Metalloproteins

J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 DOI 10.1007/s00775-009-0515-7

POSTER PRESENTATION

Metalloproteins

P501 1Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Mechanisms of isoniazid activation inferred Germany, 2Department of Chemistry, Bielefeld University, 33615 Bielefeld, from resistance mutation catalase-peroxidases (KatGs) Germany, 1 1 Christine E. Fairchild , Reza A. Ghiladi 3EMBL Hamburg, 22603 Hamburg, Germany, 1 Department of Chemistry, NC State University, Raleigh, 4Max Planck Institute of Biophysics, 60438 Frankfurt am Main, NC 27607, USA. [email protected] Germany. [email protected] Mycobacterium tuberculosis catalase-peroxidase (KatG) activates the [Fe]-hydrogenase is one of three types of enzymes known to activate pro-drug isoniazid (INH) to the isonicotinoyl acyl radical, which then molecular hydrogen (H2). It catalyzes the generation of a hydride that combines with NADH, forming an INH–NADH adduct capable of is required to reduce methenyltetrahydromethanopterin (methenyl- + inhibiting InhA, an enzyme involved in cell wall biosynthesis. H4MPT ) to methylene-H4MPT, which is an intermediary step of Inability to form mycolic acid, which comprises a protective layer of methanogenesis in many methanogenic archaea. Recently, the crystal the cell wall, causes the observed bactericidal activity. However, structure of its reconstituted holoenzyme was solved and revealed the drug-resistant strains of tuberculosis (TB) can arise from mutations in novel structure of the mononuclear iron complex in the active site. KatG. Thus, before novel drug therapies can be developed, a new We report here on the structure of the C176A mutated holoenzyme, understanding of the underlying causes of drug resistance is needed. which was crystallized in the presence of dithiothreitol (DTT) Over 25 different resistance mutation KatGs have been expressed, (Fig. 1). The active site iron center of the enzyme is octahedrally purified to, or near, homogeneity, and characterized for catalase and coordinated by one DTT sulfur, one DTT oxygen, two carbon mon- peroxidase activities as well as their ability to form the INH–NADH oxide ligands, the pyridinol nitrogen and the pyridinol formylmethyl adduct using the following: peroxide (tBuOOH and the hydrogen carbon in acyl-iron ligation (Fig. 2). This result prompted us to peroxide generating enzymatic system glucose/glucose oxidase), reinterpret the iron complex structure in the wild-type enzyme and superoxide (xanthine/xanthine oxidase), and no exogenous oxidant. would suggest an alternative open coordination site for H2 binding. No correlation was found between adduct formation and either cat- alase or peroxidase activity. Mutated residue location plays a role in available pathways of INH activation and causes of resistance. These results as well as further characterization with stopped-flow UV-vis- ible spectroscopy will be presented.

Fig. 1 Overall structure

Fig. 1 Resistance mutations studied in KatG

P502 The crystal structure of C176A mutated [Fe]-hydrogenase suggests an acyl-iron ligation in the active site iron complex Takeshi Hiromoto1, Kenichi Ataka2, Oliver Pilak1, Sonja Vogt1, Marco Salomone Stagni3, Wolfram Meyer-Klaucke3, Eberhard Warkentin4, Rudolf K. Thauer1, Ulrich Ermler4, Seigo Shima1 Fig. 2 Active site iron complex 123 S186 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224

P503 1School of Chemistry and Molecular Biosciences, The University of Substrate and metal ion modulate the catalytic Queensland, Brisbane 4072, Australia, 2Research School Of Chemistry, The Australian National University, mechanism of GpdQ: a promising bioremediator Canberra 0200, Australia. [email protected] for organophosphate nerve agents and pesticides. The organophosphate-degrading enzyme from Agrobacterium tum- Kieran S. Hadler1, Fernanda Ely1, Natasa Mitic1, efaciens (OPDA) is a metallohydrolase which is able to degrade Lawrence R. Gahan1, David L. Ollis2, James A. Larrabee3, highly toxic organophosphate pesticides and nerve gas agents into Gerhard Schenk1 less or non-toxic compounds. The reaction mechanism employed 1School of Chemistry and Molecular Biosciences, The University of by OPDA is not yet fully understood, but there are two currently Queensland, St Lucia 4072, Australia, accepted hypotheses involving either (a) a metal ion-bridging or (b) 2Research School of Chemistry, Australian National University, a terminally coordinated hydroxide/water nucleophile. In this work, Canberra 0200, Australia, we have investigated the role of the coordinated hydroxide/water 3Department of Chemistry and Biochemistry, Middlebury College, molecules in the nucleophilic reaction. The pH dependence of the Middlebury 05753, USA. [email protected] kinetic properties of several metal ion derivatives of wild type The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes OPDA, including Co(II), Zn(II) and Cd(II), yielded catalytically is a promiscuous binuclear metallophosphatase with a remarkable relevant pKa values that implicate a metal ion-bridging water ability to hydrolyze all classes of phosphate ester substrates. Notably, molecule in the reaction mechanism. The Co(II) derivative, how- it is capable of degrading the toxic byproducts of the hydrolysis of ever, has shown a second relevant protonation equilibrium (pKa VX (a powerful nerve agent) and a range of organophosphate pesti- 10.1), which is ascribed to a terminally metal ion-bound water. The cides (e.g. paraoxon and demeton). Therefore, GpdQ is of interest for 1.65 A˚ resolution structure of OPDA, crystallised in presence of its potential application as versatile enzymatic bioremediator [1]. the very slow substrate diethyl 4-methoxyphenyl phosphate (EPO) Recently, we have shown that GpdQ employs an unusual catalytic facilitated the capture of an OPDA structure with both bound mechanism where the formation of the catalytically competent substrate and product present in the active site. Preliminary results binuclear enzyme is induced by substrate and is regulated by the indicate that upon substrate binding, the bridging hydroxide, which coordination flexibility of an asparagine ligand [2]. In order to is also present in the free enzyme, is displaced to a terminally investigate the individual stages of this mechanism, stopped flow metal ion-bound position, where it is favourably placed to carry out fluorescence has been utilized, looking specifically at the pre-steady a nucleophilic attack on the substrate phosphorous atom. In addi- state features. In addition, the steady-state kinetic behaviour for a tion, kinetic analyses, as well as the crystal structures of the Y257F number of metal-derivatives of GpdQ has been measured in order to and R254H mutants have demonstrated the significance of a understand the role of the metal ions in catalysis. hydrogen-bonding network in the second coordination sphere in orienting the substrate for hydrolysis.

P505 Structural and catalytic models for phosphoesterases Lawrence R. Gahan1, Gary Schenk1, David Ollis2 1School of Chemistry and Molecular BioSciences, The University of Queensland, St Lucia, QLD 4072, Australia, 2Research School of Chemistry, The Australian National University, Canberra, ACT, Australia. [email protected] The prolonged and widespread agricultural application of organo- phosphate pesticides (OPs) has contributed to increased agricultural production and to environmental problems related to run-off and subsequent contamination of water sources. OP-degrading com- pounds are of interest therefore for environmental detoxification, References but also as agents for protection against bioterrorism; nerve gas 1. Ghanem E, Li Y, Xu C, Raushel FM (2007) Biochemistry agents VX and sarin are both OPs. Our interest is in models for the 46:9032 active sites of metallohydrolases, including the purple acid phos- 2. Hadler KS, Tanifum EA, Yip SH-C, Mitic´ N, Guddat LW, phatases (PAP), the glycerophosphodiester degrading enzyme from Jackson CJ, Gahan LR, Nguyen K, Carr PD, Ollis DL, Hengge Enterobacter aerogenes (GpdQ) and the structurally related OP- AC, Larrabee JA, Schenk G (2008) J Am Chem Soc 130:14129 degrading triesterase from Agrobacterium radiobacter (OpdA). Here, we focus on GpdQ, a universal phosphoesterase, shown to have activity towards phosphomono-, di- and tri-esters (including OPs). GpdQ shares six of seven donor ligands with PAP (Figure), P504 the notable difference being the lack of a tyrosine ligand. GpdQ Catalytic mechanism of the reaction catalyzed also shares five donor ligands with OpdA, with the metal centers coordinated by four histidines, an aspartate and a lysine. The native by the binuclear organophosphate-degrading enzyme metal ion composition of GpdQ is still unknown, but enzymatic from Agrobacterium tumefaciens activity can be reconstituted with Zn2+,Co2+ and Mn2+. We report Fernanda Ely1, Paul Carr2, Luke Guddat1, David Ollis2, the synthesis and characterization of new biomimetic systems as Lawrence Gahan1, Gary Schenk1 structural models for GpdQ.

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osteoporosis [2]. Therefore, a thorough understanding of the mecha- nism is crucial. Our aim on one hand is to find a suitable model system to mimic the active site of PAP. We have developed a new ligand based on cyclam with two distinct coordination sites that may form (hydr)oxo-bridged dinuclear complexes. The coordination chemistry of this ligand is illustrated.

