemistry 86 (2001)

Biogenesis of the Tyr-Cys cofactor and the role of the pro-sequence in the copper- containing tyrosyl radical enzyme galactose oxidase

Melanie Rogers a, Susan Firbank b, Peter Knowles b, Mike McPherson b, Simon Phillips b, Dave Dooley a ~Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA (e-mail.- mrogers@copper, ehemistry.montana.edu), bAstbury Centre for Structural Biology,

University of Leeds, Leeds, LS2 9JT, UK.

Biogenesis of organic cofactors via the post-translational modification of intrinsic amino acid residues is currently generating great interest in bioinorganic chemistry. One such novel cofactor is found in galactose oxidase, where the side chains of Y272 and C228 are autocatalytically crosslinked on exposure to J ""~, copper (II) and dioxygen, generating a thioether bond z. Galactose oxidase expressed in either Aspergillus nidulans or Pichia pastoris under rigorous metal-free conditions generates an unprocessed protein form containing a 17 anfino acid N-terminal pro-sequence which also lacks the Tyr-Cys cofactor. The crystal structure of this form has been determined (see S. Firbank abstract) 2. / The rate of formation of the Tyr-Cys moiety on addition of CuSO4 in the presence of excess ("~1(II) dioxygen has been monitored via optical spectroscopy. The possible occurrence of a tyrosyl radical during processing has been investigated using EPR spectroscopy. The anaerobic copper complex has been characterized using several spectroscopic techniques to provide details on the geometry and electronic structure of the copper site prior to processing. A second form of galactose oxidase lacking the pro-sequence has been over-expressed in Pichia pastoris under metal-free conditions. This form has allowed the role of the pro-sequence in the generation of the Tyr-Cys cofactor to be probed via optical, CD and fluorescence spectroscopy. 1. Rogers et al. JACS 122:990-991 (2000). 2. Firbank et al. PNAS In press (2001)

Chemical and biological characterization of some dicarboxilate platinum complexes

Anna Romaniewska, Janina Kuduk-Jaworska, Katarzyna Waszkiewicz Faculty of Chemistry University of Wroctaw; 14 F. Joliot-Curie St.; 50-383 Wroctaw; Poland (e-mail. aniar@wchuwr, chem. uni. wroc.pl)

According to the common opinion about less toxicity of the moderately labile platinum complexes, we synthesised the neutral complexes with general formula [cis-Pt(A)2X], where A = ethylenediamiane or 1-alkylimidazole and X = L-, D-, or DL-malate dianion. The chelating anionic malate ligands were chosen because of they cause a weak susceptibility to hydrolysis and other substitution reactions.Complexes were studied using chemical analysis, HPLC and spectroscopic methods: NMR, IR, Raman, UV-Vis. (1),(2) Potential toxic properties were estimated using Elman's test (checking reactivity with glutatione) and with ATP-ases, which play important role in renal function. The complexes are well soluble in water and in vitro exert significant antiproliferative activity. (3) They seem to be helpful in radiotherapy. (4) In vivo studies on BDFl-mice were carried out on chosen complexes. One of tested compounds, (bis(l- ethyloimidazole)(L)malatoplatinum(II)), revealed significantly less toxicity than carboplatin. Further in vivo studies are in progress.

1 J.Kuduk-Jaworska, K. Waszkiewicz, Trans. Met. Chem.; 25; 4, 2000. 2 H. Baranska, J.Kuduk-Jaworska, K. Waszkiewicz, J. Mol. Struct.; 550-551; 2000. 3 A.Opolski et al.; Anti-Cancer Drugs; 11; 2000. 4 J.Kuduk-Jaworska, et al., Chem.-Biol. Interact.; 129; 2000.

We acknowledge receipt of financial support from the KBN, Grant No 3T09A 12619.

