abstracts poster presentations (n)

Journal of Inorganic Biochemistry 86 (2001) 251

The role of central metal at the interaction of metal loporphyrins with DNA.

S. G. Haroutiunian~ Y. B. Dalyan, V. I. Vardanyan Yerevan State University, Al.Manoogian st.l , 375025, Yerevan, Armenia (e.mail . '[email protected])

For both chemical and clinical reasons the study ofporphyrins and modified porphyrins and their interactions with duplex DNA is an important area of research. Porphyrins comprise an important class of compounds whose chemical and photochemical properties are widely exploited in both medical and biological applications. Due to their ability to accumulate in malignant cells, porphyrins have found utility as diagnostic agents for determination of the tumor design. This peculiarity of porphyrins as well as their photosensitivity have enabled their use in successful photodynamic treatments of tumors. Some porphyrins also been shown to exhibit anti-viral activity.

Many of the important biological and medically relevant properties of porphyrins are dependent on the type of peripheral substituents. For example, some of us previously reported synthesis of water-soluble meso-tetra (4-N- oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their metallocomplexes with Co, Cu, Ni, Zn, which had been shown to have both antibacterial and antifungal activities. Although the anti tumor and viral activities of modified porphyrins have been established, the mechanisms of their interactions with biological macromolecules, in particular with duplex DNA, have not been thoroughly characterized. It has been shown, that the 2N -, 3N -, and 4N - positions of peripheral substituents on the pyridylic ring influences interactions of porphyrin with duplex DNA. In addition the type of the central metal in metallocomplexes of porphyrins is known to strongly influence mechanisms of binding duplex DNA.

By the uv/visible and circular dichroism (CD) spectroscopies methods the interactions of TOEPyP(4), TOEPyP(3) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA was investigated. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by interaction) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni . Results indicate that (4-N- oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.

This investigation was supported by grant CRDF # AB2-2006.

Tertiary structure of DNA in aqueous solution in the presence of platinum containing compounds

S.G. Haroutiunian~ Y.B. Dalyan, T.S. Haroutunyan, A.A. Papoyan Yerevan State University, Al.Manoogian st.l , 375025, Yerevan, Armenia (e .mai l :[email protected])

The characterization of the dependence of the high molecular DNA melting temperature (Tm) from the concentration of cis-diaminedichloriplatinum (II)(cis-DDP), shows that the effect of cis-DDP on DNA markedly differs indifferent ranges of ligand concentration (rb). Three intervals can be recognized: 1. 10 "5 M < r b < 10 -4 M: monotonous increase of melting temperature of DNA on 4-5 degrees; 2. 10 .4 M < rb < 10 .3 M:monotonous downturn of melting temperature of DNA; 3. 10-3M < rb: steep decrease of the melting temperature of DNA. The first two concentration ranges were considered in detail in this work [1,2]. The change in Tm in the first region appeared to be rather surprising. The observed changes of helix-coil transition parameters cannot be explained in term of local alterations of the double helix caused by local, kinetically inert, bidentate and monodentate platinum binding to DNA sequences. The analysis of the melting curve profiles of DNA with high GC-contents suggests the possibility of formation of ring structures in high-molecular DNA. In principle, the possibility of formation of ring structures of DNA can be also considered, The investigation on the three.dimensional structure of DNA is carried out also at extreme pH values.

1. Haroutiunian S.G., Dalian E.B.,Morozov V.F.,Mamasaldalissov Eu.Sh., Shahinian M.S., Akhrem A.A., Lando D.Yu., Messori L., Orioli P. - Inorganica Chimica Acta, 275-276, 510-514 (1998) 2. Haroutiunian S.G., Mamasachlissov Eu.Sh., Morozov V.F., Dalian E.B., Khachikian R.E., Vardevanian P,O., Karapetian A.T., P.Orioli., Bruni B. - Biophysics (Russia), 42,372-376 (1997)

This investigation was supported by grant CRDF # AB2-2006.

252 Journal of Inorganic Biochemistry 86 (2001)

A combined in-situ X-ray Spectroscopy/X-ray diffraction study of the mechanism of 13-hematin formation

I an H a r v e y a and T i m o t h y J. E g a n b

"Syncrotron Radiation Department, CLRC Daresbury Laboratory, Warrington, WA4 4AD, U.K. bDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa. (e-mail: i.harvey@dl, ac.uk)

During its blood stage, the malaria parasite uses host hemoglobin as a food source, releasing and oxidizing heme to hematin. To detoxify the hematin, the malaria parasite, at least in part, converts it to a highly insoluble substance known as hemazoin. This substance is now known to be the same as [3-hematin. Previously we have shown that the formation of [3-hematin in acidic acetate solutions occurs via rapid precipitation of amorphous hematin, followed by slow conversion to crystalline [3-hematin 1.

