learning from metalloenzymes: new homogeneous catalysts ... 41a(1) (lecture abstracts).pdf ·...

Indian Journal of Chemistry Vol. 42A, January 2002, pp.73-97

Lecture Abstracts

Learning from metalloenzymes: New homogeneous catalysts for the aerobic oxidation of alcohols and amines

Phalguni Chaudhuri & Karl Weighardt*

Max-Planck-Institut fUr Strahlenchemie, Stiftstrasse 34-36, D-45470 Mlilheim an der Ruhr,Germany

The copper containing metalloproteins galactose oxidase and the amine oxidase are enzymes, which catalyze efficiently the aerobic oxidation of primary alcohols and primary amines according to Eqs 1 and 2, respectively .

(1)

(2)

The mechanisms of both enzymes have been elucidated in great detail. Both operate via a ping-pong type mechanism where the oxidized catalyst is reduced by the substrate yielding (a) aldehydes or (b) aldehyde and ammonia in the first half cycle ("ping") whereas the reduced forms react with dioxygen in the second half cycle ("pong") generating (a) H20 2 and the regenerated catalyst and (b) ammonia, hydrogen peroxide and the regenerated catalyst. The structures of both enzymatic systems are known. Therefore, the mechanisms are established at the molecular level.

It is rather surprising that reactions 1 and 2 are not known in synthetic organic chemistry, i.e., no catalyst has been described for these reactions until very recently. This is a challenge for inorganic and organic chemists since both reactions are potentially very interesting.

References 1 Wang Y, DuBois J L, Hedman B, Hodgson K 0 & Stack T D P, Science 279 (1998) 537 .. 2 Chaudhuri P, Hess M, FlOrke U & Wieghardt K, Angew Chern Int Ed, 37 (1998) 2217. 3 Chaudhuri P, Hess M, WeyhermUller T & Wieghardt K, Angew Chern Int Ed, 38 (1999) 1095. 4 Chaudhuri P, Hess M, MUller J, Hildenbrand K, Bill E, WeyhermUller T & Wieghardt K, J Arn chern Soc, 121 (1999) 9599.

An inorganic approach to drug design

J A Cowan

Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA

We have demonstrated copper aminoglycosides to be highly efficient cleavage catalysts for DNA and RNA targets. Such catalysts mediate both oxidative and hydrolytic pathways and their cleavage reactions display enzyme-like Michaelis-Menten kinetic behavior. An unusual double-strand cleavage of DNA has been observed. Degradation of RNA viral motifs have been demonstrated in vitro, and the efficacy of such molecules against an in vivo target has also been established using a novel in vivo assay. In this lecture I will develop an understanding of the chemical mechanisms underlying the cleavage of RNA and DNA targets, recognition strategies, mechanisms of cellular delivery, and the design of in vivo assays.

74 INDIAN J CHEM, SEC A, JANUARY 2002

Iron-sulphur cluster biosynthesis

Michael K Johnsonl *, Jeffery N Agarl, Carsten Krebs2, Boi Hanh Huynh2, Pramvadee Yuvaniyama3,

Valerie L Cash3, Limin Zheng3

, Jevetson Frazzon3 & Dennis R Dean3

IDepartment of Chemistry and Center for Metalloenyzme Studies, University of Georgia, Athens, GA 30605, USA; 2Department of Physics, Emory University , Atlanta, GA 30322, USA;

3Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 , USA

Iron-sulphur clusters are ubiquitous prosthetic groups in biology, occurring in more than 120 different types of proteins, but relatively little is known about their biosynthesis. Azotobacter vinelandii has been used as a model system to investigate Fe-S cluster biosynthesis, since it has nif genes that specifically target nitrogenase cluster biosynthesis and isc genes that are used for general cluster biosynthesis and widely conserved in both prokaryotic and eukaryotic organisms. A. vinelandii NifS and IscS have been shown to be pyridoxal 5'-phosphate containing cysteine desulphurases, which form complexes with NifU and IscU, respectively, and supply the sulphur for transient cluster assembly on the NifU or IscU scaffold. A. vinelandii NifU has been shown to be a modular protein, with an N-terminal iron binding domain (NifU-l ) corresponding to IscU, and a C-terminal [2Fe-2S]2+.+ domain (NifU-2)1. To eliminate the spectroscopic background of the permanent [2Fe-2S]2+.+ cluster, the N-terminal NifU-l domain was overexpressed and purified from E. coli. The addition of ferric citrate to NifU-l resulted in a UV -visible absorption spectrum indicative of a rubredoxin-like iron site and resonance Raman spectra were consistent with a tetrahedrally ligated ferric site with at least three cysteine ligands I. In vitro cluster assembly in A. vinelandii NifU-l and IscU, using NifS or IscS, ferric citrate, cysteine, and mercaptoethanol is initially marked by the appearance of a UV -visible absorption spectrum indicative of a [2Fe-2S]2+ cluster and this was confirmed by resonance Raman and Mossbauer studies2.3. More detailed IscSmediated cluster assembly studies with IscU revealed sequential cluster assembly with the initial product containing one [2Fe-2S]2+cluster per dimer, converting first to a form containing two [2Fe-2S]2+ clusters per dimer, and finally to a form that contains one [4Fe-4Sf+ cluster per dimer4

• Subsequent Mossbauer studies have confirmed NifS-mediated assembly of both [2Fe-2S]2+ and [4Fe-4S]2+ clusters in holo NifU. The ability to assemble both [2Fe-2S]2+ and [4Fe-4S]2+ clusters in NifU and IscU supports the proposal that this ubiquitous protein provides a scaffold for NifSlIscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins. Moreover, the results also indicate that the mechanism for [4Fe-4S]2+ cluster biosynthesis involves reductive coupling of two preformed [2Fe-2S]2+ clusters.

References I Agar J N, Yuvaniyama P, Jack R F, Cash V L, Smith A D, Dean D R & Johnson M K, J Biollnorg Chem, 5 (2000) 167. 2 Yuvaniyama P, Agar J N, Cash V L, Johnson M K & Dean D R, Proc Natl Acad Sci USA, 97 (2000) 599. 3 Agar J N, Zheng L, Cash V L, Dean D R & Johnson M K, JAm chem Soc, 122 (2000) 2136. 4 Agar J N, Krebs B, Frazzon J, Huynh B H, Dean D R & Johnson M K, Biochemistry, 39 (2000) 7856.

Structure and functions of metalloproteins in postgenomics era

Lucia Banci CERM, University of Florence, Italy

The continuous advancements in decoding the genome of human and many other organisms is now opening several fundamental questions to be addressed by the scientists. One of the most relevant advancements deals with the elucidation of the structural and functional properties of the proteins encoded in the genome. This is quite a challenging task which requires, in order to be successful, combined experimental and computational efforts. In particular, suitable and efficient tools for the structural and dynamical characterization should be developed.

Within this frame, we have undertaken the solution structure determination and dynamical characterization, through NMR spectroscopy, of some proteins of various c1askes, such as copper-transport proteins and heme

Lecture Abstracts 75

proteins. The relationship between these features and the function has been analyzed, with the goal of individuating key properties of the various investigated classes.

Voltammetric studies of active sites in proteins

Fraser A Armstrong

Inorganic Chemistry Laboratory, Department of Chemistry, Oxford University, South Parks Road, Oxford OXI 3QR, England

Protein film voltammetry provides a powerful way to observe redox-active sites in proteins and to determine the mechanisms and complexities of electron transfer l

. The protein under investigation is immobilised on a suitable electrode as an adsorbed electroactive film and electrons are driven into and out of the active sites by applying a variable potential. Signals are obtained from extremely small sample quantities (monolayer coverage or less), and from these it is possible to detect and characterize labile active sites and to deconvolute complex reaction sequences2-7. The signal itself provides a handle for the active site and enables its presence and reaction progress to be monitored under strict conditions of applied potential, even under very reducing or oxidising conditions8

,9.

Cyclic voltammetry experiments may be carried out at high scan rates, > 103 volts per second in favourable cases, thus accessing coupled reactions that occur in the sub-millisecond time domain2-7. JO-12. Catalytic activity can be studied as a function of potential (driving force) and correlated with redox transitions occurring at active sites9

, 12-17. It seems likely that certain enzymes can regulate their ET activity in feedback response to the environmental potential; this may be particularly important for enzymes that are normally bound in a membrane l3,14.

Herein we present the concept and illustrate its application to some complex problems in biological redox chemistry. Recent studies include electron transfer in flavoenzymes I2-15. 17 redox properties and catalytic mechanisms of mutant forms of cytochrome c peroxidase9

, and the mechanism of long-range, redox-coupled proton transfer in a small protein that serves as a model for proton pumping enzymes6

.7.

References 1 Heering et al., Chem Soc Rev, 26 (1997) 169.

2 Butt, et aI. , JAm chem Soc, 119 (1997) 9729.

3 Hirst et ai., JAm chem Soc, 120 (1998) 11994.

4 Camba & Armstrong, Biochemistry, 39 (2000) 10587.

5 JeJken et al. , J Am chern Soc, 122 (2000) 12186.

6 Hirst et al., JAm chern Soc, 120 (1998) 7085.

7 Chen et aI., Nature, 405 (2000) 814.

8 Duff et al., J Am chern Soc, /18 (1996) 8593.

9 Mondal et al,. JAm chern Soc, 120 (1998) 6270.

10 Hirst & Armstrong, Anal Chern, 70 (1998) 5062.

11 Annstrong et al. Faraday Discuss, 116 (2000) 191.

12 Jones et aI., J Am chern Soc, 122 (2000) 6494.

13 Sucheta et al., Nature, 356 (1992) 361.

14 Hirst et al., JAm chem Soc, 118 (1996) 5031.

15 Hirst et al., J Am chern Soc, 119 (1997) 7434.

16 Heering et aI., J phys Chem B, 102 (1998) 6889.

17 Pershad et al., Biochim Biophys Acta, 1412 (1999) 262.

18 Heffron et aI., Biochemistry, in press.


Electron transport in mitochondrial NADH : Ubiquinone oxidoreductase (Complex I)

Judy Hirst

MRC Dunn Unit, Wellcome Trust / MRC Building, Hills Road, Cambridge, CB2 2XY, United Kingdom, jh @mrcdunn. caln.Clc. uk

Complex I (NADH:Ubiquinone oxidoreductase)2 is one of the key enzymes of the membranebound respiratory chain. First, it is the "entrypoint" for electrons from NADH and second, it is one of the three "proton-pumping" complexes, creating a proton gradient across the inner mitochondrial membrane. Of the five complex enzymes constituting this membrane-bound respiratory chain, Complex I is both, the largest being composed of (probably) 43 unlike polypeptides (7 encoded by mitochondrial DNA), and the least well understood. There is as yet no detailed structural data available for Complex I, and comparatively little is understood about how the energy liberated by electron transfer from NADH to Ubiquinone is coupled to vectorial proton translocation.