Journal of Inorganic Biochemistry 86 (2001) 405

Synthesis of l igands des igned for the formation of axial phenolate -meta l bonds as model for Galactose oxidase

Stela Marls de Moraes Romanowski, Francielen Tormena and Antonio Sfilvio Mangrich Department of Chemistry, Universidade Federal do Paran6(UFPR) Caixa Postal 19081, CEP.-

81531-990, Curitiba, Paran6-Brazil, Fax.55-41-3613186, (e-mail.stela@quimica.ufpr. br)

Metals at metallobiosites are often found in environments chemically or geometrically distinct, so unsymmetrical mononucleating ligands are desirable targets for modelling studies concerning to those metal centers. The geometry around the monomeric copper(II) center in galactose oxidase (GOase) can be described as square pyramidal comprised by one tyrosyl free radical oxygen atom, two histidine nitrogen atoms and one acetate oxygen atom at the plane and by a ~rosine oxygen atom occupying the apical position of the pyramid. In order to obtain a closer ..... theti i°site m Oase ave ma e t° p e are ree I

ligands with their respective complexes with an axial phenolate oxygen-copper(II) bond and .... ~ . . . . with phenolic moieties substituted that could influence the redox properties of the models. In this work, we described the preparation and characterization of two substituted free -,,o._FX_z,2,2 I unsymmetrical mononucleating ligands belonging to a same series - H3BPETEN: N,N'-bis(2- ] ~c-&-o hydroxybenzyil)-N-(2-pyridylmethyl)-N'-(2-hydroxyethyl)-ethane- 1,2-diamine and I ~ . . . . I H3BNBPETEN: N•N•-bis(2-hydr•xy-5-nitr•benzy•)-N-(2-pyridy•methy•)-N•-(2-hydr•xyethy•)-ethane-••2-diamine - and of their respective copper(II) complexes - [Cu(H3BPETEN)](NO3)2 and [Cu(HNBPETEN)]. The complexes were characterized by conductivimetry, electrochemistry, IR, UV-Vis and EPR spectroscopies. From our results, we observed that the most easily one-electron oxidizable ligand is H3BPETEN that have not substitutions by an electron-withdrawing group such as NO2 in H3BNBPETEN. So, the complex [Cu(HNBPETEN)] had a reduction potential more catodically shifted than [Cu(H3BPETEN)](NO3)2. The former complex also presented a stronger ligand field observed by the EPR spectroscopy ( A / = 174 vs. 118 G ) which is in agreement with the more energetic dI-ld transition found in its electronic spectrum. UFPR/TN, PADCT

A bacterioferri t in from the sulphate reducing bacter ium Desulfovibrio desulfuricans A T C C 27774, with a novel haem cofactor.

C.V. Romao,. a; da Costa, P.N. a; Macedo, S. a,b; Mitchell, E. b; Matias, P.M. a; Melo, E. a; Liu, M.-Y. b, Xavier, A.V. a; Lindley, P. b; LeGall, J. a,c., Carrondo, M.A. a; Saraiva, L.M a., Teixeira, M. a

Instituto de Tecnologia Quimica e Biol6gica, Universidade Nova de Lisboa, Apt 127, 2780-156 Oeiras, Portugal (e- mail. cmromao@itqb.unl.pO

ESRF, BP-220, F-38043 Grenoble Cedex, France c Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA

A bacterioferritin was isolated from the anaerobic bacterium Desulfovibrio desulfuricans ATCC 27774 ~. It contains a haem quite distinct from the haem B: iron-coproporphyrin III, the first example of such a prosthetic group in a biological system 2. The haem has a reduction potential of +140mV (pH 7.6), a value unusually high compared to that of other bacterioferritins (ca.-200 mV). The structure of this bacterioferritin has been determined at 1.95A resolution. The protein assembles in a 24-mer forming a hollow sphere with a cavity of around 80A diameter. The 24-mer is made up of 12 dimeric units, with each dimer containing one haem and two di-iron centres with one centre located in each unit of the dimer. This is the first time that the structure of the native di-iron centre in any BFR has been determined. The molecular biology studies showed that bacterioferritin and rubredoxin-2 genes form a dicistronic operon which reflects the direct interaction between the two proteins 3. Indeed, bacterioferritin and rubredoxin-2 form in vitro a complex, as shown by the significant increase of the anisotropy and decay times of the fluorescence of rubredoxin-2 tryptophan(s), when mixed with bacterioferritin. In addition, rubredoxin-2 donates electrons to bacterioferritin. This is the first identification of a physiological electron donor to a bacterioferritin and shows the involvement of rubredoxin-2 in iron metabolism.