In this study we have used the combined X-ray spectroscopic/X-ray diffraction facility of the Material Science Station 9.3 at the Synchrotron Radiation Source 2, CLRC Daresbury Laboratory (U.K.) to follow the EXAFS and diffraction of this biomineralization reaction in-situ and in real time. The direct structural data show conclusively that the formation of [3-hematin in acidic acetate solutions occurs as previously described 1. This facility has been used for a number of years by chemists and material scientists to study simultaneously both long and short-range structure of usually solid materials under conditions where chemical, structural or phase changes occur. This experiment demonstrates the feasibility of using such facilities to collect structural data on 'wet' bio-inorganic reactions in real time and is, to our knowledge the first use of such a facility in bio-inorganic chemistry.

1. Egan T., Mavuso W. and Ncokazi, K., Biochem., 40, 203-213 (2001) 2. http://srs.dl.ac.uk/xrs/Stations/Descriptions/stat9 3.html

Chiral recognition of helical metal complexes by cyclodextrins

Hideki Hasegawa, Koji Kano Department of Molecular Science and Technology, 610/0321, Japan (e-mail.'[email protected])

Doshisha University, Kyotanabe, Kyoto

Chiral recognition of M(phen)3n+ (M = metal ion, phen = 1,10-phenanthroline) 1H NMR and capillary zone electrophoresis (CZE). The enantiomers (A and A) of Ru(phen)32+ were separated by CZE using a phosphate buffer at pH 7 containing heptakis(6-carboxymethylthio-6-deoxy)-[3-cyclodextrin (per-CO2--I3-CD) (c~ = 1.83) and hexakis(2,3,6-tri-O-methyl)-a-CD (TMe-c~-CD) (co = 1.05). The binding constant (K) for the A-Ru(phen)32+ complex of per-CO2--[3-CD (K = 1250 M-l) in 0.067 M phosphate buffer at pD 7.0 is 2.1-times larger than that for the A-enantiomer 590 M-l). The thermodynamic parameters for complexation of Ru(phen)32+ with per-CO2--13-CD were determined by van't Hoff plots. Complexation of Ru(phen)32+ with per-CO2--[3-CD shows a large and positive

by cyclodextrins has been studied by

A-Ru(Phenls 2+ A-Ru(Phen)3 2*

entropy change. The positive AS values might be ascribed to dehydration from the hosts and the guests upon complexation. The -AH value for A-Ru(phen)32+ (-AH = 11.4 kJ mol-1) is larger than that of A-Ru(phen)32+ (-AH = 4.41 kJ mol-1). According to ROESY spectra, A-Ru(phen)32+ penetrates more deeply into the cavity at the CO2- group side of per-CO2--[3-CD than A-Ru(phen)32+. Therefore, van der Waals interactions may act more effectively between A-Ru(phen)32+ and per-CO2--[3-CD than between A-Ru(phen)32+ and per-CO2--[3-CD. In the case of TMe-ct-CD, the K value of A-Ru(phen)32+ (K = 108 M-1) is larger than A-Ru(phen)32+ (K = 54 M-1). Complexation of Ru(phen)32+ with TMe-c~-CD shows negative entropy changes. ROESY spectrum suggests that cationic Ru(phen)32+ is shallowly included into the cavity of TMe-c~-CD at secondary OCH3 group side through van der Waals interactions.


Development of a low frequency FTIR electrochemical difference technique using diamond optically transparent electrodes: demonstration of method with

inorganic model compounds

Shannon Hayrno .n..d a, Jerzy K. Zak b, Jason K. Stotter a, Wen-Yuan Hsieh c, Warwick Hillier a, Neil A. Law a, James E. Butler d, Vince L. Pecoraro c, Greg M. Swain a, and Gerald T. Babcock a. "Department o f Chemistry, Michigan State University, 48824-1322, East Lansing, MI, USA bDepartment o f Chemistry, Silesian Technical University, 44-10, Gliwice, Poland CDepartment o f Chemistry, University o f Michigan, 48109-1055, Ann Arbor, MI, USA dChemistry Division, Naval Research Lab, Code 6174. Washington, D.C. 20375-5000

The low frequency (far-IR) region of the spectrum reports on metal-ligand vibrations of metalloenzyme and bioinorganic compound active sites. Our goal is to access this region and indentify redox-dependent vibrations that arise from metal-substrate ligand and metal-cofactor modes. Spectroelectrochemistry offers a well-controlled way to monitor absorbance changes as a function of oxidation state. This poster focuses on the development of boron-doped diamond as an electrode material suitable for transmission spectroelectrochemical measurements. The use of boron-doped diamond (BDD) as an electrode material is a relatively new area of research. BDD is highly electrically conductive, possesses a wide working potential window, and demonstrates well-behaved electrochemical kinetics for a variety of redox systems. As an optical material, diamond is mechanically strong and able to transmit from the visible down to the far-IR (low frequency). Zak, et al have demonstrated the usefulness of BDD as an optically transparent electrode for the UV/Vis region. 2 We will present our efforts to extend the application of this method into the infrared region. The first systems we will investigate with this design are a series of inorganic Mn dimeric compounds, which serve as models of the PSII active site. We will report our findings on the effect of Mn oxidation state on the Mn core structural modes.