Schematic

Representation of Complex II

Complex I contains a number of redox active cofactors. A non-covalently bound FMN constitutes the active site, catalysing the oxidation of NADH, and a number of iron-sulphur clusters provide "stepping-stones" for moving electrons through the protein. These redox active cofactors are amenable to study by protein-film voltammetry and the work directed towards using voltammetric techniques to understand how electron transfer in Complex I is controlled, and how the available (redox) energy is coupled to proton translocation is outlined herein.

References 1 Grigoriff N, J molec Biology, 277 (1998) 1033. 2 Walker J E., Quart Rev Biophysics, 25 (1992) 253.

Molecular interactions in chromium(III)-collagen and collagenase systems: Towards a new molecular insight into stabilization of collagen

T Ramasami*, R Gayathri & Rama Rajaram

Central Leather Research Institute, Chennai, Madras 600 020, India

Chrornium(III) salts are employed industrially in the stabilization of skin. Although industrial application methodology is well understood and stabilized, molecular insight into the processes leading to the stabilization of collagen has defined earlier attempts. Chromium(III) ion serves to stabilize collagenous matrices against dimensional changes under heat as well as degradation by collagenase. Rat-tail tendon has been a known source of relatively homogenous source of collagen. A bundle of collagen fibers from rat tail tendon has now been treated with select series of chromium(III) complexes of the formulation 1, 2 and 3. The resulting collagenchrornium(III) products have been evaluated for stability against thermal stress as well as enzymatic degradation by collagenase.


The ability of chromium(III) to induce long-range order in soluble collagen has been examined using atomic force microscopy. Samples of collagenase have been interacted with the three chromium(III) complexes 1-3, and the ability of the metal ion to inhibit the activities of the enzyme has been examined. Changes in conformation of collagen as well as collagenase on interaction with the select series of chromium(III) complexes have been investigated. The degradation of collagen by collagenase after interactions with chromium(III) has been examined by monitoring the release of hydroxyproline in solution after hydrolysis. There is now convincing evidence for the (chromium) species-specific influence on the stability of collagen and mode of inhibition of collagenase. Whereas the species 1, inhibits the activity of collagenase competitively, complexes 2 and 3 exhibit through non-competitive and un-competitive binding modes. Complex 1 is observed to stabilize collagen against the degradation by collagenase as well as the dimensional changes under the action of heat more effectively than complexes 2 and 3. In other words the melting behaviour of collagen is remarkably dependent on the nature of chromium(III) species employed for stabilization. A molecular basis for the changes in the observed stability of collagen due to metal-protein and metal-proteinase interactions has now been attempted. Experimental results are discussed in terms of a new unified theory of stabilization of collagen by chromium(III) against heat and enzymatic degradation.

The inorganic chemistry of biological methanogenesis: Computer modeling of coenzyme F430

Abhik Ghosh

University of Tromso, Norway and San Diego Supercomputer Center, USA

The last step of methanogenesis by methanogenic archaea is catalyzed by a unique nickel tetrapyrrole-containing enzyme methylcoenzyme M reductase. In the active form of the enzyme, the nickel tetrapyrrole cofactor, called F430, is present in the unusual Ni(l) oxidation state. Interestingly, a proposed reaction mechanism of this enzyme also involves a Ni(III)-CH3 reactive intermediate. Computer modeling studies, of F430 have uncovered a fascinating interplay of factors such as skeletal stereoisomerism and tetrapyrrole conformation that explain the stabilization of Ni(l) and putative Ni(III) intermediates.

The work is in progress and in various stages of publication. For an early account, see: Wondimagegn T & Ghosh A, JAm chem Soc, 122(2000) 6375.

Reactivity of copper(I) complexes with dioxygenliodosylbenzene from bioinorganic perspectives: Aromatic ring hydroxylation and exogenous

substrate reactivity

R N Mukherjee

Department of Chemistry , Indian Institute of Technology, Kanpur 208 016, India

The activation of molecular oxygen by copper plays a central role in synthetically useful stoichiometric and catalytic oxidative conversions of organic molecules and in biological systems. In biological systems, monophenols (tyrosine) are hydroxylated to a-diphenol and further oxidized to an a-quinone by tyrosinase, which is a monooxygenase having a dinuclear copper center as an active site similar to that of hemocyanin, a dioxygen carrier protein. One goal in bioinorganic copper chemistry is to elucidate basic patterns of CUn-02 binding, structure, associated spectroscopy, and reactivity, in varied copper-ligand environments. Studies of synthetic systems that model the protein active site often have used dinucleating ligands with m-xylyl spacers


that undergo hydroxylation upon oxygenation of their dicopper(l) complexes. This is a case of intramolecular ligand oxidation. A balanced ligand design that precludes deleterious bimolecular reactions, yet allows substrate binding to the copper centers, is an attractive and intuitive means of converting these copper-oxygen intermediates into synthetically useful oxidants. Intuitively, another means of generating such intermediates could be to investigate reactions between Cu(I) complexes and iodosylbenzcne, which have generally not been investigated seriously, in favour of reaction with O2• New insights into copper-oxygen intermediates with a novel class of designed ligands and their reactivity toward exogenous substrates will be discussed herein

References I Solomon E I, Sundaram U M & Machonkin T E, Chern Rev, 96 (1996) 2563. 2 Kitajima N & Mora-oka Y, Chern Rev, 94 (1994) 737. 3 Ghosh D, Lal T K, Ghosh S & Mukherjee R,} chern Soc Chern Cornrnun, (1996) 13. 4 Gupta R & Mukherjee R, Inorg chirn Acta, 263 (1997) 133. 5 Ghosh D & Mukherjee R, Inorg Chern, 37 (1998) 6597. 6 Gupta R & Mukherjee R, Tetrahedron Lett., 41 (2000) in press. 7 Gupta R & Mukherjee R, submitted for publication.

Receptors for anions and cations

V G Anand, S Jayaprakash Narayanan & T K Chandrashekar* Department of Chemistry, Indian Institute of Technology, Kanpur 208 016, India

Studies on the design and synthesis of receptor molecules capable of selectively binding and transporting substrates (neutral, anionic and cationic) are currently being pursued to develop artificial membranes permeable to the bound species. Expanded porphyrin systems by virtue of increased cavity size and aromatic nature are capable of binding a variety of substrates depending on the nature of the porphyrin and the cavity size. Recently there are number of reports on the use of expanded porphyrins as molecular receptors for various substrates. Specifically, expanded porphyrins such as sapphyrin, rubyrins and smaragdyrins, in their protonated form bind variety of anionic and neutral substrates and it has been shown that they act as carriers for transporting different ionic and neutral species. Syntheses and receptor properties in solid and solution phase of a few expanded porphyrins such as sapphyrins, rubyrins and smaragdyrins will be highlighted herein.

References I Sridevi B, Narayanan S J, Chandrashekar T K, Englich U & SengeKarin R, Chern Eur}, 6 (2000) 2554. 2 Sridevi B, Narayanan S J, Rao Rohini, Chandrashekar T K, Englich U & Senge Karin R, Inorg Chern, 39 (2000) 3669. 3 Srinivasan A, Anand V G, Narayanan S J, Pushpan S K, Chandrashekar T K, Sugiura K I & Sakata Y, } org Chern, 64 (1999) 8693. 4 Narayanan S J, Sridevi B, Chandrashekar T K, Vij Ashwani & Roy Raja,} Arn chern Soc, 39 (1999) 9053. 5 Narayanan S J, Sridevi B, Chandrashekar T K, Vij Ashwani & Roy Raja, Angew Chern In! Ed Engl, 37 (1998) 3394.

Crystal structures of bovine milk xanthine dehydrogenase/oxidase and mechanism of conversion from the dydrogenase to the oxidase

Takeshi Nishino Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602,

Japan

Xanthine oxidoreductase enzymes have been isolated from a wide range of organisms, from bacteria to man, and catalyze the hydroxylation of a wide variety of purine, pyrimidine, pterin, and aldehyde substrates. All of these enzymes have similar molecular weights and composition of redox centers: these proteins are homodimers of molecular mass 290 kDa and each subunit contains one molybdopterin cofactor (Mo-pt), two [2Fe-2S] centres,


and one flavin adenine dinucleotide (FAD). The mammalian enzymes, which catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, are synthesized as the dehydrogenase form (XDH) and exist mostly as such in the cell but can be readily converted to the oxidase form (XO) by oxidation of sulphydryl residues or by proteolysis. XDH shows a preference for nicotinamide adenine dinucleotide (NAD) reduction at the FAD reaction site, while XO fails to react with NAD and exclusively uses dioxygen as its substrate leading to the formation of superoxide anion and hydrogen peroxide.

The hydroxylation of xanthine takes place at the Mo-pt. In contrast to other hydroxylases, XDH utilizes a water molecule as the ultimate source of oxygen incorporated into the product. The electrons introduced into the enzyme via molybdenum from xanthine substrate are rapidly distributed to the other centres via intramolecular electron transfer. The reduction of the natural oxidant substrate N AD occurs at the FAD site.

The crystal structures of the dimeric bovine milk xanthine dehydrogenase and oxidase were solved at 2.1 A and 2.5 A resolution, respectively. The structures showed that the two iron sulphur centres were located in the N-terminal 20 kDa domain, FAD in the intermediate 40 kDa domain and the molybdenum centre in the Cterminal 85 kDa domain, consistent with the previous predictions from the amino acid sequence comparison or protein chemical studies. Cleavage of surface-exposed loops causes major structural rearrangement of another loop close to the flavin ring. This movement partially blocks access of the NAD to the FAD and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme. The mechanism of the conversion will be discussed from the structural and mechanistic studies.