H N N N N N

Acknowledgements: This work was funded by grants from the Aus- N tralian Research Council (DP0558652 and DP0664039). N N N N H

P506 On the other hand, we present the results of computational studies Iron release from human transferrin in the absence concerning the mechanism of the enzyme. Different approaches, with and without the involvement of the protein backbone were chosen. of chelators involves five kinetic steps at acidic pH 1,2 1 Rajesh Kumar , A. Grant Mauk References 1 Centre for Blood Research, University of British Columbia, 1. Mitic N, Smith SJ, Neves A, Guddat LW, Gahan LR, Schenk G Vancouver, BC V6T 1Z3, Canada, (2006) Chem Rev 106:3338 2 Thapar University, Patiala 147004, Punjab, India. 2. Moss DW, Raymond FD, Wile DB (1995) Crit Rev Clin Lab Sci [email protected] (1995) 32:431 Kinetics of iron release from diferric human transferrin (Fe2Tf) has been studied in the presence of non-synergistic anions at the pH of cellular iron release (4.2 B pH B 5.4) in the absence of chelators. Fe3+ release involves five kinetically detectable steps. Step 1 (most P508 rapid) involves pH-linked decarboxylation of Fe2Tf. Subsequent iron release from both sites is controlled by slow H+ transfers and anion Role of the amino acid residues in the active site interactions. In Step 2, the N-lobe gains 1 H+ with kinetic linkage to of human indoleamine 2,3-dioxygenase 3+ the binding of one anion. In Step 3, Fe is released with linkage to Masaki Horitani1, Erisa Kometani1,2, Hiroshi Sugimoto1, + 7 binding of 2 H with rate-constants k2N of 1.7(6) 9 10 and Yoshitsugu Shiro1 7 -2 -1 - 2- 9(1) 9 10 M s in the presence of Cl and SO4 , respectively. 1RIKEN Spring-8 Center, Harima Institute, Hyogo 679-5148, Japan, Step 3 is also linked to release of the anion with an equilibrium 2Graduate School of Life Science, University of Hyogo, -2 -3 - constant K2S of 3.5(3) 9 10 and 4.6(4) 9 10 M for Cl and Hyogo 678-1297, Japan. [email protected] 2- + SO4 , respectively. In Step 4, the C-lobe gains 1 H with kinetic Indoleamine 2,3-dioxygenase (IDO) which contains iron protopor- linkage to anion binding. In Step 5, iron release from the C-lobe is phyrin IX, i.e. heme b, as the prosthetic group in the active site is + 4 linked to the binding of 2 H with rate constants, k3‘ of 7(2) 9 10 ubiquitously expressed the mammals. IDO catalyzes the cleavage of 5 -2 -1 - -2 and 8(2) 9 10 M s in the presence of Cl and SO4 , respec- the pyrrole ring of L-tryptophan by insertion of molecular oxygen and tively. Step 5 is also linked to anion release with an equilibrium produces N-formylkynurenine. We previously reported the structure -2 -3 constant K2S of 1.4(4) 9 10 and 8(2) 9 10 M in the presence of of human IDO was decided and proposed that the precise geometry - -2 Cl and SO4 , respectively. Supported by a Postdoctoral Fellowship between tryptophan and iron-bound di-oxygen was essential for the (RK), a grant (AGM) from the CBS-CIHR Partnership program, and a catalytic reaction [1]. However, the detail of the reaction mechanism travel fund TU&DST. has not been understood yet. In this study, we performed mutation analysis on the amino residues at loop region and polar and aromatic amino residues nearby active site. Comparison of kinetic parameters, dissociation constants for L-tryptophan and resonance Raman spectra P507 for wild type and these mutants will be discussed in detail. Towards the mechanism of purple acid phosphatases: coordination chemistry of a cyclam-based dinucleating ligand and computational studies on the enzyme Marta Zajaczkowski, Peter Comba Institute of Inorganic Chemistry, University of Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany. [email protected] Purple acid phosphatases (PAP) are heterodinuclear enzymes that catalyze the hydrolysis of phosphomonoesters. Their biological functions are divers and still are not fully understood [1]. In mam- malians, there was found a correlation between high PAP levels and

123 S188 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224

Reference were interpreted on the basis of the experimental and DFT calculated 1. Sugimoto H, Oda S, Otsuki T, Hino T, Yoshida T, Shiro Y (2006) vibrational shifts. Proc Natl Acad Sci USA 103:2611–2616

P511 P509 Redox and metal-regulated spatial subunit Magnetic circular dichroism study of a spectroscopic arrangement of the oligomer for porphobilinogen model for dicobalt(II) enzyme active sites which have synthase activation both 6-coordinate and 5-coordinate Co(II) Noriyuki Nagahara1, Nori Sawada1, Fumio Arisaka2, James A. Larrabee, W. Rainey Johnson Kaoru Mitsuoka3, Masayasu Minami1 Department of Chemistry and Biochemistry, Middlebury College, 1Department of Environmental Medicine, Nippon Medical School, Middlebury, VT 05753, USA. [email protected] Tokyo, Japan, 2- 2 [Co2(l-OH)(l-Ph4DBA)(TMEDA)2(OTf)] (1), where Ph4DBA is Graduate School of Bioscience and Biotechnology, Tokyo Institute the dinucleating bis(carboxylate) ligand dibenzofuran-4,6-bis(diphe- of Technology, Yokohama, Japan, nylacetate), contains one 6-coordinate Co(II) and one 5-coordinate 3Biomedicinal Information Research Center, National Institute of Co(II) bridged by two carboxylates and one hydroxide. This structural Advanced Industrial Science and Technology, Tokyo, Japan. motif is similar to the active sites in methionine aminopeptidase from [email protected] Escherichia coli (MetAP) and glycerophosphodiesterase from The oligomeric state of human porphobilinogen synthase (PBGS) is Enterobacter aerogenes (GpdQ). homooctamer, which consists of conformationally heterogenous The magnetic circular dichroism (MCD) spectrum of the dicobalt(II) subunits in the tertiary structure under air-saturated conditions. When form of MetAP shows two major d-d transitions at 495 nm, due to 6- PBGS is activated by reducing agent with zinc ion, a reservoir zinc coordinate Co(II) and at 567 nm due to 5-coordinate Co(II). The ion coordinated by Cys223 is transferred in the active center to be MCD spectrum of GpdQ has d-d transitions at 495 and 574 nm, due coordinated by Cys122, Cys124, and Cys132 (1). The latter zinc ion to 6- and 5-coordinate Co(II), respectively. These assignments were serves as an electrophilic catalysis. We investigated a conformational made based on ligand field arguments because no MCD data were change associated with the PBGS activation via reduction using available for mixed 6/5-coordinate models. The two major peaks in analytical ultracentrifugation, negative staining electron microscopy, the MCD spectrum of 1 are at 505 and 569 nm, which confirm the native PAGE, and enzyme activity staining. The results are in good peak assignments in MetAP and GpdQ. agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f0. Thus, the metal and redox- regulated PBGS activation is closely linked to conformational change MCD Spectrum of 1 200 of the oligomer.

100

0

-100

-200

-300 569 nm Intensity, mdeg -400

-500 505 nm -600 400 500 600 700 800 Wavelength, nm Reference 1. Sawada N, Nagahara N, Sakai T, Nakajima Y, Minami M, P510 Kawada T (2005) J Biol Inorg Chem 10:199–207 Visible and infrared spectroelectrochemistry of metalloporphinones Michael D. Ryan, Florentina Tutunea P512 Chemistry Department of Marquette University, PO Box 1881, Milwaukee, WI 53201, USA. [email protected] Crystal structure of the Michaelis complex of aldoxime Porphinediones are involved in the dissimilatory reduction of nitrite. dehydratase The presence of a carbonyl group on the porphine ring makes it can Hitomi Sawai1, Hiroshi Sugimoto2, Yasuo Kato3, excellent marker for observing the extent to which there is additional Yasuhisa Asano3, Yoshitsugu Shiro2, Shigetoshi Aono1 electron density on the ring. The reduction of iron, manganese and 1Okazaki Institute for Intergrative Bioscience, 5-1 Higashiyama, zinc porphinones was examined to characterize their products using Myodaiji, Okazaki 444-8787, Japan, visible and infrared spectroscopy. The visible spectra of MII(Pone) 2RIKEN SPring-8 Center, 1-1-1 Sayo, Hyogo 679-5148, Japan, and M(Pone)- have been obtained for M u Fe, Mn and Zn. The 3Biotechnology Research Center, Toyama Prefectural University, Zn(Pone)- complex would be expected to be a ZnII-pi anion radical. 5180 Kurokawa, Imizu T939-0393, Japan. [email protected] These spectra were then compared to the iron and manganese species. Aldoxime dehydratase (Oxd) catalyzes the dehydration of aldoximes Infrared spectroelectrochemical spectra were then carried out for (R–CH = N–OH) to the corresponding nitriles (R–C : N). Oxd these three metal complexes, and their results compared with DFT adapts a heme as the active site to which the substrate directly binds calculations. The electronic structure of the M(Pone)- complexes to proceed the reaction. The enzymatic activity is regulated by the