I. Granger, M.C., Witek, M., Xu, J., Wang, J., Hupert, M., Hanks, A., Koppang, M.D., Butler, J.E., Lucazeau, G., Mermoux, M., Strojek, J.W., Swain, G.M. Anal. Chem., 72, 3793 (2000).

2. Zak, J.K., Butler, J.E., Swain, G.M. Anal. Chem., 73,908-914 (2001).

Excision and replacement of the radical cofactor and proposed electron transfer pathway from a heme enzyme by sensitizer-linked substrates

Anna-Maria A. Hays a'b, Robin Rosenfeld a, Harry B. Gray b, and David B. Goodin a aDept, o f Molecular Biology, The Scripps Research Institute, La Jolla, CA bDept.of Chemist ry , The Cal i fornia Insti tute of Technology , Pasadena, CA 91 125 (e-mail: haysam@scripps, edu)

Building upon a technique using photosensitizers-linked substrates (1,2), the proposed electron transfer pathway (W191-G192-AI93-A194) that bridges the electron donor (cyt c) and the oxidized radical center (W191) of Cytochrome c Peroxidase (CcP) has been excised, forming an artificial channel and cavity into which complementary sensitizer- linked substrates (SLS) can be introduced. Biochemical characterization of this channel protein shows that binding specificity within the terminal cavity is limited to heterocyclic cations. These cations are then tethered with different amino acid linkers, varying placement of potential H-bonding interactions between the tether and cavity walls. These studies have illustrated that SLS probes, homologous to the native Trp'+-Giy-Ala-Ala, bind to the channel protein demonstrated by both reversible and kinetic trapping assays, yet are highly dependent on the cationic species and nature of the tether. Crystallographic studies are currently underway to further characterize SLS candidates and protein interactions. Photoinduced oxidation or reduction using SLS probes in CcP will allow rapid and direct placement of oxidizing or reducing equivalents into the active site, thus providing a novel approach for providing insight into the kinetics and energetics of redox transitions and elucidating electron transfer pathways in enzymes.

1. Wilker, J.J., Dmochowski, I. J., Dawson, J.H., Winkler, J.R., Gray, H.B. (1999) Angew. Chem. Int. Ed. 38, 90-92 2. Dmochowski, I.J., Crane, B.R., Wilker, J.J., Winkler, J.R., Gray, H. B. (1999) PNAS 96, 12987-12990.

The National Institutes of Health is acknowledged for f'mancial support.


Design of a copper binding site into a heme peroxidase as a model for the heme- a3-CuB center of cytochrome c oxidase

Anna-Maria A. Hays ~'b, Murphy, J.T., Williams, P.A., and David B. Goodin a "Dept. o f Molecular Biology, The Scripps Research Institute, La Jolla, CA 92111, bDept, of

Chemistry, The California Institute of Technology, Pasadena, CA 91125 (e-mail. haysam@scripps, edu)

Cytochrome c peroxidase (CcP) provides a striking parallel to the mechanism by which the dinuclear heme-a3-CUB site of cytochrome c oxidase (COX) reduces O2 to H20 and therefore is used as a protein-based model to construct a triple His site to mimic the a3-CuB site on the distal heine face of CcP. Spectroscopic analysis of this mutant, MP1.3, suggests there is a high-affinity Cu 2+ binding site (KdL - 0.5 ~M), as well as a second binding event (Kd2 = 25 ~tM) involving two additional coppers similar to a Cu z+ site reported by Liu et. al. (1). Furthermore, EPR studies demonstrate that binding of Cu 2+ to MP1.3 results in a dipolar interaction between heine Fe and Cu z+ as seen by a spin state conversion of the heine to low spin, changes in the paramagnetic properties of Cu 2+, and accelerated Cu 2+ relaxation rates. Thus, MP1.3 has been successfully engineered to contain a high affinity site for Cu 2+, placing it near enough to the heme to be rapidly relaxed, but not directly spin coupled. To probe the role of each of the His residues in this complex, second shell interactions are being altered and redox active residues introduced and evaluated for effects on Cu 2+ binding and interaction thereof with the heine group of CcP.

1. Sigman, J.A., Kwok, B.C. Gengenbach, A., and Lu, Y. (1999) J. Am. Chem. Soceity, 121, 8949-8950.

The National Institutes of Health is acknowledged for financial support.