Synthetic prospect of the molybdenum-cofactor of structurally characterized sulphite oxidase

Sabyasachi Sarkar Department of Chemistry, Indian Institute of Technology Kanpur 208 016, India

The revelation of the domain of Moco and its coordination arrangement in chicken liver sulphite oxidase by xray crystallography I does provide vital clues in regard to the early failures to isolate pure Moco from its apoprotein and also to synthesize a structural analogue of Moco. Based on the precrystallographic information of the protein2, we have earlier reported a synthetic analogue of the oxidized form of sulphite oxidase bearing cis{Mo VI02 (S2-dithiolene)2} moiety3. The recent X-ray crystal structure of sulphite oxidase I confirms that our synthetic model though fully functional was not exactly structural analogue due to the possession of one extra dithiolene against one thiolate ligation. Known that it has not yet been possible to structurally characterize the fully oxidized form of sulphite oxidase, we have embarked upon to duplicate the core structural features of Moco as has been structurally found in native protein I. The fully oxidized form of sulphite oxidase bearing Mo (VI)/Fe (III) oxidation states of the respective Moco and heme b5 is reported to react with traces of sulphite impurity inadvertently present in the precipitant, lithium sulphate, resulting in the reduced form of the protein to crystallize out l

. The two-electron reductive half reaction would lead to the final oxidation states of these prosthetic groups as Mo (V)lFe (II) resulting in the core formulation of the Moco as {Mo Vo (Sz-pterin)(Scys)(OH) }(I) moiety which remained deeply buried within the protein I. The inherent problem for a low molecular synthetic analogue of I with a monomeric Mo (V) core structure without the protective protein environment will respond to dimerization involving dimer-monomer equilibrium typical for Mo (V) chemistr/. Using maleonitriledithiolate (rnnt) and thiophenolate (SPh) as models for the respective pterin-ene-dithiolate and cysteinylligands of Moco of the native sulphite oxidase, we synthesized [Bu4h[{Mo Vo (S2-mnt)(SPh) hOl The chemistry of this species in relevance to sulphite oxidase will be presented.

References I Kisker C, Schindelin H, Pacheco A, Wehbi W A, Garrett R M, Rajagopalan K Y, Enemark J H & Rees D C, Cell, 91 (1997) 973. 2 (a) Pilato R S & Stiefel E J, in Bioinorganic catalysis,edited by J Reedjik.(Marcel Dekker Inc., New York,)1993, p173. (b) Enemark J

H & Young C G, Adv Inorg Chern, 40 (1994) 1.


3 (a) Das S K, Chaudhury P K, Biswas D & Sarkar S, J Am chern Soc, 116 (1994) 9061. (b) Chaudhury P K, Das S K & Sarkar S, Biochem J, 319 (1996) 953.

4 Gamer C D & Bristow S, Molybdenum enzymes, edited by T G Spiro (Wiley-Interscience, New York), 1985,p. 343.

Spectroscopic studies of tungsten-containing formaldehyde ferredoxin oxidoreductase and glyceraldehyde-3-phosphate ferredoxin oxidoreductase from

hyperthermophilic archae a

Ish K Dhawan , Roopali Roy, Michael W W Adams & Michael K lohnson*

Departments of Chemistry and Biochemistry & Molecular Biology and the Centre for Metalloenzyme Studies, University of Georgia, Athens, GA 30602 USA

Compared to the firmly established and essential role of molybdenum in global cycles, the biological role of tungsten has just started to emerge. It has now been established that growth of certain hyperthermophilic archaea is dependent upon tungsten '. Till date, three tungsten-containing aldehyde-oxidizing enzymes-aldehyde ferredoxin oxidoreductase (AOR), formaldehyde ferredoxin oxidoreductase (FOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) have been purified from hyperthermophilic archaea pyrococcus furiosus (Pf) and thermococcus litoralis (TI) (ref. 2).

In this work, the electronic, vibrational and structural properties of the Wand [4Fe-4S] centres of Pf and Tl FORs3 and Pf GAPOR are compared using the combination of EPR, resonance Raman and VTMCD spectroscopies. The spectroscopic and redox properties of as-isolated Pf and TI FORs are similar to those of the Pf AOR4 exhibiting low-, mid- and high-potential W(V) species, except that Pf FOR does not exhibit a highpotential W(V) species. As-isolated Pf and Tl FORs shows 6 - 8 fold enhancement in activity when incubated with excess Na2S under reducing conditions and the sulphide-activated FORs are inactivated by cyanides. The spectroscopic and redox properties of the sulphide-activated Tl FOR are quite distinct to those of the as-isolated enzyme, with loss of the low-potential species and changes in high-potential species that are reversed by exposure to air. Both high-potential species disappear upon addition of cyanide. The spectroscopic and redox properties of the W centre in Pf GAPOR are quite different to those of Pf AOR and Pf and Tl FORs. Dyemediated EPR redox titrations show no evidence for any high-potential W(V) species and these W(V) resonances have gay values much lower than any of those observed in tungsten-containing AORs and FORs. The structural origin of the different spectroscopic properties for the W centres in FOR and GAPOR will be discussed.

References I Kletzin A, Adams M W W, FEMS Microbial Rev, (1996) 5. 2 Johnson M K, Rees D & Adams M W W, Chern Rev, 96 (1996) 2817. 3 Dhawan I K, Roy R, Koehler B P, Mukund S, Adams M W W & Johnson M K, J Biollnorg Chern,S (2000) 313. 4 Koehler B P, Mukund S, Conover R C, Dhawan I K, Roy R, Adams M W W & Johnson M K, JAm chern Soc, 118 (1996) 12391. 5 Roy R, Mukund S, Schut G J, Dunn D M, Weiss R & Adams M W W, J Bacterial, 181 (1999) 1l71.

The reaction mechanism of xanthine oxidase and related enzymes

Russ Hille

Department of Molecular and Cellular Biochemistry, The Ohio State University, 333 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210-1218, USA

The reaction carried out by xanthine oxidase and their related molybdenum-containing enzymes represents a unique solution to the problem of biological hydroxylation, in which water, rather than dioxygen is the source of oxygen atom incorporated into product and reducing equivalents are generated rather than consumed. The


crystal structures for several members of this family of enzymes have been determined, including that for aldehyde oxidoreductase from desulfovibrio gigas, CO dehydrogenase from oligotropha carboxydovorans, and xanthine oxidase from bos taurus, which provides a structural context to understand the overall reaction mechanism. In conjunction with earlier results using X-ray absorption spectroscopy, the crystallographic information indicates that the active site of xanthine oxidase possesses an Lmo v10S(OH) centre in a distorted square pyramidal coordination geometry, with L representing a unique enedithiolate ligand to the molybdenum contributed by a pyranopterin cofactor. Results of rapid reaction kinetic studies in conjunction with EPR and ENDOR are described, providing detailed insight into the chemical sequence of events that lead to substrate hydroxylation. In addition, computational approaches to understanding reaction mechanism are discussed that provide additional information concerning the chemistry of the reaction. Our results are consistent with a reaction mechanism initiated by nucleophilic attack of the active site Mo-OH on the C-8 carbon of substrate, with concerted hydride transfer from C-8 to the Mo=S group of the molybdenum centre. This is accompanied by a tautomerization of substrate that must be accommodated by the enzyme in order to proceed the catalysis effectively. Glu 1261 , a highly conserved amino acid residue among members of this group of enzymes, participates as a general base catalyst, abstracting a proton from the Mo-OH at the onset of the reaction. We find no evidence for transient formation of a Mo-C bond in the course of the reaction.

Interaction of nitric oxide with cobalamin

P T Manoharan Department of Chemistry, Indian Institute of Technology - Madras, Chennai 600 036, India.

E-mail: [email protected] (or) [email protected]

The production of NO from L-arginine metabolism is an essential determinate of the innate immune system, important for non-specific host defence as well as tumour and pathogen killing. NO is also involved in vasodilation and neuro-transmission in the living organisms. It has been reporteq that hydroxocobalamin produced concentration dependent reductions of the relaxant action of NO and endothelium-dependent, NO mediated relaxant action of acetylcholine in rat aortic rings. Furthermore, cobalamin appears to quench NO mediated cell proliferation and acts as an NO scavenger by forming nitrosocobalamin leading to suspect deleterious effects of cobalamin in respect of NO. The existing reports 1,2 on the interaction of cobalamin with nitric oxide has built-in contradiction's and there are no definite proofs offered for the complex formation of NO with cobalamin3

. A systematic study on the interaction of nitric oxide with cobalamin in its +2 and +3 oxidation states using a variety of spectroscopic techniques provides a positive proof for the definite formation of cobalamin-NO complex4

. However, the reaction of nitric oxide with cobalamin in its +3 oxidation state is much slower than that with +2 oxidation state. Also, a mechanism for the formation of such complexes clearly emerges out of these detailed studies. Even more interesting is the fact that NO taken up by cobalamin can be eventually given back to -SH related molecules such as glutathione, cysteine and the ~-93 sulphhydryl groups of Hb. It should be noted in this context that glutathione is present in the biological medium where such reactions take place5

. Detailed optical, EPR, FT-IR and fluorescence studies bring out the complexities involved in the uptake and release of NO by cobalamin.

References I Rochelle L G, Morana S J, Kruszyna H, Russell M A, Wilcox D E & Smith R P , JEPT, 275 (1995) 48.

2 Kruszyana H, Magyar J S, Rochelle L G, Russell M A, Smith R P & Wilcox D E,,lEPT, 285 (1998) 665 .

3 Brouwer M, Chamulitrat W, Ferruzzi G, Sauls D L & Weinberg J B, Blood, 88 (1996) 1857.

4 Kundu T K, Chandramouli G Y R, Nagababu E, Rifkind J M, Sharma Y S , Boss G R & Manoharan P T, (Communicated, 2001 ).

5 Ramasamy S, Chandramouli G Y R, Chandrasekaran Swarnalatha Y, Rifkind J M & Manoharan P T, (To be Communicated, 2001 ).


Engineering excited-state polarization and collective oscillator behavior in multichromophoric systems: New insights for the design of biomimetic

light-harvesting and excitation transfer assemblies

H Tetsuo Uyeda, Victor S-Y Lin, Scott A Williams, Thomas Troxler & Michael J Therien* Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323.