123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S189 control of the coordination mode of the substrate dependent on the change in the oxidation state of the heme. The ferric Oxd forms a dead-end complex with the substrate, where the oxygen atom of the substrate is coordinated to the ferric heme iron. In the ferrous Oxd, the nitrogen atom of the substrate is coordinated to the heme iron, which is the Michaelis complex of Oxd. We succeeded to prepare crystals of the resting state (substrate-free ferrous form) and the Michaelis complex (substrate-bound ferrous form) by using X-ray cryoradiolytic reduction of the dead-end com- plex in Oxd from Rhodococcus sp. N-771 (OxdRE). The crystal structures of active forms with/without substrate reveal the mecha- nism of substrate recognition and the catalysis of OxdRE. P514 IV Chiral distortion in a Mn (salen)(N3)2 derived from Jacobsen’s catalyst as a possible conformation model for its enantioselective reaction Takuya Kurahashi, Hiroshi Fujii Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Myodaiji, Okazaki, Aichi 444-8787, Japan. [email protected] IV The Mn (salen)(N3)2 complex (1) from Jacobsen’s catalyst is syn- thesized, and the X-ray crystal structures of 1 as well as the starting III Mn (salen)(N3)(CH3OH) complex (2) are determined in order to investigate the conformation of the high-valent MnIV(salen) molecule Fig. 1 a Different coordination mode of the substrate in ferric and in comparison with that of MnIII(salen). The asymmetric unit of the ferrous Oxd. b X-ray crystal structure of OxdRE at 1.6 A˚ resolution crystal of 1 contains four complexes, all of which adopt a nonplanar stepped conformation effectively distorted by the chirality of the dii- mine bridge. The asymmetric unit of 2 also contains four complexes. Two of them show a stepped conformation of a lesser degree, but the other two adopt a bowl-shaped conformation. Comparison of the P513 structural parameters shows that the Mn center in 1 is coordinated from Differences in coordination states of replaced tyrosine both sides by two external axial N3 ligands with significantly shorter residues and quaternary structures among hemoglobins bond length, which could induce greater preference for the stepped conformation in 1. The CH3CN solution of 3 shows circular dichroism M probed by Resonance Raman Spectroscopy with a significantly strong band at 275 nm as compared to 2, suggesting 1 2 3 Masako Nagai , Yayoi Aki , Teizo Kitagawa that 1 may adopt a more chirally distorted conformation also in solution. 1Research Center for Micro-Nano Technology, Hosei University, Koganei 184-0003, Japan, 2Graduate School of Medicine, Kanazawa University, Kanazawa 920-0942, Japan, 3Toyota Physical and Chemical Institute, Aichi-gun 480-1192, Japan. [email protected] Hemoglobins (Hbs) M are naturally occurring mutants in which the proximal (F8) or distal (E7) histidine (His) are replaced by tyrosine (Tyr) in the a or b subunits. The heme of the abnormal subunits is oxidized in vivo. Among the four Hbs M, only abnormal subunit in Hb M Saskatoon (bE7Tyr) can be reduced by methemoglobin Reference reductase. To gain an insight into a cause of the difference, we 1. Kurahashi T, Fujii H (2008) Inorg Chem 47:7556–7567 examined the coordination state of E7- and F8-Tyrs and Fe–O(Tyr) bonding by UV and visible resonance Raman (RR) spectroscopy. Hbs M Iwate (aF8Tyr), M Boston (aE7Tyr) and M Hyde Park - (bF8Tyr) exhibited two extra UVRR bands at 1,603 and 1,167 cm P515 1 arising from deprotonated Tyr, but Hb M Saskatoon displayed the UVRR bands of protonated Tyr at 1,620 and 1,175 cm-1 in Oxygen stability and crystal structure of Tetrahymena addition to those of deprotonated Tyr. Evidences for the bonding of truncated hemoglobin both kinds of Tyr to the heme in Hb M Saskatoon were provided Jotaro Igarashi1, Ariki Matsuoka2 by visible RR spectroscopy. These results indicate that bE7Tyr of 1Institute of Multidisciplinary Research and Advanced Materials, Hb M Saskatoon is in equilibrium between protonated and depro- Tohoku University, Sendai 980-8577, Japan, tonated forms, which is responsible for facile reducibility. MetHbs 2Fukushima Medical University, Fukushima 960-1295, Japan. M Iwate, M Boston and M Milwaukee (bE11Glu) exhibited fre- [email protected] quency shifts for Tyr RR bands and intensity enhancement for Trp Truncated hemoglobins (trHbs) are distributed from bacteria to uni- RR bands compared with those of metHbA. This is characteristic cellular eukaryotes and play roles in oxygen transport and nitric oxide of the T quaternary structure. In contrast, metHbs M Saskatoon and detoxification. trHbs are known to exist in the ciliates Tetrahymena, M Hyde Park exhibited R-type UVRR spectra similar to metHb A. but their structures and functions are poorly understood. To explore

123 S190 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 the structure–function relationship of T. pyriformis trHb (Tp trHb), known as HasA to acquire heme from their hosts. Heme taken up by especially with reference to the stability of bound oxygen, we per- heme acquisition system A (HasA) is delivered to the specific outer formed kinetic analyses and solved the crystal structures of the Fe(II), membrane receptor, HasR. In this study, we have investigated HasA Fe(II)–O2, and Fe(III) complexes. from Pseudomonas aeruginosa. Although the crystal structure of The autoxidation rate of Tp trHb was comparable to that of sperm HasA from P. aeruginosa has not been determined at the moment, the whale myoglobin (Mb) but was slower than that of Paramecium three-dimensional structure of the corresponding hemophore from caudatum trHb. The oxygen dissociation rate constant of Tp trHb was S. marcescen indicated that the heme iron is legated to Tyr-75 and smaller than those of sperm whale Mb and P. caudatum trHb. These His-32 (Fig. 1). The overall sequence identity of the HasA from kinetic findings are consistent with data on other type I trHbs from S. marcescen and P. aeruginosa is 44%, and that the common resi- Mycobacterium tuberculosis and Chlamydomonas eugametos. The dues appear to be involved in interactions with heme. three-dimensional structure of the Fe(II)–O2 complex of Tp trHb was solved at 1.7 A˚ resolution. Tyr25 and Gln46 were hydrogen-bonded to the dioxygen molecule bound to heme. Tyr25 donated a hydrogen bond to the terminal oxygen atom, whereas Gln46 hydrogen-bonded to the proximal oxygen atom bound to the heme iron. In addition, Tyr25 was hydrogen-bonded to Gln46 and Gln50 residues. To understand the role of hydrogen-bonding in Tp trHb, we constructed Tyr25Phe, Gln46Glu, and Gln50Glu mutants. Both autoxidation and V O oxygen dissociation rate constants were markedly increased in the V mutants. We will discuss the hydrogen-bonding network in Tp trHb N N with reference to oxygen binding and nitric oxide detoxification. Fe NN N P516 P P Theoretical study of dioxygen induced inhibition N of [FeFe]-hydrogenase Martin T. Stiebritz1, Markus Reiher1 1Laboratory of Physical Chemistry, ETH Zurich, Wolfgang-Pauli-Str. Fig. 1 10, 8093 Zurich, Switzerland. [email protected] Hydrogenases comprise a variety of enzymes that catalyze the reversible oxidation of molecular hydrogen. Out of this group [FeFe]- Has A from P. aeruginosa exhibited the absorption maximum at hydrogenase shows the highest activity for hydrogen production and 407 nm when the heme iron was in the ferric state. The EPR spectrum is therefore of great interest in the field of renewable energies. showed signals at g = 2.78, 2.19, and 1.75, indicating the presence of Unfortunately, this comes with the flaw of a generally very high six-coordinated low-spin ferric heme iron ligated with His and Tyr. sensitivity against molecular oxygen that irreversibly inhibits this We did not observe any pH-dependent spectral changes. The addition enzyme [1, 2]. While many studies have already addressed the of exogenous ligand (e.g. cyanide, azide, imidazole) to ferric HasA mechanism of hydrogen formation by [FeFe]-hydrogenase little is did not alter the coordination of heme iron. However, the Soret band known about the molecular and mechanistic details leading to enzyme was shifted from 407 to 424 nm upon the mixing of dithionite and inactivation by O . 2 cyanide with HasA. Furthermore, the ferrous-CO spectrum of HasA In order to elucidate this process, we performed density functional showed the Soret absorbance at 411 nm. On the basis of our obser- theory calculations on several possible O -adducts of the catalytic 2 vations, we discuss which ligand is readily replaced in the release of center—the so called H-cluster—and show that the direct interaction heme from HasA. of the [2Fe]H subsite is an exothermic and specific reaction in which O2 most favorably binds in an end-on manner to the distal Fed. Based on the results, we propose a protonation mechanism that can explain the irreversibility of dioxygen-induced enzyme inactivation by water P518 release and degradation of the ligand environment of the H-cluster Metallothioneins expressed in the lumen and stroma [3]. of thylakoids of a thermophilic cyanobacterium References Thermosynechococcus elongatus 1. Vincent KA et al (2007) Chem Rev 107:4366–4413 Hiroshi Nakano1, Takashi Manabe1, Hidenori Hayashi1,2, 2. Baffert C et al (2008) Angew Chem Int Ed 47:2052–2054 Miwa Sugiura1,2 3. Stiebritz MT, Reiher M (2009) Inorg Chem (submitted) 1Graduate School of Science and Technology, 2Cell-Free Science and Technology Research Centre, Ehime University, Matsuyama 790-8577, Japan. [email protected] P517 Zinc ion is an essential element acting as a cofactor in more than 300 enzymes. However, high concentrations of Zn2+ are lethal for the Redox-dependent changes in the coordination cells. In cyanobacteria, two detoxification systems are known: (1) a of heme acquisition system A metallothionein SmtA that binds Zn2+ in Synechococcus PCC 7942; Shin-ichi Ozaki, Akira Nakahara, Chihori Sakaguchi (2) a Zn2+ exporter ZiaA that excretes Zn2+ from cells in Synecho- Department of Biological Sciences, Yamaguchi University, Yoshida, cystis PCC 6803. SmtA is a small soluble protein, which consists in Yamaguchi 853-8515, Japan. [email protected] 56 amino acids and contains a Zn4Cys9His2 cluster [1]. SmtA is Bacteria have developed some iron-scavenging systems to survive. responsible to elevated concentration of Zn2+ in the cells, and con- Some Gram-negative pathogens secrete a small protein hemophore sequently the cells survive at concentration up to 14 lMZn2+ [2].