Zinc-mediated methylation signaling of the Escherichia coli Ada protein: metal-substitution and structure

Chuan He, Lijing Sun, Gregory, L. Verdine* Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, 02138, Cambridge, MA, USA (e-mail: [email protected], edu)

The N-terminal domain of the Escherichia coli Ada protein (N-Ada) repairs methylphosphotriesters in DNA by direct transfer of the methyl group selectively to Cys69, a zinc-activated cysteine residue. The methylation of Cys69 increases the binding affinity of Ada to certain promoter DNA and [ o B 72 turns Ada into a transcriptional activator that activates transcription of several ~ 38 s s .

o o S-z~/'2 f2~ methylation resistant genes. To elucidate the mechanism of N-Ada, we have examined ..p~ \ /.. / the effects of metal substitutions on DNA repair and sequence specific DNA binding, o" O-CH~ S - - / r~/ Specifically, cobalt(II)-substituted N-Ada was overexpressed and purified. The DNA ~ HaNt + repair activity, DNA binding affinity and spectroscopic properties of Co-N-Ada were studied; the results of these analyses will be presented. In order to stabelize the ~ N-Ada20 unmethylated N-Ada on DNA for structural studies, a novel strategy to form covalently trapped protein-DNA complex through disulfide cross-linking was employed. The covalent N-Ada/DNA complex was successfully prepared by incubating a mutant N-Ada having an engineered surface exposing cysteine with a double- strained DNA having a modified thiol tether. Structural studies on this covalently trapped complex and the more stable methylated N-Ada/DNA complex will be presented as well.

C. He thanks the Cancer Research Fund of the Damon Runyon-Walter Winchell foundation for a postdoctoral fellowship.

Reactivities of monoporphyrinate lanthanide complexes with some tripodal ligands and shift bases

Hongshan H e a'b, Anxin Hou" Jianpin G u o a and W. K. W o n g a

aDepartment of Chemistry, Hong Kong Baptist University, Waterloo road, Kowloon Tong, Hong Kong (email.'[email protected])

bDepartment of Applied Chemistry, National Huaqiao University, Quanzhou, 362011, P.R. China

Bis(porphyrinate) lanthanides have been investigated extensively, however only a few studies were focussed on the monoporphyrinate lanthanide complexes. Our previous studies ~ indicated that the cationic monoporphyrmate lanthanide could catalyze the cyclotrimerization of phenyl isocynate efficiently. Recently we found that cationic monoporphyrinate lanthanide complexes (Fig 1) prepared by the reaction of Ln[N(SiMe3)2]3.x[LiCI(THF)3] with porphyrin free bases ~'2 can react with other ligands, giving some more stable or some novel complexes. Here we will report the reactivity of the [Ln(Por)(H20)3] toward the ligands NaLoR (Sodium(cyclopentadieny)tris(dialkyphosphito)cobalte,R = Me,Et), KTp R ( Potassiun hydrotris(3,5-dimethylpyrazol-l-yl)borate, R = H, Me),4,4'-dipyridine, Zn-schiff base, etc. The structures of the product determined by the x-ray crystallography revealed that the reaction product were quite different depending mainly upon the nature of porphyrins and ligands.

(~ O)3 -~+ ~ cI-

L_ - ~ A @ ~ Porphydn ring

1. Wong, W K, Zhang L L,Wong W T, Xue F, Mak, T C, J. Chem.Soc.,Dalton Trans, 3053(1999)

2. Wong, W K, Zhang L L,Wong W T, Xue F, Mak, T C, J. Chem.Soc.,Dalton Trans, 615(1999)

Authors thank the Natural Science Foundation of Fujian Province (D001009), Research Foundation of Huaqiao University and Hong Kong RGC for fmancial support.


Spectroscopic characterization of two atypical class II l peroxidases from tea

Hendrik A. Heefing", Marcel A.K. Jansen b, Florence Schneider-Belhaddad c, Andrew T. Smith c, Roger N. F. Thorneley b and Giulietta Smulevich a aDipartimento di Chimica, Universit~ di Firenze, Via G. Capponi 9, 50121 Firenze, ITALY

(e-mail: h.a.heering@tnw, tudelft, nI) bDepartment of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, U.K. CSchool of Biological Sciences, University o f Sussex, Falmer, Brighton BN1 9RQ, U.K.