1 .Romao, C.V., Regalia, M., Xavier, A.V., Teixeira, M., Liu, M.Y., and LeGall, J. (2000) Biochemistry 39: 6841-6849. 2. Romeo, C.V., Louro, R., Timkovich, R., Lubben, M., Liu, M.Y., LeGall, J., Xavier, A.V., and Teixeira, M. (2000) FEBS Lett 480: 213-216. 3. da Costa, P.N., Romgto, C.V., LeGall, J., Xavier, A.V., Melo, E., Teixeira, M., Saraiva, L.M. (2001), Mol Microbiol, in press.

406 Journal of Inorganic Biochemistry 86 (2001)

Engineering of metallothionein-3 neuronal activity into the inactive isoform metallothionein-1: Structural implications

Ntiria Romero-lsart a, Laran T. Jensen b, Oliver Zerbe c, Dennis R. Winge b, Milan Va~fik ~ "Institute of Biochemistry, University of Ziirich, Winterthurerstrasse 190, CH-8057 Ziirich, Switzerland (e-mail. n uria@bioc, unizh, ch). bDepartment of Biochemistry, University of Utah Health Science Center, Salt Lake City, 84132 Utah. U.S.A. CDepartment of Pharmacy, ETH, Winterthurerstrasse 190, CH-8057 Ziirich, Switzerland.

In the central nervous system, metallothioneins (MTs), low molecular-weight (6-7 kDa) metal- and cysteine-rich proteins, occur in the isoforms MT-1, MT-2 and MT-3. Despite the about 70 % sequence identity between MT-3 and the other two isoforms, only MT-3 impairs the survival and neurite formation of cultured neurons) Moreover, it could be shown that the N-terminal 13-domain of the protein (13MT-3) is responsible for its bioactivity. 2 Relative to MT-1/-2, 13MT- 3 contains one-Thr insert at position 5 and a novel motif in the MT family, i.e. Cys(6)-Pro-Cys-Pro(9). Biological and structural studies on the double mutant P7SP9A-MT-3 established the necessity of both proline residues for the bioactivity of MT-3 and revealed their profound effect on the unprecedented cluster dynamics of its 13-domain. 3

To explore further the underlying basis for differential bioactivity of MT isoforms, the unique conserved motif of I3MT-3 was genetically engineered into mouse MT-1. Biological studies showed that whereas the incorporation of the two prolines alone has no effect on activity, the additional insertion of Thr5 gives rise to a bioactive MT-1 form. Temperature- dependent 1~3Cd NMR studies and 1H-~13Cd HSQC saturation transfer experiments with the l l3Cd(II) reconstituted wild- type and mutant MT-1 forms, revealed that the gain of MT-3 neuronal activity is paralleled by an increase of the conformational flexibility and cluster dynamics in the N-terminal 13-domain of MT-1.

1.Uchida Y., Takio K., Titani K., Ihara Y., Tomonaga M., Neuron, 7, 337-347 (1991). 2.Sewell A. K., Jensen L. T., Erickson J. C., Palmiter R. D., Winge D. R., Biochemistry, 34, 4740-4747 (1995). 3.Hasler D. W., Jensen L. T., Zerbe O., Winge D R., Vagfik M., Biochemistry, 39, 14567-14575 (2000).

Ca 2+ function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Annette Rompel b, Taka-aki Ono a, Hiroyuki Mino a, Noriyuki Chiba a ~' Laboratory for Photo-Biology (1), RIKEN Photodynamics Research Center, The Insti tute of

Physical and Chemical Research, 519-1399 Aramaki-Aoba, Aoba, Sendai 980-0845, JAPAN b Ins t i tu t f i i r Biochemie, Westfiilische Wilhelms-Universitiit , Wilhelm-Klemm-Str. 2, 48419,

Miinster, GERMANY (e-mail . rompela@nwz, uni-muenster, de)

Effects of adding monovalent alkali metal cations to Ca2+-depleted photosystem II membranes on the biochemical and spectroscopic properties of the oxygen evolving complex (OEC) were studied. ~ The Ca2+-dependent activity of oxygen evolution was competitively inhibited by K *, Rb +, and Cs +, of which the ionic radii are larger than the one of Ca 2+. The activity was not inhibited by Li + and Na +, of which the ionic radii are smaller than the one of Ca 2~. None of the cations restored oxygen evolution. CaZ+-depleted membranes without metal cation supplementation showed normal $2 multiline EPR signal and a S2QA- thermoluminescence (TL) band with an essentially normal peak temperature after illumination under conditions for single turnover of PS II. Membranes supplemented with Li ÷ and Na + showed properties similar to those of the Ca2+-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K ÷, Rb +, and Cs ÷ was elevated to around 38 °C, and no $2 EPR signals were detected. The K+-induced high-temperature TL band and the normal S2QA band were interconvertible by the addition of K ÷ or Ca 2+ after illuminating the membranes. These results indicate that the ionic radii of the cations, occupying CaZ+-binding site, crucially affect the properties of the Mn cluster.