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From the amino acid sequence, SmtA seems to locate only in stroma The results were shown in Table 1. Activities are expressed relative to in the cyanobacterial cells, although protective mechanism should be that of PhADH containing Fe2+. In previous study, it was revealed necessary also in the thylakoid lumen. A thermophilic cyanobacte- that the wild type of PhADH had zinc and iron. The result suggested rium Thermosynechococcus elongatus was isolated from a hot spring, that only iron was essential to show the catalytic activity. In addition, which contains high concentration of heavy metals. To examine the we constructed the three mutants (H267A, H271A, and H279A). We effects of localization of metallothionein on Zn2+-resistance, we will report the effects of these three histidine residues on enzymatic constructed T. elongatus mutants, which express smtA homologue of activity of PhADH. Synechococcus PCC 7002 in the stromal space and the thylakoid lumen. In T. elongatus stroma, a metallothionein was induced by addition of 10 lMZn2+. When the SmtA homologue was expressed in the thylakoid lumen, Zn2+-bound homologue was detected at P520 molecular mass of *5700 with MALDI-TOF MS. The recombinant Effect of mutation around the substrate channel cells achieved resistance to high concentrations of Zn2+ such as of thermophilic cytochrome P450 on the direct electron 2+ 1.25 mM. Such results will be discussed in term of cellular Zn donating system with NADH traffic and application to bioremediation. Keisuke Matsuda1, Hirotoshi Matsumura2, Nobuhumi Nakamura1, Masafumi Yohda1, Hiroyuki Ohno1 References 1Department of Biotechnology and Life Science, Tokyo University of 1. Blindauer CA, Harrison MD, Parkinson JA, Robinson AK, Cavet Agriculture and Technology, Koganei, Tokyo 184-8588, Japan, JS, Robinson NJ, Sadler PJ (2001) PNAS 98:9593–9598 2Department of Biomaterial Sciences, Graduate School of Agriculture 2. Turner JS, Morby AP, Whitton BA, Gupta A, Robinson NJ and Life Science, The University of Tokyo, Bunkyo-ku, (1993) J Biol Chem 268:4494–4498 Tokyo 113-8657, Japan. [email protected] Cytochrome P450s are a ubiquitous family of monooxygenases, which carry a thiolate ligated ferriheme cofactor to metabolize sub- strates of varying size and chemical functionality. Two successive P519 electrons are required for the oxygen activation in the monooxygen- Dependence of metal ions on enzymatic activity ation reactions, which are derived from NAD(P)H and delivered of alcohol dehydrogenase from hyperthermophilic generally via one or more redox partner protein(s). However, our previous study revealed that the thermophilic cytochrome P450 from archaeon Pyrococcus horikoshii OT3 the Sulfolobus tokodaii strain 7 (P450st) could catalyze the epoxi- 1 2 Haruka Mishiba , Hirotoshi Matsumura , dation of styrene using NADH as a direct electron donor. In this 1 1 1 Nobuhumi Nakamura , Masafumi Yohda , Hiroyuki Ohno study, we have constructed the P450st mutant eliminated loop region 1 Department of Biotechnology and Life Science, Tokyo University of (Leu151-Glu156), which located around the substrate channel of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan, P450 st (Fig. 1). It is expected that this mutation makes NADH easily 2 Department of Biomaterial Sciences, Graduate School of Agriculture accessible to heme iron of P450st. In fact, the catalytic activity of the and Life Science, The University of Tokyo, Bunkyo-ku, mutant using NADH as a direct electron donor was higher than that of Tokyo 113-8657, Japan. [email protected] wild-type P450st. The effect of this mutation on affinity of NADH NAD(P)-dependent alcohol dehydrogenases (ADHs; EC 1.1.1.1) will be discussed. belong to the oxidoreductase family, which catalyze the intercon- version reaction of alcohols with the corresponding aldehydes using NAD(P) as the cofactor. The enzymes can be divided into three groups; group I, group II and group III ADHs. The group III ADHs have been little studied so far. In previous study, we succeeded to express with Escherichia coli and purify the group III ADH derived from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhADH). In this study, we examined enzymatic activities of PhA- DHs containing various metal ions.

Table 1 Dependence of metal ions on PhADH activity Added metal ions Activity (%)

Ca2+ 0 Mg2+ 0 Mn2+ 230 Fe2+ 100 Ca3+ 82 Co2+ 110 Ni2+ 240 Cu2+ 0 Zn2+ 0 Fig. 1 Structure of the substrate channel of P450st. Leu151-Glu156 are highlighted in red

123 S192 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224

P521 Bilirubin oxidase from Myrothecium verrucaria (BOD; EC 1.3.3.5) pH dependence of structure and enzymatic activity classified in multicopper oxidase families could catalyze dioxygen to water around the neutral pH condition. BOD receives electrons at the of thermophilic cytochrome P450 type I Cu site from electron donating substrates. Then the electrons 1 1 2 Shohei Hayakawa , Keisuke Matsuda , Hirotoshi Matsumura , are transferred to the type II Cu/type III Cu cluster, where dioxygen is 1 1 1 Nobuhumi Nakamura , Masafumi Yohda , Hiroyuki Ohno reduced to water. BOD has been often applied to the biocathode of 1 Department of Biotechnology and Life Science, Tokyo University biofuel cells. DET of BOD at graphite and gold electrodes has been of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan, reported previously. However, the gold electrode showed only few 2 Department of Biomaterial Sciences, Graduate School of Agriculture current density. and Life Science, The University of Tokyo, Bunkyo-ku, In this study, we investigated DET of BOD at an AuNPs-electrode. In Tokyo 113-8657, Japan. [email protected] comparison with the polycrystalline gold electrode, the AuNPs-elec- Cytchrome P450s (P450s) are heme-containing monooxygenases trode showed high catalytic density (Fig. 1). We also report the DET involved in a variety of oxidative reactions. P450s are potentially of the other multicopper oxidases. useful catalysts for the production of chemicals which are difficult to synthesize by usual organic synthesis methods. Thermophilic cyto- chrome P450s, which have high stability and activity even under the extreme conditions, are particularly useful. We have investigated the character of thermophilic cytochrome P450 from Sulfolobus tokodaii 0 strain 7 (P450st), and then, have revealed high thermostability of P450st, so far. Here, we report pH dependence of structure and poly AuE activity of P450st. No changes in UV-visible spectra were observed 200 for different acidic pH values. Although it was observed that some spectral changes occurred at basic pH, the changes were reversible. 400 The pH dependences of catalytic reactions were examined through the peroxide shunt pathway. Both reactions of ethylbenzene hydroxyl- AuNPs ation and styrene epoxidation were activated at basic solutions 600 (Fig. 1).

0.1 0.2 0.3 0.4 0.5 0.6 0.7

Fig. 1 Linear sweep voltamograms for BOD immobilized on an AuNPs-electrode and on a polycrystalline gold electrode under air- saturated conditions with stirring at 1000 rpm

P523 Redox responses of D-fructose dehydrogenase on nanoparticles modified electrodes Masato Suzuki, Kenichi Murata, Kazuki Kajiya, Nobuhumi Nakamura, Hiroyuki Ohno Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. [email protected] The direct electron transfer (DET) reaction of fructose dehydrogenase (FDH) from Gluconobacter sp. at a silver nanoparticles (AgNPs)- modified electrode was examined. We fabricated an AgNPs-modified electrode (AgNP/AgE) in a simple manner by casting only AgNPs Fig. 1 The yields of product by ethylbenzene hydroxylation at onto a supporting Ag electrode. Then AgNP/AgE was covered with 2- various pH mercaptoethanol (MET) to obtain the MET-modified AgNP/AgE (MET-AgNP/AgE). P522 Cyclic voltammetry (CV) was performed using the MET-AgNPs/AgE Construction of a biocathode based on direct electron as a working electrode in the 100 mM acetate buffer containing FDH and D-Fructose. The catalytic oxidation current was observed and transfer of multicopper oxidases the current began to increase at a potential around -100 mV Kazuki Kajiya, Kenichi Murata, Nobuhumi Nakamura, (vs. Ag/AgCl). Hiroyuki Ohno The structure of the heme c-domain at the electrode surface was Department of Biotechnology and Life Science, Tokyo University of analyzed by surface-enhanced resonance Raman spectroscopy Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. (SERRS) with an excitation wavelength of 413.1 nm. In the SERRS [email protected] spectrum of FDH adsorbed on the AgNPs electrode at -300 mV, the The third generation biosensors and biofuel cells are constructed by -1 oxidation marker band m4 was observed at 1,362 cm , and the spin direct electron transfer (DET) reaction between enzymes and elec- and coordination marker bands m2 and m3 were observed at 1,592 and trodes. To obtain high current density, suitable electrodes and 1,495 cm-1, respectively. All these bands have their corresponding immobilization methods of enzymes are required for individual redox bands at the same frequencies in the resonance Raman spectrum of enzymes. Especially, nano-materials such as carbon nanoparticles and FDH solution, indicating that the native structure of the heme gold nanoparticles (AuNPs) have been utilized to increase current c-domain of FDH is maintained at the surface of the MET-AgNP/ density. AgE. When the electrode potential was raised at -50 mV, the

123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S193 oxidation state peak of heme c was observed. This result suggests that 1Graduate School of Engineering, Nagoya University, the electron transfer to the surface of the electrode occurs through the Nagoya 466-8603, Japan, heme c-domain. 2Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan, 3Center of Quantum Science and Technology under Extreme Conditions (KYOKUGEN), Osaka University, Osaka 560-8531, P524 Japan. [email protected] Control of the stability of novel heme Fe–N-terminal Nitrogenase is a metalloenzyme, which catalyzes a reduction reaction amino group coordination bond in denatured of atmospheric dinitrogen to ammonia. This dinitrogen activation cytochrome c reaction could occur efficiently in a biological condition, Nitrogenase 1 2 1 has attracted much attention in recent years. Over the past years, Hulin Tai , Toratane Munegumi , Naoki Watanabe , extensive experimental and theoretical studies have been conducted, Kiyofumi Irie1, Shigenori Nagatomo1, Yasuhiko Yamamoto1 1 however, details of the reaction mechanisms are still unclear. Department of Chemistry, University of Tsukuba, Recently, inorganic model complexes were synthesized, which well Tsukuba 305-8571, Japan, 2 reproduced the structural features of the native metal centers ([8Fe- Department of Materials Chemistry and Bioengineering, 7S] clusters) [1, 2]. These inorganic model complexes are important Oyama National College of Technology, Oyama 323-0806, Japan. for understanding of the fundamental properties of [8Fe-7S] clusters. [email protected] In this study, electronic structures of the inorganic model complexes In the denatured states of H. thermophilus (HT) cytochrome c552 (cyt are investigated by using the broken-symmetry DFT methods. Iron c552) and P. aeruginosa (PA) cyt c551, and their mutants, the N-terminal charge states and spin structures are discussed [3, 4]. amino group of the polypeptide chain is coordinated to heme Fe in place of the axial Met, the His–Nterm form being formed (Fig. 1) [1, 2]. The coordination of the N-terminal amino group to heme Fe leads to loop formation by the N-terminal stretch preceding the first Cys residue bound to the heme. The His–Nterm form was shown to be rather stable and hence it can influence the stability of the denatured state. We have investigated the stabilities of the His–Nterm forms emerging upon gua- nidine hydrochloride (GdnHCl)-induced unfolding of the proteins. The stability of the His–Nterm form was found to be affected by the N-terminal stretch, such as its length and the N-terminal residue. With a given N-terminal residue, the stability of His–Nterm form is higher for a Fig. 1 Molecular structures of inorganic [8Fe-7S] model complexes 9-residue-N-terminal stretch than an 11-residue one. In addition, with [1, 2] a given length, the His–Nterm form with an N-terminal Gly or Glu is more stable by a few kJ mol-1 relative to that with an N-terminal Asn. References 1. Ohki Y et al (2003) J Am Chem Soc 125:4052 2. Ohki Y (2007) J Am Chem Soc 129:10457 3. Shoji M et al (2006) Int J Quantum Chem106:3288 4. Shoji M et al (2007) Chem Phys Lett 446:228