New classes of porphyrin-based chromophore arrays have been prepared from ethyne-elaborated porphyrin synthons through the utilization of metal-mediated cross-coupling methodologies. These systems feature porphyrin pigments wired together via ethynyl- and butadiynyl-linkage motifs; topological connectivities of this type afford exceptional electronic interactions between the chromophores which are evident in their spectroscopy, photophysics and ultrafast excited state dynamics. Time resolved fluorescence anisotropy measurements demonstrate that appropriately designed synthetic multichromophoric systems can exhibit low energy fluorescent excited states in which the transition dipoles of the pigment building blocks are exquisitely correlated in defined phase relationships; impressively, the polarized nature of these singlet excited states can be maintained over timescales that are long with respect to the archetypal strongly coupled chromophore assemblies that figure prominently in the biological light harvesting apparatus. Analysis of corresponding fluorescence intrinsic decay rate and quantum yield data show that ethyne- and butadiyne-bridged multiporphyrin species that manifest high excited-state anisotropies display exceptional superradiance enhancement factors: such photophysics derive from the fact like the strongly-coupled pigment assemblies of the biological antennae apparatus; these conjugated chromophore arrays behave as collective oscillators and feature large Frank-Condon barriers for intersystem crossing between their respective S I and T I states. These pigment systems can thus be utilized to provide mechanistic insight into fast electron and energy transfer reactions of biological significance, as well as serve as platforms for the development of exceptional optoelectronic materials.

Core modified porphyrin building blocks and unsymmetrical porphyrin arrays

M Ravikanth*, D Kumaresan & Neeraj Agarwal Department of Chemistry, Indian Institute of Technology, Powai, Mumbai 400 076, India

Unsymmetrical porphyrin arrays are suitable models for mimicking energy transfer processes of photosynthesis. Selective excitation of one porphyrin unit and energy transfer from that unit to another porphyrin unit is feasible in unsymmetrical porphyrin arrays. Recently, few unsymmetrical porphyrin dimers such as porphyrin - chlorin and porphyrin - corolle have been synthesized in order to obtain long-lived charge transfer states. Interestingly, there is no report on unsymmetrical arrays containing core modified porphyrins. Core modification of porphyrin rings by introducing thiophene, furan, selenophene and tellurophene in place of pyrrole leads to novel core modified porphyrins which exhibit interesting properties in terms of both aromatic characteristics and their ability to stabilize metals in unusual oxidation states. The talk will feature the design and synthesis of different core modified porphyrin building groups with N3S, N2S2, N30 and N20 2

porphyrin cores and their use in the construction of unsymmetrical porphyrins containing N4 and core modified porphyrin cores. 1,2 The energy transfer from N4 porphyrin unit to core modified porphyrins also will be presented.

Lecture Abstracts

References 1 Ravikanth M, Tetrahedron Lett, 41 (2000) 3709. 2 Ravikanth M, Agarwal Neeraj & Kumaresan D, Chern Lett, (2000) 836.

Geometric and electronic structure contributions to function in bioinorganic chemistry: Active sites in non-heme iron enzymes

Edward I Solomon

Department of Chemistry, Stanford University, Stanford, CA 94305 [email protected]

83

Non-heme iron active sites are found in a wide range of enzymes which perform different biological functions requiring dioxygen. These reactions often involve dioxygen activation by a ferrous active site which is generally difficult to study with most spectroscopic methods. A new spectroscopic methodology has been developed utilizing variable temperature and variable field magnetic circular dichroism (VTVH MCD) which enables one to obtain detailed insight into the geometric and electronic structure of the non-heme ferrous active site and probe its reaction mechanism on a molecular level. This spectroscopic methodology will be presented and applied to a number of key mononuclear non-heme iron enzymes leading to a general mechanistic strategy for O2 activation. These studies will then be extended to consider the new features present in the binuclear non-heme iron enzymes and applied understand the mechanism of the two electron/coupled proton reaction of dioxygen binding to hemerythrin and structure/function correlations over stearoyl-ACP 119 desaturase, ribonucleotide reductase and methane monooxygenase. Electronic structure/reactivity correlations for O2 activation by nonheme iron enzymes will also be considered.

References I Neese F, Zaleski J M , Loeb-Zaleski K & Solomon E I, JAm chem Soc, 122 (2000) 11703. 2 Yang Y, Baldwin J, Ley B A, Bollinger J M &, Solomon E I, JAm chem Soc, 122 (2000) 8495 3 Solomon E I, Brunold T, Davis M I, Kemsley J N, Lee S-K, Lehnert N, Nesse F, Skulan A J, Shan Y-S & Zhou J, Chem Rev, 100

(2000) 235. 4 Brunold T, Solomon E I, JAm chem Soc, 121 (1999) 8277 5 Brunold T & Solomon E I, JAm chern Soc, 121 (1999) 8288. 6 Zhou J, Gunsior M, Bachmann B 0 , Townsend C A & Solomon E I, J Am chern Soc, 120 (1998) 13539 7 Kemsley J N, Mitic N, Zaleski K Loeb, Caradonna J P & Solomon E I, JAm chern Soc, 121 (1999) 1528. 8 Yang Y-S, Broadwater J S, Fox B G &. Solomon E T, JAm chern Soc, 121 (1999) 2770 9 Neese F & Solomon E I, JAm chem Soc, 120 (1998) 12829.

10 Brunold T C, Tamura N, Kitajima N, Moro-oka Y & Solomon E I, JAm chem Soc, 120 (1998) 5674 II Solomon E I, Zhou J, Neese F & Pavel E G, Chemistry and Biology (1997) 795. 12 Pavel E G, Kitajima N & Solomon E I, J Am chern Soc, 120 (1998) 3949.


The catalytic mechanism of cytochrome oxidase

Denis L Rousseau

Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461

Cytochrome c oxidase, the terminal enzyme in the electron transfer chain, catalyses the reduction of oxygen to water in a multiple step process by utilizing four electrons from cytochrome c. The enzyme contains four redox centres consisting of CUA (a binuclear copper centre), heme a (a low spin heme group), and a heme a3 -- CUB binuclear centre where the oxygen reduction process takes place. To study the reaction mechanism, the resonance Raman spectra of the intermediate states were measured during single turnover of the enzyme' . By measuring the change in intensity of lines associated with heme a, the electron transfer steps were determined and found to be biphasic. The time dependence for the oxidation of heme a and for the measured formation and decay of the oxy, the ferryl ("F") and the hydroxy intermediates were simulated by a simple reaction scheme. In this scheme, the presence of the "peroxy" ("P") intermediate does not build up a sufficient population to be detected because its decay rate is too fast in buffered H20 at neutral pH. A comparison of the change in the spin equilibrium with the formation of the hydroxy intermediate demonstrates that it is high spin. A large change in the frequency of the vibrational mode associated with the formyl substituent upon reduction of heme a3 was detected in a quinol oxidase, acidianus ambivaleni. Based on these experiments, a new model for proton translocation is proposed involving protonation/deprotonation of histidine that coordinates to the CUB atom form the binuclear centre.

References I Han S, Takahashi S & Rousseau D L, J bioi Chern, 275 (2000) 1910. 2 Das T K, Gomes C M, Teixeira M & Rousseau D L, Proc Nat Acad Sci, USA 96 (1999) 9591.

Some new aspects of the reaction mechanisms in heme peroxidases as studied with their site-directed mutants and new rapid solution mixing and

freeze-quench devices

1sao Morishima*, Manabu Teramoto, Masato Shintaku, Motomasa Tanaka, Satoshi Takahashi & Koichiro 1shimori

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan

Peroxidases are members of hemeproteins that catalyze one-electron oxidation of various substrates by utilizing hydrogen peroxide as an oxidant. They react with hydrogen peroxide to yield a reaction intermediate, compound I, in which one oxidant equivalent is commonly stored as a ferryl iron and another oxidation equivalent forms a porphyrin or protein radical. One of the typical peroxidases, horseradish peroxidase(HRP), bears a radical centre on tyrosine near heme and a cytochrome c peroxidase(CcP) has the tryptophan radical in the heme vicinity . To examine the regulation mechanism of the radical centres in peroxidases, we introduced Tyr or Trp residue in the two positions of HRP. One of the positions is Phe179, whose relative position to the heme is rather similar to that of Tyr385 in PGHS. Another location is Phe221, in which introduction of Trp was demonstrated to induce the Trp radical as observed for CcP in our previous work'. HRP F179Y mutant was found to yield the typical porphyrin radical, followed by subsequent transfer to the Tyr site, as confirmed by the time resolved UV -vis and ESR spectra combined with the rapid-mixing/freeze-quench technique. The F179W mutant, in contrast, generated only the porphyrin radical. The F221 Y mutant afforded the compound I spectra indistinguishable from those of WT HRP. Consequently, Tyr radical was observed only in the F179Y, while Trp radical was formed only in the F221 W mutant. These observations suggest that distance is not the only factor to determine the radical transfer in peroxidases. Other factors such as orientation of the phenyl ring and the stability of the radicals including dielectric environment around the heme and the Tyr or Trp residue would be crucial.

Lecture Abstracts 8S

In an attempt to clarify the formation mechanism of compound I in HRP, we developed a new freeze-quench device which can interrupt chemical reactions within -1 ms after mixing two parent solutions as well as a new solution mixing device for measuring short-lived (-SOils) reaction intermediate with UV -Vis spectra. Our results indicated that the heterolytic cleavage of the 0-0 bond in compound a could be formulated as Fe3+H20 2 rather than Fe3+OOH. We suggest that the lifetime of compound a at room temperature is shorter than SOils. Furthermore, the EPR observation of compound I frozen immediately after the mixing leads us to propose a pH dependent conformational change in the heme pocket of compound I.

Reference 1 Morimoto A, Tanaka M, Takahashi K, Ishimori K, Hori H & Morishima I, J bioi Chem, 273 (1998) 14753.

Conformational stability of horseradish peroxidase

K Chattopadhyay & S Mazumdar*

Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005 , India

The plant peroxidase, horseradish peroxidase (HRP) consists of a protoheme prosthetic group at the active site. The same heme prosthestic group is present in the oxygen binding protein myoglobin. The structure and stability of the heme cavity are however widely different in these two proteins, mainly because of differences in the interactions between the heme and the surrounding amino acids. Detailed circular dichroism and fluorescence studies at different pH have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme c (HRPc) to understand these interactions'. The change in the CD at 222 nm region corresponds to changes in the overall secondary structure of the enzyme, while that at 400 nm region (Soret region) corresponds to changes in the tertiary structure around the heme in the enzyme. Temperature dependence of the tertiary structure around heme also affected the intrinsic tryptophan fluorescence emission spectrum of the enzyme.