Two novel class III tea peroxidases with atypical substrate specificity were characterized by resonance Raman (RR), electronic absorption and FTIR spectroscopies. TcAPXII has a very high specific ascorbate peroxidase activity associated with stress response. TPRX1 has an unusual preference for hydrophilic substrates, but not for ascorbate. The enzymes have blue-shifted electronic absorption spectra, high RR frequencies of the core size marker bands, and complex low frequency RR spectra. These characteristics are typical of a 5-coordinate quantum mechanically mixed-spin (5cQS) heme, which among the various peroxidases, is observed exclusively in class III. However, two independent v(Fe-Im) stretches were identified in ferrous TcAPXII, as in pea APX but not in class Ill, where only one v(Fe-Im) is observed. All peroxidases of class III bind benzohydroxamic acid (BHA), a substrate used to probe the aromatic-donor site of plant peroxidases. However, ferric TPRX1 has a very low affinity for BHA (Kd = 14 mM), while no spectral changes were observed for TcAPXII with BHA. The spectra of the fluoride adducts of TcAPXII and TPRX1 suggest that F is strongly H-bonded, which is a characteristic of class I in which the distal Phe of class III is substituted for Trp. Moreover, one of the conformers of the ferrous-CO adduct of TcAPXII also appears to be exceptionally strongly H-bonded, while TPRX 1 has a very low pKa of 8.5 for the alkaline transition to a strongly H-bonded hydroxo low-spin form. These results suggest that a hydrogen-donor residue is present in the active site, which appears peculiar to these tea peroxidases.

This work is supported by the Italian Ministero dell'Universit~ e Ricerca Scientifica e Tecnologica (MURST97 CFSIB) and the European Union, contract FMRX-CT98-0200.

Heme A synthase: a novel oxygenase involved in the biosynthesis of cytochrome c oxidase

Eric L. Hegg, Kenneth R. Brown, Peter H. Do, Behzad Khodaverdian, M. Scott Morrison, Brienne M. Allan Department o f Chemistry, University o f Utah, 315 South 1400 East, Salt Lake City, UT 84112- 0850, USA (email. Hegg@ehemistry. utah.edu)

Cytochrome c oxidase, the terminal oxidase in all plants, animals, and aerobic yeasts, catalyzes the reduction of OE to HE0. In the process, a proton-motive force is generated which is utilized to synthesize ATE The site of OE reduction is an unusual heterobimetaUic center consisting of a copper ion and a heine. Interestingly, this heme molecule is not a typical heme (heme B or protoheme), but rather a derivative known as heme A. The conversion of heme B to heme A requires two important modifications, both of which are absolutely required to obtain active cytochrome c oxidase. The first modification is the replacement of a vinyl group with a farnesyl moiety, and the second is the oxidation of a methyl group to a formyl substituent.

Heine A synthase (HAS), which catalyzes the second transformation, is especially intriguing. A typical oxygenase would be expected to oxidize the methyl substituent to an alcohol; a dehydrogenase would then be required to further oxidize the alcohol to an aldehyde. In performing both reactions, heme A synthase not only catalyzes

? H2 HO--( ;H (

H 3 C ; ~

Hc-%

;H 2

;H 2

;H 3

CH=CH2

/~--CH3

;H2 ~.H 2

COOH COOH Heme A

a key step in the biosynthesis of cytochrome c oxidase, it also represents an unusual class of heme-containing oxygenases. We have cloned the HAS gene from B. subtilis into various expression vectors and transformed the plasmids into E. coil Significantly, the gene product is active in both the absence and presence of an N-terminal tag as determined by heme analysis of whole cells via HPLC. Side products of the reaction have been isolated and identified, thus providing important mechanistic insight into this novel oxygenation reaction.

Journal of ]norganic Biochemistry 86 (2001) 257

Synthesis and characterization of biologically relevant heme/Mn and Cun-S-S-Cu n complexes using the ligand tris(2-pyridylmethyl)amine

Matthew E. Helton, Partha P. Paul, Zoltfin Tyekl/tr, Kenneth D. Karlin Department of Chemistry, The Johns Hopkins University, Baltimore, MD, 21218, US

The tripodal tetradentate ligand tris(2-pyridylmethyl)amine (TMPA) has been used to investigate small molecule active site analogues of a number of metal containing enzyme systems. Manganese peroxidase (MnP) is a manganese and heme-containing enzyme secreted from the white rot fungus Phanerocheate chrysosporium whose function is to biodegrade lignin. Ecologically, there is great interest in fmding a cleaner, more efficient method for removing lignin

• e w ( I I I l l ) from wood. To model the reactivity and coordination chemistry of the MnP active site, n (heme)Fe ' --- MnH(TMPA) complexes have been generated. Synthetic strategies will be presented for tethering a TMPA moiety from the side of a partially fluorinated tetraarylporphyrin, along with subsequent metallation reactions to yield Fe°LIlX)/Mn ~J complexes. The spectroscopy and implied reactivity of these tethered Fe{H'm)/Mn H complexes, as well as the untethered analogues, with small molecules (i.e. 02, HzO2) will be discussed. Furthermore, as part of our foremost efforts in developing copper coordination chemistry of relevance to copper proteins such as nitrous oxide reductase (NOR), we have examined the reactivity of elemental sulfur ($8) with copper ion centers that have TMPA coordination. The results of these studies will be presented which consist of the synthesis and spectroscopy of the structurally characterized ~t-1,2 end-on disulfide bridging complex [(TMPA)CuH-S-S-CuII(TMPA)](C104)2.