1. Ono, T.-a., Rompel, A., Mino, H., Chiba, N., Biophysical Journal, submitted.

The MSWWF and the JSPS is acknowledged for t'mancial support.

Journal of Inorganic Biochemistry 86 (2001) 407

Phenols nitration by peroxidases in the presence of nitrite and hydrogen peroxide: nitrogen dioxide or peroxynitrite?

Raffaella Roncone a, Enrico Monzani a and Luigi Casella a Depar tmen t o f Genera l Chemistry , Univers i ty o f Pavia, Via Taramel l i 12, 27100 Pavia, ITALY

It is well documented that reactive nitrogen species derived from nitrogen monoxide are involved in many pathological conditions. In this study the attention is mainly focused on the nitration reaction of tyrosine derivatives carried out by peroxidases in the presence of nitrite and hydrogen peroxide. A large range of phenol and nitrite concentrations (from physiological to molar concentration) was studied. The results indicate that nitrogen dioxide cannot be the only nitrating agent and suggest the presence of two simultaneously operative pathways, one proceeding through enzyme generated NO2" and another through a more reactive species (possibly peroxynitrite) generated by reaction of hydrogen peroxide with enzyme-bound nitrite. The importance of the two pathways depends on peroxide and nitrite concentrations, with the N02 ° driven reaction most important at lower nitrite and higher oxidant concentrations. Comparison of the nitration rates at physiological nitrite concentration with data extrapolated from the study at various nitrite concentrations indicates that lactoperoxidase activates mainly the peroxynitrite driven mechanism. With horseradish peroxidase the process involving NO2 ° perdominates in physiological conditions and that involving peroxynitrite prevails at high nitrite concentration.

This work was supported by the Italian CNR through the Target Project "Biotechnology" and by the European INTAS and COST Chemistry programmes.

Calix[6]arene based Cu(I) complexes. Supramolecular control of a biomimetic receptor

Yannick Rondelez, Olivier S6n6que, Olivia Reinaud Laborato ire de Chimie et B ioch imie Pharmaco log iques et Toxicologiques, CNRS UMR 8601, Universi td Rend Descar tes , 45 rue des Saints-Pdres , 75270 Paris Cedex 06, France.

The specific characteristics of a metallo-enzyme stem from a combination of the well-suited electronic properties of the metallic site and the structural properties of the organic pocket in which it is buried. Calix[6]arenes are able to act as molecular hosts. Thus by connecting them to a metal center, they can play the role of the substrate binding pocket of a metallo-enzyme. Indeed, the fixation of a cuprous center on an adequately functionnalized ealixarene yields complexes presenting an introverted free binding site, access to which is controlled by a funnel. The supramolecular interactions between the calixarene, the metal ion and the organic guest can be controlled through variations of the ligand's structure. Aspects including affinity, conformation and kinetic features of these receptors were studied.

@,

o o

I he host (gray] is distorled by its guest (black).

1. Blanchard, S.; Le Clainche, L.; Rager, M.-N.; Chansou, B.; Tuchagues, J.-P.; Duprat, A. F.; Mest, Y.; Reinaud, O. Angew. Chem. Int. Ed. Engl. 37, 2732-2735 (1998).

2. Rondelez, Y.; S6n6que, O.; Rager, M.-N.; Duprat, A.; Reinaud, O. Chem. Eur. J., 6, 4218-4226 (2000).

408 Journal of lnorganic Biochemistry 86 (2001)

Oxygen activation by glucose oxidase

Jus t ine P. Ro th , Jud i th P. K l i n m a n Department of Chemistry, University of California, Berkeley, California 94720.