P526 DFT and ONIOM(DFT:MM) studies of myo-inositol Fig. 1 Ligand exchange upon unfolding of HT and PA oxygenase Hajime Hirao1, Keiji Morokuma1 1Fukui Institute for Fundamental Chemistry, Kyoto University, These results provide a novel insight into the stabilizing interactions Kyoto 606-8103, Japan. [email protected] in the denatured cyts c that will facilitate elucidation of the folding/ A nonheme diiron enzyme myo-inositol oxygenase (MIOX) catalyzes unfolding mechanisms of the proteins. unique ring-opening, four-electron oxidation of myo-inositol to D-glucuronate, presumably playing a critical role in the pathogenesis References of various diabetic complications. Although recent kinetic, spectro- 1. Tai H, Kawano S, Yamamoto Y (2008) J Biol Inorg Chem 13:25– scopic, and crystallographic studies have led to significant advances 34 in our understanding of MIOX, there remains much to be clarified. 2. Tai H, Munegumi T, Yamamoto Y (2009) Inorg Chem 48:331– We therefore performed computational studies on mouse MIOX using 338 density functional theory and hybrid ONIOM QM/MM methods. Our calculations showed that formation of a (superoxo)Fe(III)Fe(III) intermediate, which requires charge transfer from Fe(II) to dioxygen, is facilitated by nearby Lys257, and that the intermediate favors P525 ferromagnetic over antiferromagnetic coupling of the Fe(III) and the DFT studies on the electronic structures in inorganic superoxide site. The H/D kinetic isotope effect values obtained from [8Fe–7S] model complexes frequency calculations for the whole enzyme agreed well with the experimental value, supporting a mechanism in which, a hydrogen Mitsuo Shoji1, Yasutaka Kitagawa2, Takashi Kawakami2, 3 2 3 atom is abstracted from the substrate by the (superoxo)Fe(III)Fe(III) Shusuke Yamanaka , Mitsutaka Okumura , Kizashi Yamaguchi intermediate. Details are discussed in light of the results of our

123 S194 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 calculations, with particular emphasis on the effect of protein P528 environment. Dose His131 at the orifice of the catalytic cavity transiently binds a zinc ion in human porphobilinogen synthase? Nori Sawada1, Noriyuki Nagahara1, Yoko Endo2, Yoshiaki Nakajima2, Tomoyuki Kawada1 1Department of Hygiene and Public Health, Nippon Medical School, Tokyo 113-8602, Japan, 2Clinical Research Center on Occupational Poisoning, Tokyo Rosai Hospital, Tokyo, 143-0013, Japan. [email protected] Human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is a metallo- enzyme, containing two zinc-binding sites per subunit. As a distal zinc- binding site at the orifice of the catalytic cavity, His131 and Cys223 have been considered to transiently coordinate a zinc ion. As a proximal zinc- binding site at the bottom of the catalytic cavity, Cys122, Cys124 and Cys132 coordinate a zinc ion, which was catalytically essential. We unexpectedly found the mutant PBGS from human Hep3B cell, in which, His131 was substituted with arginine. Enzymatic activity of the mutant PBGS was lower than that of wild-type enzyme without change in the total number of coordinated zinc ion. To clarify whether His131 P527 binds the distal zinc ion, we constructed mutant enzymes, H131A Synthesis and properties of zinc myoglobin appending (His131 ? Ala) and C223A (Cys223 ? Ala), and performed zinc ion a benzenesulfonamide moiety as carbonic-anhydrase analysis, enzyme kinetic study for wild type and two mutant PBGSs in the presence of 2-mercaptoethanol and 20-fold excess molar of zinc ion. inhibitor Replacement of Cys223 with Ala did not change the enzymatic activity, Asami Nakagawa, Hiroshi Takashima, Keiichi Tsukahara but the total number of zinc ion was decreased. On the other hand, Department of Chemistry, Faculty of Science, Nara Women’s replacement of His131 with Ala decreased the enzymatic activity, but the University, Nara 630-8506, Japan. [email protected] zinc content did not change. It is concluded that Cys223 binds a distal Photoinduced electron-transfer reaction of hemoprotein complexes zinc ion and, however, it does not contribute to the catalysis. On the has been one of the important research subjects. We have prepared other hand, His131 does not bind a distal zinc ion and, however, it chemically modified myoglobin (Mb), whose cofactor is replaced by contributes to the catalysis in some way. zinc(II) porphyrin derivative, to construct an artificial photoinduced reaction system.

H O P529 N O O N Inhibition and activation of carbonic anhydrase H O N N SO2NH2 I and II activities by effective ocular hypotensive agents Zn (timolol and latanoprost) N N Junzo Hirose, Toshiyuki Hata, Yukio Ono CO2H 1Faculty of Pharmacy and Pharmceutical Science, Fukuyama University, Fukuyama, Japan. [email protected] ZnPPSA Several types of agents such as beta-blockers (e.g. timolol), prosta- Carbonic anhydrase (CA) is a metalloenzyme which catalyses the glandins (e.g. latanoprost) and carbonic anhydrase (CA) inhibitors are convention of carbon dioxide to carbonate and hydrogen ions. Ben- clinically used in the treatment of glaucoma. CA inhibitors are often zenesulfonamide derivatives have been developed as enzyme used as the adjunctive agent to anti-glaucoma agents such as timolol inhibitors for CA. In this study, we have synthesized zinc porphyrin and latanoprost. The effects of timolol and latanoprost on CA activ- having a benzenesulfonamide moiety (ZnPPSA) and reconstituted it ities were investigated by the enzyme kinetics. In our results, into apoMb. ZnPPSA was synthesized via seven steps and ZnMbSA latanoprost was a noncompetitive inhibitor (Ki = 0.24 mM), but was successfully obtained by cofactor reconstitution of ZnPPSA to timolol noncompetitively activated CA activity (KA [ 3 mM). apoMb. The absorption spectral change of ZnMbSA in addition of CA AutoDock simulations of timolol and latanoprost with CA clearly suggests the formation of ZnMbSA-CA complex. Interactions showed that latanoprost binds to the zinc ion, which is located at the between ZnMbSA and CA are discussed. bottom of the narrow cavity of CA, by the long chain of latanoprost (Fig. 1a) and that timolol binds to the entrance of the active site cavity of CA in a region where the proton shuttle residue, His 64, is placed (Fig. 1b). The results of AutoDock simulations strongly substantiate the observations obtained by the enzyme kinetics.

Reference 1. Sugimoto A, Hirose J, et al (2008) Biol Pharm Bull 31:796–801

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used pBEX as an expression vector and totally synthesized cDNA encoding CYP2D6. The expressed CYP2D6 was extracted from E. coli and purified by column chromatography. The carbon monoxide difference spectra for the purified CYP2D6 exhibited Soret maximum at 450 nm, with no evidence of cytochrome P420 formation. Thus, it is clear that we could purify the enzyme in the active form. Then we investigated the drug binding and metabolizing properties of CYP2D6. Several marketed drugs, for example, dextromethorphan and cimetidine, were examined, but not every drug was metabolized by this enzyme. To distinguish substrates for CYP2D6, we focused on the heme iron coordination state. In the presence of enough amounts Fig. 1 The surface models of latanoprost-CA I (a) and timolol-CA I of drugs, UV-vis difference spectra and resonance Raman spectra (b) complexes which are calculated by AutoDock simulation could reveal the effect of drug binding. In case of substrates, the coordination states were found to change clearly and the enzyme kinetic parameters (Km and Vmax) were calculated by Michaelis– P530 Menten plots. Building new electron transfer pathways in Ru-modified cytochrome cb562 Heather R. Williamson, Harry B. Gray, Jay R. Winkler P532 Beckman Institute, California Institute of Technology, Pasadena, Electrochemical studies of a bacterial nitric oxide CA, USA. [email protected] synthase Previous work on electron transport in Ru-modified cytochrome b562 [1] has demonstrated the importance of structure on the distance Charlotte A. Whited1, Katherine D. Lavoie2, Michael G. Hill2, decay constant, b. Two of nine mutants examined exhibited slower Jay R. Winkler1, Harry B. Gray1 electron transfer than predicted based on their donor-acceptor dis- 1Beckman Institute, California Institute of Technology, Pasadena, tances. Notably, Beratan et al. [2] have developed a theoretical CA 91125, USA, ‘‘dominant-pathway’’ framework that accounts for these slower 2Department of Chemistry, Occidental College, Los Angeles, reactions. We are testing the Beratan model by measuring and ana- CA 90041, USA. [email protected] lyzing electron transfer rates in a series of Ru-modified mutants of Nitric oxide synthases are responsible for biological production of the cytochrome cb562. One goal is to determine the role of thioether links signaling molecule nitric oxide. These enzymes produce nitric oxide to the heme in tuning distant electronic couplings. from arginine in two turnovers using a heme-thiolate active site. A mutant of a bacterial enzyme from the thermophile Geobacillus References stearothermophilus has been expressed [1]. This mutant contains only 1. Winkler JR, Di Bilio AJ, Farrow NA, Richards JH, Gray HB one solvent-exposed cysteine residue, Cys84, which is in close (1999) Pure Appl Chem 71:1753–1764 proximity to the heme. Cys84 was used to covalently attach the 2. Prytkova T, Kurnikov IV, Beratan DN (2007) Science 315:622– protein to an electrode, affording direct electron transfer to the heme 625 active site of the enzyme. Cyclic voltammetry and redox titrations have been conducted on this enzyme in order to characterize its electron transfer properties.