The authors greatly acknowledge the National Institutes of Health for financial support.

The Mechanism of Metal Ion Containing [I-Lactamases

Lars Hemmingsen a, Lars Olsen a, Jens Antony a, Ulf Ryde b "Department of Mathematics and Physics, The Royal Veterinary and Agricultural University,

Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark, (e-mail: [email protected], dk, [email protected])

b Department of Theoretical Chemistry, University of Lund, Chemical Center, P.O.B. 124, S-221 O0 Lund, Sweden

Metal ion containing 13-1actamases provide bacteria with a defense mechanism against a broad spectrum of antibiotics, cleaving a C-N bond of the lactam ring ~. This poses a serious health threat to the general public. Understanding the catalytic mechanism of the enzymes at the atomic level is in itself interesting, and provides a basis for the design of inhibitors. In this work we have applied first principle quantum mechanical methods to model different possible reaction pathways, and compared calculated activation energies with experimental kinetic data. Model systems including different amino acids in the active site are tested, and a likely model for the actual reaction pathway is presented 2.

1. Wang Z., Fast W., Valentine A.M. and Benkovic S.J., Curr. Op. Chem. Biol., 3,614 (1999) 2. Olsen L., Antony J., Ryde U., and Hemmingsen L., in preparation


Purification and characterization of components of the nos (nitrous oxide reductase) gene cluster from Achromobacter cycloclastes

Shannon M. Henery, John A. Bollinger, and David M. Dooley Department of Chemistry and Biochemistry, Montana State University, Bozeman, 59715, USA

Nitrous oxide reductase (N2OR) is a homodimeric enzyme that catalyzes the reduction of nitrous oxide to dinitrogen, completing the denitrification pathway. Two recent crystal structures have shown that the enzyme contains six Cu ions per monomer. These Cu sites comprise a binuclear CuA electron transfer center and a novel, tetranuclear, sulfide-bridged CuZ site. Assembly of the CuZ site requires accessory proteins that are coded for by the nos gene cluster. The genes coding for these ancillary proteins show the conserved motifs and transmembrane regions found in all members of the superfamily of ABC transporters. We describe purification and characterization of NosX, a

Kp,,[(3Oa31

.~7,oI t 2555

Bg/~] ~ [532)

,n~e ,,nosY ~nncN nne7 nnc~

nosL nosF nosCluster

9485 bp

periplasmic flavoprotein with FAD as the cofactor. NosX has a calculated molecular weight of 29,181 Da and a calculated pI of 6.24. Inductively Coupled Plasma analysis showed the protein to contain no metal. While the function of NosX is not known, it is known to be required for N20 utilization in Sinorhizobium meliloti. In addition, a recent study indicates that mutations introduced into both the nosX and nirX genes of Paracoccus denitrificans eliminate nitrous oxide reduction capabilities by causing production of N2OR lacking the CuA site. We have also partially purified NosD, a 46,000 Da periplasmic protein that co-purifies with NzOR and a protein believed to be NosL. This result supports previous work indicating that NosD is necessary for assembly of the CuZ site of NzOR and indicates that NosI. may play a role in Cu insertion into NosD or N2OR.

Binuclear copper(I) complexes of composition [Cu2(SR)2(NR')2]: structural diversity and relation to the CUA site of cytochrom-c oxidase

Gera ld H e n k e l a, J6 rg S c h n e i d e r a, B j6 rn L i p p o l d a, T h o m a s M e y e r a

"Department of lnorganicChemistry, Gerhard-Mercator-Universitdt, 47048 Duisburg, German)'. (e-mail: [email protected])

In an approach to model the CUA site of the cytochrome-c oxidase which has been shown to contain cysteine bridges by EXAFS spectroscopf and subsequently by X-ray diffraction, 2 we were able to synthesize several novel Cu(I) complexes bearing a compositional relationship with CUA. In this series of complexes the bifunctional ?