The results suggested that melting of the tertiary structure to a pre-molten globule form of the enzyme takes place at 4SoC, which is much lower than the temperature (Tm=74°C) at which depletion of heme from the heme cavity takes place. The melting of the tertiary structure was found to be associated with a pKa of -S indicating that this phase possibly involves breaking of the hydrogen bonding network of the heme pocket keeping the heme moiety still inside it. The stability of the secondary structure of the enzyme was also found to decrease at pH below 4.S. A 'high temperature' unfolding phase was observed which was however, independent of pH. The stability of the secondary structure was found to drastically decrease in the presence of DTT (dithiothreitol) indicating that the 'high temperature' form is possibly stabilised due to inter-helical disulphide bonds. Depletion of Ca2+ ions resulted in a marked decrease in the stability of the secondary structure of the enzyme.

Reference 1 Chattopadhyay K & Mazumdar S, Biochemistry, 39 (2000) 263.

Proton-conducting channels in cytochrome oxidase

Robert B Gennis

Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA

Cytochrome oxidase catalyzes the reduction of dioxygen to water, and converts the available free energy to a transmembrane proton motive force. This is done in two ways. Charge separation across the membrane is generated by the enzyme having the source of electrons and protons used for the chemical reaction which originates on opposite sides of the membrane. The electrons arrive at the buried active site from cytochrome c, via the CuA and heme at metal redox centres. The protons are directed to the enzyme active site by protonconducting channels, taking protons from the side of the membrane opposite to the cytochrome c binding site.


This results in the equivalent of transferring four full charges across the membrane per dioxygen molecule that is reduced to two waters. A second mechanism used by the enzyme is a proton pump, which transfers an additional four protons across the membrane per dioxygen reduced. In order to accomplish this, protons are directed to the active site by key functional elements of the enzyme--the proton-conducting channels. These have been defined by the use of site-directed mutagenesis on the bacterial oxidases, and are also observed in the X-ray structures of the oxidases. There are two channels, called the K-channel and the D-channel. These are utilized by the enzyme during different parts of the catalytic cycle. New data resulting from the collaboration of Dr A Konstantinov and our group has demonstrated that the K-channel is required for protonic equilibrium between the enzyme active site and the bulk aqueous phase when the enzyme is in the oxidized state. After the reaction of the reduced enzyme with dioxygen or upon reaction of the oxidized enzyme with hydrogen peroxide, in either case forming an oxygenated heme intermediate, the K-channel is closed. These data will be presented.

Cytochrome c oxidase - electron entry and proton pathways

Bernd Ludwig

Molecular Genetics, Biocenter University of Frankfurt, Germany

Cytochrome c oxidase couples electron transport to the generation of an electrochemical gradient across the mitochondrial inner membrane, or the cytoplasmic membrane of bacteria. The structure of the four-subunit enzyme from paracoccus denitrificans has been determined from 3-D crystals and is easily amenable to sitedirected mutagenesis to study its structure/function relationship.

Interaction with its substrate, cytochrome c, is mainly governed by electrostatic interactions of oppositely charged side chains on either partner protein, but hydrophobic residues participate in the fine tuning of the electron transfer complex as well, as shown by a set of specific mutants. In particular, an exposed tryptophan residue on subunit II of oxidase has been characterized as the entry point of electrons reaching the CUA centre. With the availability of a soluble fragment of the genuine (bacterial) donor, cytochrome C552 of P. denitrificans, we are able to compare ET kinetics employing both the commonly used horse heart and the bacterial cyt c.

There is ample evidence for two different proton pathways operating; a total of eight protons are being taken up during a complete oxygen cycle from the inside to reach the binuclear centre of oxidase for water formation, and to be translocated all across the membrane. While a functional assignment to either pathway is still controversial, specific mutants along the presumed D-channel show partial or complete uncoupling and further track down the extension and exit region of the D-pathway.

Rational molecular design of a catalytic site: Engineering of catalytic functions to the myoglobin active site framework

Yoshihito Watanabe

Insti tute for Molecular Science, Myodaiji, Okazaki, 444-8585, Japan

Proteins that contain the heme prosthetic group are responsible for different types of catalytic actIVIty. Understanding the mechanisms through which a particular type of catalytic activity is favoured over the others remains a significant challenge. Recently, the most common strategy for structure-function studies for a particular enzyme has involved substitution of amino acid residues by site-directed mutagenesis followed by investigations of the effect of the substitution on the catalytic activity of that system. We have taken significant departure from this common strategy. Instead, we seek to convert a non-enzymatic hemoprotein into one that is capable of catalytic activity. In doing so we expect to gain an understanding of the general structural requirements for particular enzymatic functions .


Comparison of X-ray crystal structures of myoglobin and peroxidases reveals differences in arrangement of amino acid residues in the heme pockets. On the basis of these structural differences and the reaction mechanism of peroxidases, we have rationally designed several myoglobin mutants in order to convert myoglobin into a peroxidase-like enzyme. We have discovered that the location of the distal histidine in the active site provides a critical balance between the formation and subsequent decay of the oxo-ferryl porphyrin radical cation (compound I), a catalytic species for one- and two-electron oxidation and oxygen transfer reactions. In addition, the replacement of the distal histidine to aspartic acid has been carried out to prerpare a model for chloroperoxidase. The mutants prepared in this work have permitted compound I to be observed in myoglobin for the first time. This allows us to investigate mechanistic details under single turnover conditions by use of double mixing stopped-flow spectroscopy. Furthermore, some of the mutants we have constructed might be useful as good catalysts for asymmetric oxidations.

Crystal structures and functionality of ligninolytic laccase and lignin peroxidase: A common mechanism for the tuning of the redox-potential

Klaus Piontek*, Matteo Antorini, Wolfgang Blodig & Andrew T Smith t

Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), CH-8092 ZUrich, Switzerland School of Biological Sciences, University of Sussex, Brighton, BN I 9QG, UK

Laccases (Lac) and lignin peroxidases (LiP) are used by filamentous fungi to degrade the recalcitrant biopolymer lignin which is a major constituent of woody plants. Lignin is build up by phenyl propanoid units, which are linked by various non-hydrolysable C-C- and C-O- bonds. Lignin peroxidase is a heme containing glycoprotein utilizing hydrogen peroxide as co-substrate to attain its high redox state needed for activity. Laccase on the other hand is a polyphenol oxidase which catalyses the one electron oxidation of a variety of phenolic and inorganic compounds, with the concomitant four-electron reduction of oxygen to water. The latter protein belongs to the family of blue multi-copper enzymes. Due to their enlarged substrate range, in particular in the presence of specific mediators and due to their high redox potential the fungal metalloenzymes LiP and Lac have the potentiality for the application in various industrial processes. We have been engaged in structuralfunctional work on LiP since some years and have determined the crystal structures of some isozymes and of some variants. Previously, our investigations resulted in the identification of a unique modification on Trp 171 in LiP. Using site-directed mutagenesis and protein chemistry we could show that this residue is essential for the oxidation of the natural substrate veratryl alcohol (VA), but not for artificial substrates. Furthermore, we could define the interaction site of V A with LiP and moreover the exact site of radical formation during the redox cycle. Our work also provides a solution to the long-sought way how electrons are transferred from the buried heme to bulky substrates like lignin, which can not approach the heme co-factor. More recently, we have extended our interest towards fungal laccases, which resulted in the first crystal structure determination of a laccase in its glycosylated, fully functional form, containing a full complement set of coppers. In the first part of this lecture the crystal structures of the oxidized form and of the ascorbate-reduced form of laccase from Trametes versicolor will be presented. The latter was trapped upon flash freezing with the substrate, which was found in a pocket in proximity to the type-l copper. The positioning of the substrate in the binding pocket is stabilized by several hydrophobic interactions, in particular with phenylalanins and by hydrogen bonds with His485, being one of the Tl Cu ligands, and with a carbonyl oxygen. By comparing the substrate bound Lac structure with that of ascorbate oxidase, their respective substrate specificity can be explained. Furthermore, a description of the likely structural features giving rise to the high EO value of Trametes versicolor laccasewill be presented. The second part of the lecture will address some structural and functional aspects of LiP, which were mentioned above. A previously suggested mechanism for the regulation of the redox-potential in heme peroxidases will be compared to the one proposed to function in laccases.


Characterization of the cytochrome c:Apaf-l binding interaction

Cherie Purring-Koch, Tianning Yu & George McLendon* Princeton University, Department of Chemistry, Princeton, NJ 08544

In 1996, a discovery was made that cytochrome c, a critical protein in the process of mitochondrial electron transport, also plays a crucial role in the mitochondrial pathway of apoptosis through interactions with the protein Apaf-l (Liu, et ai., 1996). This work provides a detailed characterization of the binding interaction between cytochrome c and Apaf-l, including information on the stoichiometry and free energy of cytochrome c:Apaf-l complex formation, and on the roles of dATP, pH, and ionic strength in regulating protein binding and apoptotic function (Purring, etai., 1999; Purring-Koch & McLendon, 2000; Yu, et aI., 2001).

Fluorescence polarization studies show that cytochrome c binds to Apaf-l with a 2: 1 cytochrome c:Apaf-l stoichiometry. Cytochrome c:Apaf-1 binding is extremely strong; the binding association constant is measured of the order of 1010 M· I

. Fluorescence polarization experiments also demonstrate that cytochrome c binding to Apaf-l is highly specific. While horse heart cytochrome c binds to Apaf-l with a binding constant of 1010 M· I

,

the highly conserved cytochrome c homologue in yeast has an Apaf-l binding constant at least three orders of magnitude lower (Purring, et ai., 1999).

The nucleotide dATP is necessary for the apoptotic function of cytochrome c and Apaf-l (Zou, et ai., 1999). The role of dA TP is determined using fluorescence polarization, fluorescence quenching and chemiluminescence techniques. These results show that dATP is not a necessary co-factor for cytochrome c binding to Apaf-l, however dA TP is necessary for the cytochrome c:Apaf-l complex to induce apoptosis through activation of procaspase-9. The presence of dATP causes the cytochrome c:Apaf-l complex to oligomerize to form a larger species consisting of approximately 8-10 Apaf-l molecules with associated cytochrome c. This complex, referred to as the "apoptosome", is responsible for recruitment of procaspase-9.

Ionic strength experiments demonstrate that there is a significant electrostatic component to the binding energy in the cytochrome c:Apaf-1 interaction. Increasing the salt concentration from 0 to 200 mM decreases the binding constant by about 4 orders of magnitude. Apaf-l activation of procaspase-9 is also pH dependent. Decreasing pH from 7.5 to 5.7 results in increased apoptosome formation. In vitro procaspase-9 activation assays suggest that at decreased pH, Apaf-l can activate procaspase-9 in the absence of cytochrome c (PurringKoch & McLendon, 2000).