Glucose Oxidase (GO) is a flavoenzyme that activates O2 for the metabolism of glucose. The oxidative half-reaction ultimately produces oxidized flavin and H202. Kinetic analysis indicates two reactive forms of enzyme and a pKa of 8.1, suggesting the participation of an active site histidine in catalysis. At 25 °C, Vmav/KM(02) varies from 1.6 x 106 M -t s -~

(pH 3-6) to 6.3 × 102 M -1 s -1 (pH >12). Neither kinetic solvent isotope effects nor viscosity effects are observed on V,n,JKM(02) over the entire pH range, ruling out proton transfer and diffusion-controlled reaction with 02- Oxygen-18 kinetic isotope effects (pH 5 to 9) are consistent with rate-determining 02- formation. Temperature studies reveal activation energies of 5.6 and 9.3 kcal mol ~ for low and high pH forms, respectively, and prefactors ~10 ~ M -~ s I. Outer- sphere electron transfer mechanisms are proposed to explain reactivity in both pH regimes. Since the low pH form of GO is actually 0.175 V (4.0 kcal mol 1) less reducing than the high pH form, a simple analysis based on flavin redox potentials cannot explain the results. Rather, the protonation state of the active site base must modulate the free energy barrier. The results support a view whereby catalysis is achieved through a strategically positioned point charge that increases the effective reaction driving force and/or reduces the outer-sphere reorganization energy.

The National Institutes of Heath (NIH) is acknowledged for financial support.

Flavocytochrome c3 from Shewanellafrigidimarina: investigating the electron pathway by site directed mutagenisis

Emma L. Rothery a, Katherine L. Pankhurst a, Caroline S. Miles b, Mary K. Doherty a, Graham A. Reid b, Stephen K. Chapman a. ~Department of Chemistry, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh, Scotland, EH9 3JJ. blnstitute of Cell and Molecular Biology, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh, Scotland, EH9 3JR.

Flavocytochrome c3 from the marine bacterium Shewanella frigidimarina is a small soluble fumarate reductase, the crystal structure of which has been solved to 1.8A resolution i. Electrons are shuttled, via four c-type hemes arranged to form a 40A 'molecular wire' from the surface of the protein to the non-covalently bound FAD at the active site. Heme irons are bis-histidine ligated, and low spin. The substitution of one of the ligating histidines, to an alanine, has essentially left the iron as a 5-coordinate species, with the possibility of moving into a high spin conformation. The cavity that remains is then filled in aqueous solution by water, which can be replaced by other exogenous ligands.

The reduction potentials of the heroes, as measured by potentiometric titration, have been shifted to lower values, by up to 40 mV from the wild type values 2, with a knock on effect being felt by the neighbouring hemes. Addition of imidazole to the enzyme restores 70% of wild type activity, whilst modulating the potentials of the hemes, by 20mV towards the wild type potentials.

I. Taylor, P.; Pealing, S.L.; Reid, G.A.; Chapman, S.K.; Walkinshaw, M.D., Nature Structural Biology, 6, 1108-1112, (1999).

2. Turner, K. L.; Doherty, M.K.; Heermg, H.A.; Armstrong, F.A.; Reid, G.A,; Chapman, S.K., Biochemistry, 38, 3302- 3309 (1999)

Journal of lnorganic Biochemistry 86 (2001) 409

Metal binding to members of the nuclear hormone receptor superfamily

Brian W. Rous" and H i l a r y A. G o d w i n a.

"Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208-3113, USA (email. b-rous@northwestern.edu)

The nuclear hormone receptor superfamily is an important class of transcription factors that are involved in signal transduction pathways and essential to the development of higher eukaryotes. The DNA-binding domain (DBD) of these proteins directs the sequence-specific binding to DNA. The DBD is highly conserved among the members of this protein family, and it features two Cys4 structural zinc-binding sites. Zn 2+ binding to these two sites is essential for the domain to fold properly and for the protein to function properly. The affinity of Zn 2÷ for the Cys4 metal-binding sites in the DNA- binding domain of the glucocorticoid receptor (GR DBD) was studied by using Co 2+ as a spectroscopic probe. In addition, because Pb 2+ has been shown to displace Zn 2+ from and bind tightly to Cys4 structural zinc-binding sites ~, the steroid receptor proteins are potential targets for Pb 2+. Studies are now focused on determining how Pb z+ interacts with members of the nuclear steroid hormone receptor superfamily in an effort to better understand the molecular mechanism(s) of Pb z+ toxicity.

1. Payne, J.C., ter Horst, M.A. and Godwin, H.A., J. Am. Chem. Soc., 121, 6850-6855 (1999)

The National Institutes of Health (NIH) is acknowledged for fmancial support.