guanidine-based nitrogen donor system btmgp was used in a metal-to-ligand ratio of 2:1 together ~y with thiophenolate ligands of different substitution patterns. The structural diversity of the ~ = ~ [Cu2(btmgp)(SR)2] complexes span the range from the - stretched - open-chain species ~ [Cuz(btmgp)(SC6H2-2,4,6-tBu3)2] (1) via [Cuz(btmgp)(SC6Hs-2,6-(SiMe3)2] (2) containing a cyclic CuzN2S2 core to [Cu2(btmgp)(SC6H2-2,4,6-iPr3)2] (3) which is characterized by copper atoms in different coordination environments (see Figure). The structural flexibility of these complexes in solution was probed by means of EXAFS 3 spectroscopy. These investigations show that complex 3 transforms into a species with chemically ' ~ equivalent Cu atoms containing two thiolate bridges and a tetrahedrane-like Cu2S2 frame. This frame provides a chemical basis for a proposed possible structure of CuA which is characterized by a non-planar CuzS2 frame. 1 1. Henkel G., Mtiller A., WeiBgdiber S., Buse G., Soulimane T., Steffens G.C.M., Nolting H.-F., Angew. Chem. Int. Ed. Engl. 34, 1488 - 1489 (1995) 2. a) Wata S., Ostermeier C., Ludwig B., Michel H., Nature 376, 660 - 669 (1995); b) Tsukihara T., Aoyama H., Yamashita E., Tomizaki T., Yamaguchi H., Shinzawaitoh K., Nakashima R., Yaono R., Yoshikawa S., Science 269, 1069 - 1074 (1995)


Studies on CotA, a laccase associated with the surface of bacillus subtilis endospores

Adriano O. Henriques a, Ligia O. Martins a'b, Clfiudio M. Soares a, Teresa Costa a, Manuela Pereira a, Miguel Teixeira a

~Instituto de Tecnologia Quimica e Bioldgica, U.N.L., Apt. 127, 2781-901 Oeiras, Portugal bUniversidade Lus6fona de Humanidades e Tecnologias, 1749-024 Lisboa, Portugal.

Endospores of Bacillus subtilis are encased in a proteinaceous coat that participates in the resistance of spores to a variety of chemical and physical insults. The 65kDa CotA protein is an abundant coat component, responsible for the brown pigment of sporulating colonies. Database searches and threading methods reveal significant similarities between CotA and the family of blue multicopper oxidases which i ~ includes the structurally characterized ascorbate oxidase and a fungal laccase. Based on the ~ , known data a structure of CotA (see figure) was derived by comparative modelling ~ L , ~ , . ~ . ~ ~ techniques. Wild type B. subtilis spores, but not those of a cotA null mutant, show activity ~ ~ with the phenolic and non-phenolic laccase substrates SGZ and ABTS, respectively. Single ~ ~''~" amino acid substitutions in the T1-Cu center (H497A or M502L) cause the formation of . ~ ' ¢ ~ "~-.)~.~j unpigmented colonies and of spores lacking laccase activity. Moreover, when overproduced in Escherichia coli, the purified protein shows spectroscopic and biochemical characteristics that confirms CotA as a laccase-associated with the surface of B.subtilis endospores. A remarkable property of CotA is its intrinsic thermostability; the half-life of thermal inactivation of the coat-associated and of the purified enzyme was 4 and 2h, respectively. Thus, the notorious resistance of bacterial endospores may in part result from the intrinsic stability of some of its structural components.

Spectroscopic character izat ion and mechanist ic studies o f biotin synthase

Heather L. Hernandez a;, Michele Mader Cosper at, Nathalie Y. R. Agar b, Justin B. Powlowski b, John Perkins c, Michael K. Johnson a

"Department of Chemistry, Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-2556 USA, bDepartment of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Blvd. West, Montreal, Quebec H3G 1M8 CANADA," CRoche Vitamins Inc., 340 Kingsland Street, Nutley, NJ 07110 USA (e-mail: tmmader@chem, uga. edu; Chsackett@chem. uga. edu)

Biotin synthase (BioB) is an Fe-S protein and belongs to the family of S-adenosylmethionine-dependa m radical- generating enzymes. The final step of biotin biosynthesis is mediated by BioB and entails the insertion of S into dethiobiotin. The Fe-S cluster of BioB is postulated to have a role in radical generation and is believed to be the source of the inserted S atom. The over-expression of Escherichia coli (Ec) and Bacillus subtilis (Bs) BioB in Ec C41 [DE3] in the presence a plamsid encoding for the Pseudomonas aeroginosa isc gene cluster resulted in high levels of soluble BioB (20 mg/L). Aerobically isolated BioB contains one [Fe2Sz] 2+ cluster per monomer, as previously reported, l Distinct differences are observed in the UV-visible absorption and resonance Raman spectra of the [FezS2] z+ centers in Bs and Ec BioB, and the structural origin of these difference will be discussed. Apo forms of Ec and Bs BioB have been reconstituted under strictly anaerobic conditions with an [Fe4S4] 2+ cluster, and the [Fe4S4] + cluster in the dithionite reduced reconstituted BioB has been characterized by EPR, MCD, and M/Sssbauer spectroscopies. The results of spectroscopic studies designed to address the nature of the cluster transformation occurring during the course of a single enzymatic turnover will be presented.