P531 Drug binding and metabolizing properties of human CYP2D6 Tadayuki Uno1, Shinji Uemura1, Shiori Yanagita2, Taku Yamashita1, Hiroshi Aoyama1 1Graduate School of Pharmaceutical Sciences, Osaka University, Japan, 2Graduate School of Pharmaceutical Sciences, Kumamoto University, Japan. The cytochrome P450s (CYPs) constitute a superfamily of heme- containing enzymes that catalyze the metabolism of a wide variety of endogenous and xenobiotic compounds. In human, one of the most important CYPs is CYP2D6 because it is involved in the phase I metabolism of about 25% of clinically often prescribed drugs, including neuroleptics, antidepressants, b blockers, and antiarryth- mics, and these substrates being structurally diverse. In addition, this enzyme is reported to show a number of single nucleotide polymor- phisms (SNPs) which may affect drug metabolism. The relationship between SNPs and drug metabolism, however, has not been analyzed in detail because CYP2D6 is unstable. Therefore, we tried to produce CYP2D6 in the active form and examine its properties at molecular Reference level. In order to express the enzyme in E. coli BL21 Gold (DE3), we 1. Sudhamsu J, Crane BR (2006) J Biol Chem 281:9623–9632

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P533 Acknowledgement: This work was supported by Research Grants Regulation of human tryptophanyl-tRNA synthetase Council of Hong Kong (HKU7512/05 M, HKU7043/06P, HKU2/06C, HKU1/07C, HKU7038/08P) and the University of Hong activity by heme Kong! Keisuke Wakasugi1,2 1 Department of Life Sciences, Graduate School of Arts and Sciences, References The University of Tokyo, Komaba 3-8-1, Meguro-ku, 1. Ishida S, Lee J, Thiele DJ, Herskowitz I (2002) Proc Natl Acad Tokyo 153-8902, Japan, 2 Sci USA 99:14298–14302 Precursory Research for Embryonic Science and Technology 2. Lin X, Okuda T, Holzer A, Howell SB (2002) Mol Pharmacol (PRESTO), Japan Science and Technology (JST), 4-1-8 Honcho, 62:1154–1159 Kawaguchi, Saitama 332-0012, Japan. [email protected] 2+ 3. Kim BE, Nevitt T, Thiele DJ (2008) Nat Chem Biol 4:176– Mammalian tryptophanyl-tRNA synthetases (TrpRSs) are Zn - 185 binding proteins that catalyze the aminoacylation of tRNATrp. The expression level of human TrpRS is highly upregulated by interferon- c (IFN-c). In this study, a heme biosynthesis inhibitor, succinylace- tone (SA), was found to inhibit TrpRS activity in IFN-c-activated cells without affecting TrpRS protein expression. In addition, sup- P535 plementation of lysates from the SA-treated cells with hemin fully Metallothionein model studies: zinc, cadmium, mercury restored TrpRS activity to control levels. Biochemical analyses using 1,2-benzenedithiolates with intramolecular NHS 2+ purified TrpRS demonstrated that heme could bind with Zn -deple- hydrogen bonds ted human full-length TrpRS to enhance the aminoacylation activity 1 2 1 significantly. In contrast, the Zn2+-bound form of TrpRS did not bind Taka-aki Okamura , Koji Baba , Hitoshi Yamamoto , Tetsuo Yamamoto1, Norikazu Ueyama1 heme. Further studies using site-directed mutagenesis clarified that 1 the Zn2+-unbound human H130R mutant cannot bind heme. These Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan, results provide the first evidence of the involvement of heme in 2 regulation of TrpRS aminoacylation activity. Chemical Analysis Research Center, National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan. [email protected] Metallothionein is a cysteine-based protein with a relatively low P534 molecular weight. The functions are detoxification of toxic metal Expression, purification and characterization ions such as Cd and Hg, regulation of the transport and storage of of the N-terminal domain of the human copper essential metal ions, scavenger of various radicals and active oxy- transporter (hCtr1) gen species, and so on. In metallothioneins, metals such as Zn and Cd are chelated by four cysteine thiolates with tetrahedral coordi- Xiubo Du, Xinghao Wang, Denise S. B. Chan, Hongzhe Sun nation geometry. The crystal structure of metallothionein suggests Department of Chemistry and Open laboratory of Chemical Biology, the presence of NHS hydrogen bonds to coordinating sulfur The University of Hong Kong, Pokfulam Road, Hong Kong, People’s atoms. Republic of China. [email protected] A series of Zn, Cd, and Hg 1,2-benzenedithiolates with intramolecular Copper is an essential element required for many important cellular NHS hydrogen bonds were synthesized and characterized by X-ray proteins and reactions, however, excess copper is toxic. In cells, analysis and spectral measurements. The IR spectra indicated the copper homeostasis is tightly regulated, resulting in very low free presence of the hydrogen bonds and showed that the NHS hydrogen cellular metal concentration. It has been shown previously that the bonds in Cd and Hg complexes are stronger than those in the corre- high affinity copper (Cu (I)) uptake at the plasma membrane in sponding Zn complexes, which are supported by theoretical humans is mediated by the hCtr1 protein, a three-putative trans- calculations. The experimental and theoretical results suggested that membrane protein (190 aa) [1, 2]. The methionine-rich region, which the NHS hydrogen bond influences the efficient capture of toxic Cd is regarded as potential metal-binding sites, is located in the extra- and Hg ions by metallothioneins. cellular domain in the N-terminus (hCtr1_N). Surprisingly, the widely used anticancer drug, cisplatin [cis-diammedichloroplatinum (II)], which bears a different coordination from Cu+, was also found to be R 2- transported by the protein both in yeast and mammalian [3]. H In this work, we overexpressed, purified and characterized the N-ter- O N R minal extracellular domain of hCtr1 (hCtr1_N, residues 1-55). Two S H N O independent methods, i.e. dialysis equilibrium and the protein’s pre- S M vention effect on the copper-catalyzed ascorbic acid oxidation, revealed S + S that the protein could bind 3 Cu ions per monomer. An average dis- H N K K K K 1/3 -14 N O sociation constant was determined to be D = ( 1 2 3) = 10 M H R for binding of Cu+ to hCtr1_N protein via competition with the O ligand bicinchoninic acid (BCA). The binding of Cu+ to hCtr1_N R + is reversible as the bound Cu can be released in the presence of che- (M = Zn, Cd, Hg) lating ligand, e.g. bathocuproine disulfonate bcs or BCA. Replacement of Met within the two Met-rich motifs with Ala greatly reduced the Cu+ binding affinity of the protein, also with a change of stoichiome- try(n) value. Interestingly, both the apo- and Cu+-bound hCtr1_N existed as monomer in solution based on gel filtration chromatography Reference and native electrophoresis. The implication for copper transport will 1. Baba K, Okamura T, Yamamoto H, Yamamoto T, Ueyama N be discussed. (2008) Inorg Chem 47:2837–2848

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P536 P538 Oxidative degradation of Reactive Blue 21 solutions Effect of cysteine residue on reaction of human by metal enzymes covalently immobilized myoglobin with hydrogen peroxide on aminopropyl glass beads Satoshi Nagao1, Osamu Asami1, Shun Hirota1 1 Enzo Laurenti1, Tatiana Marchis1, Andrea Barbero1 Graduate School of Materials Science, Nara Institute of Science and 1Department of Inorganic, Physical and Materials Chemistry, Technology, Nara 630-0192, Japan. [email protected] University of Torino, Torino 10125, Italy. [email protected] Myoglobin (Mb) is a well-known heme protein, which consists of 153 Reactive Blue 21 (RB21) is a copper(II)-phthalocyanine belonging to amino acids forming eight a-helices and seven non-helical segments. the class of reactive dyes, which represent about 12% of the world- The heme is coordinated by the nitrogen atom of the histidine residue, wide production of dyestuffs [1]. However, their use is particularly and dioxygen binds to the heme iron in the opposite position of the problematic because the low yield of binding with the fibres causes bound histidine. Human Mb (hMb) possesses a unique cysteine (Cys) the production of large volumes of wastewaters from exhausted baths. residue at position 110, whereas most of other mammalian Mbs do Therefore, the study of an eco-friendly way of degradation of these not. The effect of the Cys residue of Mb on the reactions with various compounds is a challenge for green chemists. In the textile and fabric molecules has been investigated. For example, reduction of the heme finishing industry, enzymatic processes have already replaced tradi- was observed for Cys-introduced mutant sperm whale metMb by tional one [2], however, their exploitation in solution is restricted by incubation under carbon monoxide atmosphere [1]. W14-peroxyl, cost and low operational stability. In this sense, immobilization Y103-phenoxyl, and C110-thiyl radicals of wild type (WT hMb) were appears to be a reasonable solution to overcome these limits. generated by an addition of H2O2 [2]. To further investigate the effect In this work, Glycine max (soybean) peroxidase and Trametes ver- of the Cys residue in Mb, we prepared Cys-depleted mutant (C110A) sicolor laccase have been covalently immobilized onto aminopropyl hMb and compared the reactions of WT and C110A hMbs with H2O2. glass beads with controlled pore diameter, in order to obtain solid and WT hMb degraded more than C110A hMb by the reaction with H2O2. reusable biocatalysts. The samples were characterized by means of However, the amount of degraded WT hMb after the reaction with several techniques (IR, ESR, UV-visible, BET, and SEM), their H2O2 decreased by addition of reduced glutathione, whereas the bleaching potential has been tested on aqueous solutions of RB21 at effect of glutathione was negligible for C110A hMb. In addition, WT different pH values, and on two exhausted dye baths coming from an hMb reacted with H2O2 faster than C110A hMb in the presence of Italian textile factory. Both the enzymes showed complete decolor- glutathione. These results indicate that degradation of WT hMb by the ation of RB21 in batch at pH 5.0 and a partial decoloration of the reaction with H2O2 is enhanced by the generated C110-thiyl radical, effluents, probably due to their heterogeneous composition. which is suppressed by electron transfer from glutathione to the Cys site. References 1. Peralta-Zamora P, Pereira CM, Tiburtius ERL, Morales SG, Rosa References MA, Minussi RC, Duran N (2003) Appl Catal B Environ. 1. Hirota S, Azuma K, Fukuba M, Kuroiwa S, Funasaki N (2005) 42:131–144 Biochemistry 44:10322–10327 2. Galante YM, Formantici C (2003) Curr Org Chem 7:1399–1422 2. Witting PK, Douglas DJ, Mauk AG (2000) J Biol Chem 275:20391–20398