Refernces 1 Liu X, Naekyung K, Yang J, Jemmerson R & Wang X, Cell, 86 (1996) 147. 2 Purring-Koch C & McLendon G, Proc Natl Acad Sci, 97 (2000) 11928. 3 Purring C, Zou H, Wang X McLendon G, J Arn chern Soc, 121 (1999) 7435.-4 Yu T, Wang X, Purring-Koch C, Wei Y &, McLendon G, J bioi Chern, 276 (2001) 13034. 5 Zou H, Li Y, Liu X & Wang X, J bioi Chern, 274 (1999) 11549.

The structural dynamics of myoglobin

Maurizio Brunori Dipartimento di Scienze Biochimiche, Universita di Roma "La Sapienza", Piazzale Aldo Moro 5, 00185 Roma, Italy

Conformational fluctuations have been invoked to explain the observation that the diffusion of small ligands through a protein is a global phenomenon, as suggested (for example) by the oxygen induced fluorescence quenching of buried tryptophans. In enzymes processing large substrates, a channel to the catalytic site is often seen in the crystal structure; on the other hand in small globular proteins, it is not known if the cavities identified in the interior space are important in controlling their function by defining specific pathways in the diffusion to the active site. This point is addressed in this paper, which reports some relevant results obtained on myoglobin,


the hydrogen atom of molecular biology. Protein conformational relaxations have been extensively investigated with myoglobin because the photosensitivity of the adduct with CO, O2 and NO allows us to follow events related to the migration of the ligand through the matrix. Results obtained by laser photolysis, molecular dynamics simulations, X-ray diffraction of intermediate states of wt type and mutant myoglobins are briefly summarized. Crystallographic data on the photochemical intermediate of mutants of sperm whale myoglobin show the photolyzed CO sitting in two of the Xe-binding cavities, removed from the heme group. These results support the viewpoint that pre-existing "packing defects" in the protein interior playa major role in controlling the dynamics of ligand binding, including oxygen, and thereby acquire a survival value.

Time resolved spectroscopy of nitrogen cycle enzyme

Roger N F Thorneley

Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH, UK

The enzymes of nitrogen cycle are of agronomic and environmental importance in terms of increased food production (nitrogen inputs), nitrogen loss from the soil by leaching leading to ground water contamination by nitrates with associated eutrophication of lakes, and denitrification that produces oxides of nitrogen that contribute to global warming.

There are now high resolution X-ray structures of several nitrogen cycle enzymes. However, the mechanisms by which they function remain unclear. Two key nitrogen cycle enzymes, nitrogenase and nitrite reductase (cyt, cd l ) and a NO binding protein (cyt c') will be used to illustrate the power of time resolved spectroscopic techniques to give new insights into the mechanism of action of metallo enzymes and proteins. A relatively new technique, stopped-flow infrared spectroscopy (SF-FfIR), will be described and the information it has yielded on small molecule binding and activation at metal sites in these systems have been reviewed I .

Nitrogenase, the enzyme that reduces dinitrogen to ammonia hydrolyses 16 equivalents of MgATP to MgADP + Pi for each N2 reduced to 2 NH3 with concomitant reduction of 2H+ to H2. Stopped-flow spectrophotometry and fluorimetry have defined the partial reactions of the nitrogenase Fe-protein cycle in which MgATP hydrolysis is coupled to electron transfer from the nitrogenase Fe-protein to the other component of introgenase, the MoFe-protein. Similarities between nitrogenase and other energy transducing systems (p21ras, actomyosin, kinesin) will be discussed.

Nitrite reductase from Paracoccus pantotrophus is a respiratory enzyme that catalyses the one-electron reduction of nitrite to nitric oxide. The enzyme is a dimer, each monomer containing c-type cytochrome centre and one active site dl haem. SF-FfIR, SF-spectrophotometry and rapid freeze EPR have detected and characterized a transient cFe(III)dIFe(II)-NO species and a subsequent electron redistribution from the d l to the c-haem2.

Cytochrome c' from Alcaligenes xylosoxidans, is a periplasmic protein containing a 5-coordinate c-type haem with histidine as a solvent exposed fifth ligand. SF-FfIR3 and protein crystallographl have shown that although NO binds initially to the distal side of the haem, a rapid isomerisation follows resulting in a final product with NO bound to the proximal side having displaced the histidine. By contrast, CO binds to the distal side. These observations may have implications for the activation of soluble guanylate cyclase induced by NO and CO binding to the haem domain.

References 1 Thomeley R N F & George S J, Time resolved infrared spectroscopy of functioning nitrogenase in nitrogen fixation in bacteria:

Cellular and molecular Biology, edited by E Triplett (Horizon Scientific Press, Wymondham, U K) 2000, pp 81 .

2 George S J, Allen J W A, Ferguson S J & Thorneley R N F, J bioi Chern (In press, 2000).

3 George S J, Andrew C R, Lawson D M, Thomeley R N F & Eady R R, JAm chern Soc (to be communicated, 2001).

4 Lawson D M, Stevenson C E M, Andrew C R & Eady R R, EMBO J (Submitted, Aug 2000).


Oxygen utilization by novel hemeproteins*

Tapan K Das

Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Ave, NY 10461

About two billion years ago, the atmospheric oxygen level increased rapidly as a result of photosynthesis by primitive organisms. Oxygen chemistry in biological systems then started and a new class of proteins and enzymes evolved that utilized oxygen as a part of the cellular metabolism of their parent organisms. One of the most well known examples of oxygen utilization is oxygen activation by respiratory heme-copper oxidases that harness energy from the dioxygen chemistry.

Hemoglobin and myoglobin in mammals are known to utilize oxygen by binding it reversibly and then transporting or storing it for various cellular processes. In recent years, hemoglobin has been discovered in almost all kingdoms of life. Although named hemoglobin, these proteins in non-vertebrates have very low sequence similarity to those in vertebrates. The most striking difference is their ligand binding properties; some of these hemoglobins bind oxygen too tightly (oxygen dissociation rate - 0.01-0.004 S·I) to serve as oxygen transporters. At this time, very little is known about the physiological function of these hemoglobins in different organisms, although a variety of novel functions including oxygen scavenging/sensing/detoxification, metabolism of oxygen/nitric oxide, and as a respiratory component have been proposed ..

We have examined several non-vertebrate hemoglobins from different phyla in an attempt to understand their biological functions. In hemeproteins, the stereochemistry of the neighbouring groups in the heme pocket primarily dictates its function. Thus, one of the most vital clues to discern hemoglobin function lies in the structure and stability of its ligand-bound complexes. A multitude of spectroscopic and kinetic technique in conjunction with single-point mutagenesis has been used to study the structure-function aspects of these hemoglobins. The physicochemical properties of these hemoglobins vary widely from one phylum to another. A comprehensive view of the possible biological functions derived from structural and kinetic data will be presented.

Characterization of spectroscopically quiet metals: Zinc in biological systems

James E Penner-Hahn*, Katrina Peariso, Daniel Tobin & Stephanie Mann

University of Michigan, Ann Arbor, Michigan 48109-1055 USA

Zinc is the most common trace element and is the only transition metal known to be required for at least one enzyme in each of the major classes of enzymatic activities. Unfortunately, characterization of Zn(II) in biological systems is challenging. Zinc is found only in the Zn(II) oxidation state, and thus, as a dlo ion, is "silent" to most traditional spectroscopies. This makes the characterization of Zn sites extremely difficult in the absence of a crystal structure. X-ray absorption spectroscopy, including both extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge structure (XANES) is the principal exception to this generalization, and both EXAFS and XANES have found frequent application in the characterization of biological Zn sites. This presentation the recent advances in Zn by EXAFS and XANES, and describes the applications of these to the characterization of the nucleophilic Zn site in a novel class of Zn methyl-transfer enzymes. In addition, recent work on in vivo characterization of Zn speciation will be described.

* This work was supported by the National Institute of Health, USA and done in collaboration with Denis Rousseau, Manon Couture, Jonathan Wittenberg, Beatrice Wittenberg, Joel Friedman, Jack Peisach, Caroline Lee (AECOM, New York), Michel Guertin (Laval Univ, Canada), Luc Moens (Univ Antwerp, Belgium), Roy Weber (Univ Aarhus, Denmark), Kiyoshi Yamauchi (Shizuoka Univ, Japan), Robert Hill (Univ Manitoba, Canada) and Daniel Goldberg (Washington Univ School of Med, SI. Louis).


Zinc binding mechanism of the nucleocapsid protein of HIV -1: An equilibrium & kinetic investigation

Yves Mely

UMR 7034 CNRS, Universite Louis Pasteur, Faculte de Phannacie, 74, route du Rhin, 67401 Illkirch Cedex, France

With the exception of spumaretroviruses, the nucleocapsid proteins of all retroviruses are characterized by one or two copies of a highly conserved retroviral-type zinc finger motif, also called CCRC motif. In the human immunodeficiency virus type 1, the nucleocapsid protein, NCp7, is characterized by two CCRC motifs that have been shown to strongly bind zinc ions in mature viruses 1. The binding of Zn2

+ to these motifs induces a sharp transition from an unfolded to a stable and highly constrained structure2

• NCp7 is critically involved in the encapsidation, the reverse transcription and the integration steps of the viral life cycle3

. Most of the functions of NCp7 strongly depend on the presence and the integrity of the zinc-saturated finger motifs. Accordingly, these motifs constitute promising targets for an antiviral chemotherapy. In order to design new strategies aimed to impair the binding of Zn2

+ to NCp7, an increased knowledge of the physicochemical basis of Zn2+ coordination

to NCp7 is necessary. To this end, we investigated the zinc binding properties of (35-50)NCp7, a peptide corresponding to the C-terminal finger motif of NCp7 (refs 4,5). This peptide is characterized by a single Trp residue that constitutes a sensitive intrinsic fluorescent probe for the binding of zinc. Using steady-state fluorescence and stopped-flow measurements, we determined the equilibrium and kinetic constants of the binding process at various pR. These parameters were compared to those of single-point mutants where one of the zinc-coordinating residues has been replaced by a noncoordinating residue (manuscript in preparation). Moreover, steady-state and time-resolved fluorescence measurements performed on these mutants provide some clues on the structure of the wild-type intermediates in the binding process. Taken together, our data allowed us to propose a mechanism of binding of Zn2

+ to the CCRC finger motif.