1. Duin, E. C.; Lafferty, M. E.; Crouse, B. R.; Allen, R. M.; Sanyal, I.; Flint, D. H.; Johnson, M. K. Biochemistry, 36, 11811 - 11820 (1997)

The National Institutes of Health (GM 62524 to MKJ and DK59730 to MMC) is acknowledged for financial support.


Scavenging NO and peroxynitrite: a new function of myoglobin?

Susanna Herold, Martin Mehl and Michael Exner Laboratory of Inorganic Chemsitry, ETH Ziirieh, Universitgitsstrasse 6, CH-8092 Ziirich, Switzerland (e-mail." herold@inorg, chem.ethz.ch)

Nitrogen monoxide (NO) is currently a species of extreme biological interest because of the variety of physiological functions which have been found to be associated with this inorganic messenger molecule. NO undergoes two major processes in biological systems: nearly diffusion-controlled reaction with superoxide (02"-) to form the powerful oxidizing and nitrating agent peroxynitrite (ONOO-) and reaction with different metal centers of proteins. We have shown that NO rapidly oxidizes oxymyoglobin (MbFeO2) to metmyoglobin (MbFe m) and that this reaction proceeds via the intermediate MbFe~IIOONO, which then decays to MbFe ltl and nitrate (eq. 1). j In addition, we have demonstrated that the reaction of peroxynitrite with MbFeO2 proceeds in two steps with the formation of the intermediate ferryl species MbFelV=o (eq. 2). 2

k=4.4 x 107 M Is-I k>500s -1 MbFeO 2 + NO ~ MbFeOONO ~ MbFeOH2 (1)

k=5.4x 104M ls-I k=2.2 x 104M I s 1 MbFeO 2 + HOONO ~ MbFelV=O + HOONO "~ MbFeOH2 (2)

Here we present detailed HPLC-analyses of the protein after complete hydrolysis as well as enzymatic digestion. Our results show that MbFemOONO does not nitrate any tyrosine residue in the protein. In addition, we demonstrate that when added in equimolar amounts, peroxynitrite nitrates less than I% of the available tyrosine residues of MbFeO2. Furthermore, we show that MbFeO2 protects free tyrosine from peroxynitrite-mediated nitration. Taken together these data suggest that myoglobin might have another function besides O2 storage in vivo, that is scavenge endogenously generated NO and detoxify from peroxynitrite.

I. Herold, S., et al. (2001) Biochemistry 40, 3385-3395.2. Exner, M. et al. Chem. Res. Toxicol. 13,287-293 (2000).

Compound II in Peroxidases: New Resonance Forms Suggested by pH Dependent Structures of Intermediates Formed in Myoglobin-Peroxide Reactions.

Hans-Petter Hersleth a, Bjorn Dalhus a, Carl Henrik G6rbitz a, K. Kristoffer Andersson b. " Department of Chemistry, University ofOslo, P. O, Box 1033 Blindern, 0315 Oslo, NORWAY (e-

mail. h.p.hersleth@kjemi, uio.no) b Department of Biochemistry, University of Oslo, P. O. Box 1041 Blindern, 0316 Oslo, NORWAY

The biological conversions of 02 and peroxides to water as well as certain incorporations of oxygen atoms into small organic molecules can be catalyzed by metal-ions in different clusters or cofactors. The catalytic cycles of these reactions pass through similar metal-based complexes in which one oxygen- or peroxide-derived oxygen atom is coordinated to an oxidized form of the catalytic metal-center. In haem-based peroxidases or oxygenases the ferryl (FetVOi form is important in the compound I and compound II complexes, which are two and one oxidation equivalents higher than the ferric (Fe m) form, respectively. In this study we report three high resolution X-ray structures of a compound II model protein, obtained by reacting hydrogen peroxide with ferric myoglobin at pH 5.2 (with 1.35 A resolution), 6.8 and 8.7. The molecular geometry is virtually unchanged compared to the ferric form, indicating that these reactive intermediates do not undergo large structural changes. The essential Fe-..O distance is 1.9 A at all pH-values. This observation, together with the hydrogen bonding of the distal histidine, suggests that the dominating compound II resonance form is possibly a hydroxyl radical-ferric iron 1 at low pH and a hydroxyl-ferryl iron at high pH, and not an oxo-ferryl form as has been suggested previously. The 1.9 A Fe-..O distance is in agreement with an EXAFS study of compound II in horseradish peroxidase.

1. Hersleth, H.-P., Dalhus, B., G6rbitz, C.H. and Andersson, K.K., J. Biol. Inorg. Chem. (submitted)

Supported by the Norwegian Research Council and the Iron-oxygen Protein Network (ERBMRFXCT980207) of EU TMR programme to K. K. Andersson.

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