Ab initio study of adenine tautomer complexes with closed- and open-shell copper ions

A.Yu Rubina a'b, V.A. Sorokin a, Yu.V. Rubin a, M.K. Shukla b, J. LeszczynskP a Molecular Biophysics Department, Institute for Low Temperature Physics and Engineering, National Academy of Science, Lenin ave.47, Kharkov, 61164, Ukraine, (e-mail: rubina@ilt.kharkov, ua) ~ Computational Center for Molecular Structure and Interaction, Jackson State University, 1400 Lynch.str, Jackson, Mississippi, 39217-0510, USA

Molecular geometry optimizations and electronic transition energy calculations were performed for two tautomers of adenine (Ade) and their complexes with closed-shell Cu (I) ion and open-shell Cu (II) ion using Gaussian 98 suite of programs. N 1, N3, N7, N9 sites of adenine tautomers were considered as binding ones to metal ions. The ground state geometry optimizations were carried out at the (U)HF/6-31+G** (for open-shell metal ions) while transition energies were calculated using the configuration interaction involving single excited configurations (CIS method). The effect of bulk aqueous solvation was studied applying the polarized continuum model (PCM) of the self-consistent reaction field (SCRF) theory. The amino group rotation phenomenon is also addressed to. Experimental UV-spectrum of adenine complexes with Cu (II) in water solution has been obtained. This spectrum shows a low-frequency shift of approximately 1000 cm-~ in comparison with those of adenine.

The most probable binding site of adenine tautomers with Cu(I) and Cu(II) ions was found to change under aqueous solvation using PCM model. Thus, the most probable binding site of Cu(I) ion in gas phase is the N7 atom of the adenine N9H tautomer while in the solvation model the site in question is N1 or N3. Analysis of excitation energies calculated for the first singlet n-n* transitions showed that the metal ion binding to the N1 or N7 atomic sites of adenine would lead to the red shift while the N9 site binding would show the blue shift as compared to the absorption spectrum of adenine.

410 Journal of Inorganic Biochemistry 86 (2001)

Hydrogen bonding to the proximal cysteine in nitric oxide synthase

Denis L. R o u s s e a u a, M a n o n C o u t u r e a, Subra ta A d a k b, Denn i s J. S tuehr b

" Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY, 10461, U.S.A.

Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, 44195, U.S.A.

Nitric oxide synthase (NOS) catalyzes the formation of NO and citrulline from L-arginine and oxygen. However, the NO so formed has been found to auto-inhibit the enzymatic activity ~. We hypothesized that the NO reactivity is, in part, controlled by hydrogen bonding between the conserved tryptophan residue (position 409 in nNOS) and the cysteine residue that forms the proximal bond to the heme. By using resonance Raman spectroscopy and NO as a probe of the heine environment, we show that in the W409F and W409Y mutants of the oxygenase domain of the neuronal enzyme (nNOSox), the Fe-NO bond in the Fe3+NO complex is weaker than in the wild type enzyme, consistent with the loss of a hydrogen bond on the sulfur atom of the proximal cysteine residue. The weaker Fe-NO bond in the W409F and W409Y mutants might result in a faster rate of NO dissociation from the ferric heine in the W409 mutants, which could contribute to the lower accumulation of the inhibitory NO-bound complexes observed during catalysis with the W409 mutants 2. The optical and resonance Raman spectra of the Fe2+NO complexes of the W409 mutants differ from those of the wild type enzyme and indicate that a significant population of a five-coordinate NO-complex is present. These data show that the hydrogen bond provided by the W409 residue is necessary to maintain the thiolate coordination when NO binds to the ferrous heine. Taken together our results indicate that the heine environment on the proximal side ofnNOS is critical for the formation of a stable Fe-cysteine bond and for the control of the electronic properties of heine-NO complexes.

[. Abu-Soud, H.M.,Wang, J., Rousseau, D.L.,Fukuto, J.M.,Ignarro, L.J . ,and Smehr, D.J. ,J. Biol. Chem. 270,22997- 23006(1995).

2. Adak, S., Crooks, C. ,Wang, Q., Crane, B. R., Tainer, J. A., Oetzoff, E. D., and Smehr, D .J . , J . Biol. Chem. 274, 26907-26911(1999).

This work was supported by NIH grants GM54806 to D.L.R. and CA53914 to D.J.S.