References I Summers M F, Henderson L E, Chance M R, Bess J W, South T L, Blake P R, Sagi I, Perez-Alvarado G, Sowder R C I, Hare D R &

Arthur L 0 , Protein Sci, I (1992) 563. 2 Morellet N, de Rocquigny H, Mely Y, Jullian N, Dememe H, Ottmann M, Gerard D, Darlix J L & Roques B P J molec Bioi, 235

(1994) 287. 3 Darlix J L, Lapadat-Tapolsky M, de Rocquigny H & Roques B P, J molec Bioi, 254 (1995) 523. 4 Mely Y, de Rocquigny H, Morellet N, Roques B P & Gerard D, Biochemistry, 35 (1996) 5175. 5 Bombarda E, Morellet N, Cherradi H, Spiess B, Bouaziz S, Grell E, Roques B P & Mely Y, (Submitted,2001).

The mechanism of protein mediated electron transfer

Gerard W Canters, Sharmini Alagaratnam, Lars leuken, Pieter van Vliet, Irene van Amsterdam & Marcellus Ubbin*

Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands

This contribution deals with two topics: A) the relation between structure and mechanistic properties of type-l copper sites and B) the construction of electron transfer (ET) maquettes and their ET properties.

A. As soon as the first 3D-structure of a blue copper protein became known, speculation started about the precise way "Nature" had tailored the blue copper site to its function as an ET centre: the coordination geometry of the copper ion could be best understood by describing it as a compromise between the preferred Cu(I) and Cu(II) geometries. Thus, the blue site presented a frozen transition state eminently adapted for the redox switches accompanying physiological turn-over. The cost of maintaining this transition state was paid for by the protein, while the high redox potential was the result of the stabilisation of Cu(I) over Cu(II) state by the protein.

Two recent types of experiments that shed new light on these ideas will be presented. Loop-directed mutagenesis experiments have shown that precise tailoring of the metal site by the protein is not necessary for


ET turnover rates at physiological speed. Further, cyclic voltammetry experiments on protein films of an azurin mutant in which a coordinating histidine has been excised and replaced by a variety of external ligands show that the coordination geometry of the copper as observed in blue copper proteins actually stablilizes Cu(IJ) over Cu(l) state. The functional picture of a blue copper protein that emerges from these and other experiments is similar to that of a cytochrome: the redox centre is accessible at the rim of a metal ligand that pierces through the protein surface. The protein surface surrounding this patch can be used to engineer specificity for encounters with partners.

B. Recent endeavours have focussed on the study of protein mediated ET by studying the structural details of the association complex of the reaction partners and by constructing homo- and hetero-dimers of redox proteins. The dimers have been realised by a) the engineering of a covalent peptide link, b) the linking of prosthetic groups by means of an organic linker (,hot wire') or c) the application of a disulphide bridge. This allows to look into the effect of the conformation of the complex on the ET process within a precisely defined construct. It appears that a range of electronic couplings can be engineered: total absence of coupling in cases which, at least on paper, should give a high ET rate, as well as strong electronic coupling across surface patches or through covalent paths leading to high intra-molecular rates. The results shed new light on the dependence of the ET process on the structural details of the association complex.

References I Jeuken L J C, et at.,J moiec Bioi, 299 (2000) 737. 2 Buning C, et at. ,i Am chem Soc. 122 (2000) 204. 3 Canters G W, et at..Faraday Discussion 116 (In press, 2000). 4 leuken 1 C, et al.,J Am chem Soc. (In press, 2000).

Structure, function and flexibility of the metal centre in some blue copper-proteins

E Danielsen* & R Bauer Department of Mathematics and Physics, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871

Frederiksberg C. Denmark www.matfys.kvl.dkl-eva

Electron transport in biological systems is a task that is often carried out by blue copper-proteins with the copper ion in a type 1 geometry. Knowledge of the details of the metal site geometry is important for understanding the regulation of the redox-potential of the electron transporting proteins, but, as it will be shown in the talk, the interplay between the charge of the metal ion and the protein can also be important for regulating the binding of the electron transporting protein to the redox-partner l

.

These conclusions rely on combined studies of the metal-sites of azurin and plastocyanin, carried out with the technique Perturbed Angular Correlations of y-rays (PAC). This technique offers a method for comparing the metal-site structure for a monovalent ion to that of a divalent ion and at the same time study the interaction of the electron transporting protein to a larger protein . In PAC, the naturally occurring metal ion is substituted by radioactive Cd or Ag and the nuclear quadrupole interaction of the radioactive nuclei is used to study the structure and dynamics of the metal-site. These studies have shown that Ag(l) and Cd(IJ) in azurin have geometries that are very similar to copper in native azurin2

• A number of mutants of azurin in which methionine has been substituted have also been studied, and the results show that though methionine does not seem to coordinate to Cd, it plays an important role in stabilising the metal-site and excluding ligands from the solvene.

Ag(l) and Cd(IJ) plastocyanin also have the characteristic geometry of a copper ion in a type 1 copper-protein, but with a very characteristic peak broadening interpreted as motion of one of the histidines in the plane. Furthermore, the results of the PAC-experiments suggest that one of the histidines has a different position in the Ag protein than in the Cd protein, and that for unbound plastocyanin, this difference relaxes on a time-scale of 20 ns, when Ag decays to Cd. Binding of Ag-plastocyanin to Photosystem I, however, stabilises the Ag metal-


site structure, so that no relaxation takes place on a time scale of 100 ns. This indicates that the metal site structure is involved in regulating how the dissociation constant depends on the charge of the metal ion.

Preliminary studies of Cd(II)-amicyanin suggest that for this protein two different geometries exist at pH 7.5, indicating that this protein is even more flexible.

References I Danielsen E, Scheller H V, Bauer R, Hemmingsen L, Bjerrum M J & Hansson 6, Biochemistry, 38 (1999) 1153l. 2 Bauer R, Danielsen E, Hemmingsen L, Bjerrum M J, Hansson 0 & Singh K, J Am chern Soc, 119 (1997) 157. 3 Danielsen E, Bauer R, Hemmingsen L, Andersen M, Bjerrum M, Butz T, Trtiger W, Canters G W, Hoitink C W G, Karlsson G,

Hansson 6 & Messerschmidt A, J bioi Chern, 270 (1995) 573.

Dioxygen activation and alkane hydroxylation by the copper methane monooxygenase

Sunney I Chan A A Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, CA 91125, USA and

Institute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan

The particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the conversion of methane to methanol in methanotrophic bacteria. Despite its discovery during the '70, its isolation and purification has resisted the attempts of many laboratories. The pMMO is apparently quite unstable and its activity is readily disrupted during isolation and purification. Recently, my laboratory at Caltech (Pasadena, CA) has been successful in purifying the pMMO isolated from methyloccocus capsulatus(Bath) which consists of a three subunit hydroxylase (45, 27 and 23 kDa) and an NADH oxidoreductase (38 kDa). The hydroxylase is a copper protein, with 15 copper ions arranged in five trinuclear copper clusters. Two of the copper clusters are associated with the 27 kDa subunit and are involved in the hydroxylation chemistry of the enzyme. The remaining three copper clusters are associated with the 45 kDa subunit, which provides a buffer of reducing equivalents in the protein and participate in electron transfer, mediating the electron input from the NADH reductase to the catalytic clusters. The E-clusters reside in the water-exposed domains of the protein and could be removed from the protein by incubation of the protein in the presence of proteases. The C-clusters remain in the membrane-bound domain following this treatment. We have recorded and simulated the EPR spectrum of the oxidized C-clusters isolated in this manner and the results are consistent with ferromagnetic ally coupled trinuclear Cu(II) clusters with a zero-field splitting of ca. 300 Gauss. The functional form of the enzyme is fully or partially reduced copper hydroxylase. The pMMO exhibits unusual substrate specificity: only small normal alkenes are hydroxylated and similar alkenes epoxidated. In addition, the chemistry is highly regiospecific as well as stereoselective. It is apparent that the substrate binds to a short and narrow hydrophobic pocket and the hydroxylation chemistry involves concerted binding of the C-H bond to an activated copper cluster and insertion of the "active" oxygen species. A mechanism for the dioxygen activation mediated by the clusters will be proposed.

Design of molecular assemblies of P450 enzymes for high-through-put screening of novel drugs

Gianfranco Gilardi Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2A Y

The cytochrome P450 enzyme family is responsible for the metabolism of a large number of drugs and xenobiotics. These enzymes are highly relevant to human and animal health as they play a key role in the pharmacodynamics of drugs. Despite their importance, structural studies are impaired by problems related to their poor interaction with electrode surfaces and their association to biological membranes. Very recently, a


soluble bacterial P450 enzyme (P450 BM3) has been found to share a high degree of homology with the human systems.

An exciting potential application of these enzymes relies in the creation of electrode arrays for high-throughput screening for propensity to metabolic conversion or toxicity of novel potential drugs. In order to achieve this goal, three issues need to be addressed: (1) the efficient electron transfer of the P450 with the electrode surface, (2) the ability of handling stable and soluble human P450 enzymes, and (3) the availability of a screening method able to identify active variants of P450 to be used to produce arrays with different properties. The first two issues have been addressed with the molecular lego approach. Mimicking the natural molecular evolution that proceeds at DNA level by modular assembly of introns and exons, flavodoxin from D. vulgaris and the soluble haem domain of cytochrome P450 from B. megaterium have been used to produce an efficient, artificial electron transfer chain. The scaffold of this soluble enzyme has been used to build in the key structural and functional elements of the human cytochrome P450 2EI. The chimeric protein containing the reductase and P450 domain has been successfully constructed and expressed. On a related front of research, a rapid screening method able to readily detect the activity of P450 enzymes in whole cells in a microtietr plate format has been developed. This method offers a powerful tool in 1) screening enzymatic activity on various substrates of biotechnological (bioremediation and biosensing) and pharmacological interest and 2) screening libraries of random mutants of NAD(P)H-dependent oxidases to allow design of new catalytic specificities of these enzymes. The validity of the screening method has been tested on a series of furazan derivatives, a class of potential drugs with activity as vasodilators and inhibitors of platelet aggregation. The strength of the method is proven by its ability to distinguish between two isomers of a furazan derivative.