P537 P539 Construction of an artificial enzyme for olefin Towards a reaction mechanism for the sulfur oxygenase metathesis Clemens Mayer, Dennis Gillingham, Donald Hilvert reductase of the thermoacidophilic archaeon Acidianus Laboratory of Organic Chemistry, ETH Ho¨nggerberg, Zu¨rich, ambivalens Switzerland. [email protected] Arnulf Kletzin1, Andreas Veith1, Kerstin Seyfart1, Tim Urich1,2, This work is supported by a Marie-Curie International Incoming Miguel Teixeira3, Carlos Fraza˜o3 Fellowship to D. G. (IIF-AEOM). 1Institute of Microbiology and Genetics, Darmstadt University of Olefin metathesis is a fundamentally new reaction in synthetic Technology, Schnittspahnstr. 10, 64287 Darmstadt, Germany, chemistry and it has quickly become established as one of the cor- 2Present address: Department of Genetics in Ecology, University of nerstones in synthetic analysis and polymer chemistry. An Vienna, Vienna, Austria, underappreciated feature of olefin metathesis is its bioorthogonality. 3ITQB, Av. da Repu´blica (EAN), 2781-901 Oeiras, Portugal. Current efforts to utilize this property are mired in the struggle to [email protected] create an active and reliable water-soluble catalyst [1]. We describe The soluble sulfur oxygenase reductase (SOR) is the initial enzyme in our efforts to create an artificial metalloenzyme by covalently linking sulfur oxidation by the archaeon Acidianus ambivalens. The globular, a metathesis catalyst to a protein scaffold. The new artificial metal- hollow 24-subunit enzyme with 432-point group symmetry [1] cata- loenzyme is optimized by mutagenesis to address selectivity problems lyzes the O2-dependent sulfur disproportionation to sulfite and H2S. in current olefin metathesis technology. The active sites in each subunit comprise a mononuclear low-poten- tial non-heme iron and a cysteine persulfide in a spacious pocket [2]. Reference Two consecutive pores provide substrate access to the active sites. 1. Jordan JP, Grubbs RH (2007) Small-molecule N-heterocyclic- The pores in the outer shell formed by chimney-like protrusions at the carbene-containing olefin-metathesis catalysts for use in water. fourfold symmetry axis have an apolar interior suggesting a substrate Angew Chem Int Ed 46:5152–5155 entry pathway [1]. Pore opening lead to an increase in activity [3].

123 S198 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224

The second pore formed by Met and Val residues provides entrance to P541 the active site cavity. Mutagenesis resulted in a decrease of enzyme How does the activator protein, P14K, function activity unless the pores remain intact. Binding of Zn2+ ions blocks the putative pathway of product exit at the threefold symmetry axis. on the maturation of cobalt-type nitrile hydratase? 1 2 3 A hydrogen-bonding network in the second coordination sphere sur- Satoshi Shimizu , Rory A. Camerom , Hiroshi Nakayama , 1 1 2 rounding the metal ion presumably contributes to the low reduction Takanori Sasaki , Miyuki Kuramoto , Don A. Cowan , 3 1 1 potential [1]. Mutagenesis of surrounding residues reduced but did not Naoshi Dohmae , Masafumi Yohda , Masafumi Odaka 1 fully abolish SOR activity [3]. Derivatives with the Fe3+ ion Department of Life Science and Technology, Graduate School of exchanged for Co2+,Mn2+,Ni2+, and Ga3+ resulted in active enzyme Engineering, Tokyo University of Agriculture and Technology, suggesting that a valence change does not occur during catalysis and Koganei, Tokyo, 2Advanced Research Center for Application Microbiology, that O2 is activated by the substrate and not by the metal [3]. We propose a reaction mechanism involving a hydroxyl-catalyzed Department of Biotechnology, University of the Western Cape, nucleophilic cleavage of cysteine-bound polysulfide as the initial step. Bellville, South Africa, 3 The resulting polysulfenic acid should act as a strong reductant for Biomolecular Characterization Team, Advanced Research Institute, RIKEN, Hirosawa, Japan. [email protected] activation of O2 and formation of polysulfonic acid as the primary product. Nitrile hydratase (NHase, EC 4.2.1.84), catalyzing the hydration of nitriles to the amides, is industrially important because it is used for References kilo-ton scale production of acrylamide. NHase consists of a- and 1. Urich T, Gomes CM, Kletzin A, Frazao C (2006) Science b-subunits having a low spin non-corrin cobalt or non-heme iron cat- 311:996–1000 alytic center with two post-translationally modified ligands, cysteine- 2. Urich T, Kroke A, Bauer C, Seyfarth K, Reuff M, Kletzin A sulfinic acid (Cys–SO2H) and -sulfenic acid (Cys–SOH). Both types of (2005) FEMS Microbiol Lett 248:171–176 NHase require their specific activator proteins, encoded downstream of 3. Veith A, Seyfarth K, Urich T, Protze J, Teixeira M, Fraza˜oC the structural gene, for their functional expression. The preliminary (2009) unpublished studies have suggested that both activator proteins assist the insertion of metals to the enzyme proteins. However, their detailed functions remain unknown. The activator protein of Co-type NHase from Bacillus pallidus RAPc8, P14K, has no known metal-binding motif but shows weak amino acid sequence homology with NHase b subunit. P540 Thus, we assumed that P14K interacted with NHase a subunit and Selective extraction of proteins from aqueous to ionic assisted the maturation of NHase. By pull-down experiments, P14K liquid phase using temperature-driven phase change was found to form a tight complex with a subunit (aP14K). When a Yuki Kohno, Kenta Fukumoto, Hiroyuki Ohno subunit was co-expressed with P14K in a Co-supplemented culture Department of Biotechnology, Tokyo University of Agriculture and medium in E. coli, aP14K incorporate a Co ion (aP14K(+Co)). The Technology, Koganei, Tokyo 184-8588, Japan. [email protected] absorption spectrum of aP14K(+Co) is very similar to that of Co-type We have reported that some ionic liquids (ILs) exhibited lower crit- NHase. These results suggest that P14K binds a-subunit and promotes ical solution temperature (LCST) type phase separation with water the Co-binding in first step of the Co-NHase maturation process. Latest [1]. These ionic liquids showed full miscibility with water upon results on the function of aP14K(+Co) will be presented. cooling but phase separated upon heating. In this study, we extracted proteins utilizing LCST-type ionic liquid/water system. We used tetrabutylphosphonium N-trifluoromethanesulfonyl leucine P542 ([P4444][Tf-Leu]) for protein extraction systems because of moderate phase separation temperature. As a model protein, we used horse Bridging ligand at the active site of oxidized [NiFe] heart cytochrome c (Cyt.c), which is a typical heme protein. An hydrogenase aqueous solution of Cyt.c was added to the ionic liquid, and the Hisao Osuka1, Shun Hirota2, Shin-ichi Terawaki1, solution was cooled at 20°C to make a homogeneous solution. The Yasuhito Shomura1, Hirofumi Komori1, Naoki Shibata1, phase separation was induced by heating the solution at 25°C. Cyt.c Yoshiki Higuchi1 was migrated from aqueous to ionic liquid phase (Fig. 1). We then 1Graduate School of Life Science, University of Hyogo, studied the higher-ordered structure of Cyt.c extracted in the ionic Hyogo 678-1297, Japan, liquid phase by resonance Raman spectroscopy. Some key marker 2Graduate School of Materials Science, Nara Institute of Science and bands suggested that the heme was in a six-coordinate and low-spin Technology, Nara 630-0192, Japan. [email protected] state in ionic liquid phase as in an aqueous solution. Hydrogenases are enzymes that catalyze the reversible oxidation of molecular hydrogen. They are found in a wide variety of microor- ganisms and play an important role in the energy metabolism. Hydrogenases are classified as [NiFe], [FeFe] and [Fe] hydrogenases according to the metal composition of the active site. X-ray crystal structures of [NiFe] hydrogenase from Desulfovibrio vulgaris Miya- zaki F showed that four cysteine sulfur atoms are coordinated to Ni and two of them are also coordinated to Fe. Fe has three more non- protein ligands (CN- and/or CO), and an additional bridging ligand exists between the two metals in the oxidized states (Ni–A: diatomic Fig. 1 Extraction of Cyt c from water phase to ionic liquid phase and Ni–B: monatomic species). No bridging ligand could be assigned in the electron density map of the X-ray crystal structure of the reduced state. Recent spectroscopic studies on the Ni–A state have Reference suggested that the atomic species of the bridging ligand should be an 1. Fukumoto K, Ohno H (2007) Angew Chem Int Ed 46:1852–1855 oxygen species (such as dioxygen or hydroperoxide). On the other