Towards the novel metallofullerene-based probes for EPR, NMR and nuclear medicinet

Vitaly K Koltover

Institute of Problems of Chemical Physics, RAS, Chernogolovka Research Centre, 142432, Moscow Region, Russia ([email protected])

Endohedral metallofullerenes (incarceranes, M@C2n) are carbon clusters that contain metal atoms (lanthanides, Sc, Y, etc.) trapped within a fullerene cage. Recently we have developed a simple extraction scheme that affords to produce M@C2n in large preparative amountsl.2. It has been suggested that M@CS2 be used for measurement of oxygen in chemical and biological systems3

. For example, La@CS2 demonstrates EPR signals that are highly sensitive to p02. Under careful deoxygenation, the solutions of La@CS2 demonstrate octet EPR signals whose individual line-width values are of about 0.01 mT (at room temperature) and increase with the air-oxygen pressure. The effects of the line-width broadening by oxygen are of reversible and linear character that makes it possible to use La@C82 for measurement of oxygen concentrations. In addition, the EPR spectral parameters of La@CS2 were found to be sensitive to its rotational diffusion that makes the basis of applications of this endometallofullerene as the new-type spin probe for studies of local viscosity and phase transitions in organized molecular systems. Furthermore, endometallofullerenes hold much promise as paramagnetic probes for NMR imaging. The Gd(III)-based chelates Gd(DTPA)2- and Gd(DOTAf are in current clinical use as contrast agents for NMR imaging. Meanwhile, Gd@Cs2 endometallofullerene has the similar magnetic properties as the chelates, namely it has a large effective magnetic moment (;::; 7 f.!B) in combination with a long electron spin relaxation time. In addition, the fullerene cage of M@CS2 can be easily modified into the water-soluble forms that provides means for synthesizing the targeted NMR relaxants and shift-agents with solitary biomedical features . Furthermore, M@C2n with the appropriate particle-emitting radionuclides inside can be created, while technological advances in the molecular biotechnology are providing targetting vectors to deliver therapeutic

t Research sponsored by the Russian Foundation for Basic Research, grant 98-03-33243, and the State Program "Current Directions in Condensed Matter Physics", Subprogram "Fullerenes and Atomic Clusters", grant 98078-99010.


doses of the ionizing radiation with high specificity for treatment of secondary or metastasis cancer tumor cells. Thus, endometallofullerenes, due to their unique physical and chemical properties, provide obvious prospects for designing the novel paramagnetic probes and therapeutic radiopharmaceuticals in the coming millennium.

References 1 Laukhina E E, Bubnov V P, Estrin Ya I, et al .. J Mater Chel11, 8 (1998) 893. 2 Koltover V K, Bubnov V P, Laukhina E E, Estrin Ya I, Molecular Materials, 13 (2000) 239 . 3 Koltover V K, Laukhina E E, Estrin Ya J, et aI. , Dokl Akad Nauk - Physical Chemistry, 353 (1997) 85.

Inorganic drug design for hormone responsive cancers

Subhash Padhye

Department of Chemistry, University of Pune, Pune 411 007

Hormone responsive cancers constitute almost fifty per cent of the twelve most common cancers in the world. Steroidal hormones are considered to be the major determinants of the proliferation and development of these cancers. They exert their action through specific intracellular protein receptors found in target tissues known as estrogen receptors (ER). The receptor proteins specific for androgens and estrogens are present in higher concentrations in malignant than in normal cells and hence offer excellent opportunities for the synthetic chemists to evolve targeted drugs by attaching cytotoxic moieties to the receptor binding canier ligand molecules.

Cunent work in our laboratory is aimed at evolving a combinational library of such targetted drug molecules consisting of a canier molecule, the linker chain and the cytotoxic metal conjugates. The synthesized molecules are evaluated for their anti proliferative activities against the ER positive and ER negative cancer cell lines in order to evaluate the efficacy of the approach. The enhanced antiproliferative activities particularly observed for the copper conjugates shall be discussed in the light of their postulated role in preventing dimerization of ER proteins resulting in inhibition of DNA binding and subsequent transcription processes.

Metal ions and the thermodynamics and kinetics of tertiary RNA folding

Tobin Sosnick*, Tao Pan, Xingwang Fang, Valerie Shelton, P Thiyagarajant, K Littrd, S Hendersont

Department of Biochemistry & Molecular Biology, Uni versity of Chicago, t Argonne National Laboratory, t Department of Physics, Uni versity of Colorado, Boulder

Divalent cations playa fundamental role in the stability and folding of tertiary RNAs including ribozymes. We have applied mUltiple spectroscopic, chemical and enzymatic probes to examine the cooperativity and stability of tertiary RNAs. We present a framework to quantify the free energy for tertiary RNA folding using Mg2+ and urea titrations. We describe the compaction process along the Mg2+-induced thermodynamic folding pathway. The kinetic pathway of this and other large RNAs is complex and often fraught with multiple kinetic traps. Intermediates can exist on certain pathways and folding can be under kinetic control. However, we show that a large ribozyme can fold all the way to the biological active state in 0.1 sec (orders of magnitude faster than previously observed) without falling into kinetic traps. We introduce the Mg2+ and urea "chevron" plots and conduct the first complete, quantitative analysis of tertiary RNA folding pathway. A folding scheme containing two kinetic intermediates accounts for all the free energy, number of bound Mg2+ ions, and surface burial of the equilibrium transition. The folding of this ribozyme is best described by a classical pathway. These results indiCate that the conformational research in tertiary RNA folding can be very fast and occur along a smooth energy landscape.


Spectroscopic and voltammetric studies on ruthenium(II) mixed ligand complexes of 1,lO-phenanthrolines and 2,2' -bipyridyl: Enantioselective conformational

transition from B to Z DNA

PUma Maheswari & M Palaniandavar*

Department of Chemistry, Bharathidasan University, Tiruchirappalli , 620024, Tamil Nadu, India

A number of metal chelates have been used as probes for DNA structure in solution, as agents for mediation of strand scission of duplex DNA and as chemotherapeutic agents. The interaction of a variety of kinetically inert Ru(1l) complexes of polypyridine or 1, lO-phenanthroline (phen) ligands with DNA has been studied with an aim to design small, non-radioactive molecules capable of selectively binding to nucleic acids . Such interactions have been characterized largely through spectroscopic and photophysical studies and determination of enantiomeric selectivity associated with binding by the metal complexes. We have used electrochemical methods' to show that B DNA discriminates the enantiomers of [Ru(phen)3]2+. We have interacted 1:2 copper(II) complexes of phen and various methyl substituted 1,1O-phenanthrolines with calf thymus DNA to show that, of all the complexes, the 5,6-dimethyl-l,1O-phenanthroline (dmp) complex reversibly bids to DNA and effects the unexpected conversion2,3 of right-handed B DNA to left-handed Z DNA. In order to throw more light on the factors affecting such interactions, we have now isolated a series of simple as well as mixed ligand dmp complexes of ruthenium(II). The racemic and I':!.- and I':!.-[Ru(dmphf+ and the mixed ligand complexes of the types [RuL2(dmp)f+ [where L = bipyridyl or phen] and [Ru(bipy)(phen)z]2+, [Ru(bipyhCphen)]2+ and [Ru(dmphCpy)zf+ have been isolated and interacted with calf thymus DNA. A variety of physical methods like absorption, emission and circular dichroism (CD) spectral, viscosity measurements and voltammetry methods have been used to monitor the interactions. Electronic absorption spectral method reveals that almost all the complexes with phen ligand show hypochromism and red shift, much higher than the other complexes possibly due to partial interaction of phen ligands. Emission measurements show enhancement in intensity for all the complexes to different extents with the maximum for [Ru(dmph]2+ complex. Interestingly, of all the complex species, only [Ru(dmph]2+ and [Ru(dmp)z(py)z]2+ induce the B to Z transition revealing that the coligands bipy and phen prefer to bind the DNA via surface and partial intercalation, respectively and hinder the complexes from effecting the transition . Obviously, the dmp ligand prefers to interact with DNA backbone. It is found that for [Ru(dmph]2+, the B to Z transition is complete at the [Ru]/[NP] value of 2. Further, 1':!.-[Ru(dmp)3]2+ brought about the complete transconformational change at the lIR value of 1 suggesting that the change is enantioselective. Cyclic voltammetric studies reveal the oxidation of guanine residue on the interaction of the complexes with calf thymus DNA.

References 1 Mahadevan S & Palaniandavar M, Bioconjugate Chern, 7 (1996) 138. 2 Mahadevan S & Palaniandavar M, J chern Soc, Chern Cornrnun, (1996) 2547. 3 Mahadevan S & Palaniandavar M, lnorg Chern, 37 (1998) 3927.

Chemistry of-OH-containing molecules and their metal ion complexes including their reactivities: Our recent efforts from lIT Bombay

Chebrolu Pulla Rao

Department of Chemistry, Indian Institute of Technology, Indian Institute of Technology, Powai, Mumbai, India

Understanding bioinorganic systems indeed demands and deserves a lot of attention in terms of the chemical studies based on appropriate model systems. Performing studies using model systems is not a new phenomenon


and is indeed a practice for a long time and continues to be supported further and strengthened to extend its utility in solving complex problems related to biological systems. In the present context, this is being referred to the role of metal ions in biological systems. Such complex problem-solving deserves inputs from different dimensions wherein chemistry plays a vital role. A primary approach for such studies must involve the design of organic molecules with requisite functional groups, understanding their coordination chemistry, and establishing the structure and reactivity aspects of the products.

In this context we have focussed our attention at a series of different types of -OR-containing molecules (such as those of alcoholic and phenolic types) which are important in the context of their coordination behaviour towards transition metal ions. Thus, the molecules chosen by our group in this regard range from simple Schiff's base, Mannich bases to poly hydroxy molecules such as saccharides and its derivatives to polyphenolic molecules such as calixarenes and its derivatives. Our group, at IIT Bombay has been working on the design and synthesis of organic part and also on the coordination chemistry aspects of these including the structure and reactivity of the products of a number of metal ions, primarily the transition metal ions. Thus, our group has developed considerable amount of coordination chemistry aspects of all these -OR-containing molecules. Correlations have been drawn based on experimental data including those of structure and spectra. This presentation will include our recent results of all these studies with appropriate discussions.

I thank the funding agencies, DST, CSIR and BRNS for their financial support.

learning from metalloenzymes: new homogeneous catalysts ... 41a(1) (lecture abstracts).pdf ·...

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