123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S199 hand, a hydrogen species (such as hydride) has been proposed to exist site, and the R2 subunit a tyrosyl radical and a diiron-oxygen cofactor, at this bridging ligand site in the reduced form. Here, we report the which are essential for initiation of the nucleotide reduction process in photoreaction of the Ni–A state, where the IR bands of the Fe ligands R1. The R2 subunit of the enzyme complex reacts with ferrous iron and were up-shifted to higher wavenumbers by illumination with light. dioxygen to generate a diferric iron–oxygen cluster and a tyrosyl radical that is essential for enzymatic activity [1]. The reduced form of the class Ib enzyme, Bacillus cereus R2, has been studied using a combination of circular dichroism (CD), magnetic circular dichroism (MCD), and vari- able-temperature variable-field (VTVH) MCD [2]. The tyrosyl radical in the active state has been studied by electron paramagnetic resonance (EPR/HF-EPR) and Raman spectroscopies. Bacillus cereus R2 Raman and HF-EPR spectra resemble most the Escherichia coli R2. Spectral features of individual ferrous iron sites have been analyzed to obtain detailed geometric and electronic structural information. The results have P543 been compared to the studies of Escherichia coli R2, mouse R2, and Crystal structure of the apo-protein of methionine p53R2, which all are of RNR class 1a. aminopeptidase from an extremely thermophilic References bacterium, Thermus themophilus HB8 1. Kolberg M, Strand KR, Graff P, Andersson KK (2004) Biochim 1 1 2 Takafumi Ando , Koichi Hashimoto , Armelle Melet , Biophys Acta Proteins Proteomics, 1699:1–34 1 3 2 Akashi Ohtaki , Keiichi Noguchi , Marie-Agne`s Sari , 2. Tomter AB, Bell CB, Rohr AK, Andersson KK, Solomon EI 2 1 1 Isabelle Artaud , Masafumi Yohda , Masafumi Odaka (2008) Biochemistry 47:11300–11309 1Department of Life Science and Technology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan, 2 Laboratoire de Chimie et Biochimie, Pharmacologiques et P545 Toxicologiques, UMR8601-CNRS, 45 rue des Sts Pe`res, 75270 Paris Cedex 06, France, A comprehensive exploration of FeMoco with density 3Instrumentation Analysis Center, Tokyo University of Agriculture functional theory and X-ray absorption spectroscopy and Technology, Koganei, Japan. [email protected] Travis V. Harris, Robert K. Szilagyi Methionine aminopeptidase (MAP) catalyses the removal of the Department of Chemistry and Biochemistry, Montana State N-terminal initiator methionine from nascent polypeptides. MAP has University, Bozeman, MT 59715, USA. [email protected] dinuclear metal center as a catalytic center. MAP incorporates Mn(II), Biological nitrogen fixation is carried out by the metalloenzyme Co(II), Ni(II), Zn(II) or Fe(II), but the biologically relevant metal has not nitrogenase, which binds, activates and reduces dinitrogen at an Fe–S been identified because the metals are lost during its purification. MAP is cluster of formula [MFe7S9X], where M is Mo, V or Fe, and X is an attracted as a target protein for anti-cancer agents because it is inhibited unknown interstitial atom (C, N or O). The most active and well by anti-angiogenesis and anti-tumor agents such as fumagillin. In this studied form of nitrogenase uses the Mo-containing active site, study, we determined the crystal structure of MAP from an extremely FeMoco, yet many questions remain regarding the structure of this thermophilic bacterium, Thermus themophilus HB8 at a resolution of cluster. Density functional theory (DFT) calculations supported by 2.25 A˚ . The crystal structure was determined by molecular replacement X-ray absorption spectroscopy (XAS) provide a powerful tool to method using the structure of MAP from E. coli. The structure of apo- address the still unknown composition, charge, spin coupling and HB8MAP closely resembled that human MAP (hMAP) and had pseudo electron density distribution in FeMoco. For example, S K-edge XAS twofold-related N and C-terminal domains. Each domains contained two features are sensitive to small changes in S (3p) covalency which we a-helices and two antiparallel b-strands, showing that HB8MAP belongs have quantified as a function of FeMoco chemical composition, to the ‘‘pita-bread’’ family. No metal was incorporated but the structure oxidation and spin state. Using DFT calibrated to XAS data, we have around the metal-binding site was conserved with hMAP. A methionine kept an unbiased view in exploring all possibilities in the multi- molecule was observed at the active site. This is the first structure of the dimensional parameter space of unknowns, including the protonation apo-protein of MAP complexed with methionine. state of homocitrate. Our results indicate that the lowest energy spin coupling state is one, which maximizes anti-ferromagnetic coupling. Furthermore, we obtained computational evidence in agreement with P544 previous XANES and EXAFS measurements for the infrequently Spectroscopic studies of the tyrosyl radical and the discussed [Mo(IV)–2Fe(II)–5Fe(III)] state with carbide anion as the binuclear iron site of the R2 subunit of ribonucleotide central atom and a protonated homocitrate group. reductase from Bacillus cereus Ane B. Tomter1, Caleb B. Bell III2, Giorgio Zoppellaro1, Anne-Laure Barra3, Edward I. Solomon2, K. Kristoffer Andersson1 1Department of Molecular Biosciences, University of Oslo, Norway, 2Department of Chemistry, Stanford University, Stanford, CA, USA, 3Grenoble High Magnetic Field Laboratory, CNRS, Grenoble, France. [email protected] Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis deoxyribonucleotides from the corresponding ribonucleotides needed for DNA synthesis and repair in all living organisms. Class I RNR is divided into three different classes and they all consist of two non- identical subunits called R1 and R2. The R1 subunit contains the active

123 S200 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224

P546 contact in our structure is between lysine 15 of CueR and a guanine in Direct electrochemistry of nitrous oxide reductase the operator. from Achromobacter cycloclastes We also present preliminary results on the crystallization of CueR in the repressor state bound to PcopA. These structures will allow us to Mika Hirasawa1, Yuji Obara1, Koyu Fujita2, David M. Dooley2, 1 complete the crystallographic characterization of the CueR mecha- Takamitsu Kohzuma nism of gene regulation. 1Institute of Applied Beam Science, Ibaraki University, Ibaraki, Japan, Reference 2Department of Chemistry and Biochemistry, Montana State 1. Ansari AZ, Bradner JE, O’Halloran TV (1995) DNA-bend University, Bozeman, USA. [email protected] modulation in a repressor-to-activator switching mechanism. Nitrous oxide reductase (N OR) catalyzes the two-electron reduction 2 Nature 374:371–375 of N2OtoN2 (Eq.1) as the final step of denitrification.

þ N2O þ 2e þ 2H ! N2 þ H2O ð1Þ

N2OR has two different types of multi-copper site; CuA and CuZ.CuA P548 is a binuclear electron-transfer site, analogous to that in cytochrome c The reduced [2Fe-2S] clusters in adrenodoxin oxidase. The unique tetranuclear copper cluster, Cu plays a role in Z and Arthrospira platensis ferredoxin share spin density N2O reduction. The redox potentials for CuA and CuZ in N2OR from Paracoccus pantotrophus were reported to be *+260 mV and with protein nitrogens, probed using 2D ESEEM *+60 mV (vs. NHE), respectively [1]. Sergei A. Dikanov1, Rimma I. Samoilova2, Reinhard Kappl3, The direct electrochemical behavior of protein molecules may be used Antony R. Crofts4,Ju¨rgen Hu¨ttermann3 to estimate both the redox potential and electron transfer kinetics of 1Department of Veterinary Clinical Medicine, University of Illinois, metallo-protein sites. The direct electrochemistry of N2OR from Urbana, IL 61801, USA, 0 2 Achromobacter cycloclastes (AcN2OR) was performed with a 4,4 - Institute of Chemical Kinetics and Combustion, Russian Academy of pyridine disulfide modified gold electrode (4-pyds/Au). N2OR dem- Science, 630090 Novosibirsk, Russia, onstrated the well defined quasi-reversible cyclic voltammogram at 3Fachrichtung Biophysics, University des Saarlandes, 66421 the 4-pyds/Au electrode, and the redox potential is 260 mV versus Homburg/Saar, Germany, 4 NHE at pH 7.0. A catalytic current for the direct N2OR electro- Department of Biochemistry, University of Illinois, Urbana, IL chemistry in the presence of N2O was observed and provides direct 61801, USA. [email protected] electrochemical direct evidence for the CuA site being an electron We have used X-band ESEEM to study the reduced [2Fe-2S] cluster donor to the CuZ site. in adrenodoxin and Arthrospira platensis ferredoxin. By use of a 2D approach (HYSCORE), we have shown that the cluster is involved in Reference weak magnetic interactions with several nitrogens in each protein. 1. Rasmussen T et al (2002) Biochem J 364:807–815 Despite substantial difference in the shape and orientational depen- dence of individual cross-peaks, the major spectral features in both proteins are attributable to two peptide nitrogens (N1 and N2) with similar hyperfine couplings *1.1 and *0.70 MHz. The couplings P547 determined represent only a small fraction (0.0003–0.0005) of the Insights into MerR mechanism of gene regulation: unpaired spin density of the reduced cluster transferred to these nitrogens over H-bond bridges or the covalent bonds of cysteine crystal structures of the CueR copper ligands. Simulation of the HYSCORE spectra has allowed us to metalloregulatory protein bound to DNA estimate the orientation of the nuclear quadrupole tensors of N1 and Monica A. Canalizo-Hernandez1, Yi Xue1, Alfonso Mondragon2, N2 in the g-tensor coordinate system. The most likely candidates for Thomas V. O’Halloran1,2 the role of N1 and N2 have been identified in the protein environment 1Department of Chemistry, Northwestern University, 2145 Sheridan by comparing magnetic-resonance data with structures of the oxidized Rd, Evanston IL 60201, USA, proteins. Differences indicate a possible role of redox-linked struc- 2Department of Biochemistry, Molecular Biology and Cell Biology, tural changes, analyzed using available structures for related proteins Northwestern University, 2145 Sheridan Rd, Evanston, IL 60201, in two redox states. USA The MerR protein family regulates the transcription of a variety of stress-response operons that play a protective role in bacteria. The P549 long and suboptimal spacing between the –35 and –10 promoter elements (i.e. 19 bp instead of 17 bp) of these promoters and the Solution trapping of oxygenated intermediates ‘DNA distortion mechanism’ are a key feature of this family of from an extradiol dioxygenase transcriptional activators. Mike Mbughuni1, Michael P. Hendrich2, John D. Lipscomb1 Here, we report the first crystal structure of a metal responsive MerR 1Department of Biochemistry, Molecular Biology and Biophysics, family member bound to DNA. The structure of CueR, the E. coli University of Minnesota, Minneapolis, MN 55455, USA, 1+ 2 copper sensor, bound to Ag and to a 23 bp copA promoter (PcopA) Department of Chemistry, Carnegie Mellon University, Pittsburgh, was solved to 2.9 A˚ resolution. The structure provides strong support PA 15213, USA. [email protected] for the mechanism of gene regulation previously proposed for this Homoprotocatechuate dioxygenase (HPCD) is a non-heme Fe(II) family of metalloregulators [1]. We observe significant remodeling of containing enzyme that activates dioxygen during a ring-cleaving the promoter characterized by unwinding of the central DNA base reaction of substituted catechols. This reaction represents a key step pairs, caused by the binding of Ag-CueR. in the microbial metabolism of aromatic compounds. We are inter- These metalloprotein induced DNA distortions results in shortening ested in understanding the O2 activation chemistry of these enzymes, of the duplex equivalent to roughly 2 base pairs. The main base-direct specifically the structures and reactivities of intermediates as they

123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S201 occur in the reaction cycle. We have previously presented optical and EPR evidence for short-lived intermediates in O2 activation phase of the reaction cycle [1, 2]. Also, intermediates have been structurally characterized by monitoring the reaction of the slow substrate 4- nitrocatechol in single crystals [3]. Transient kinetic studies of HPCD mutants have shown that a key active site acid/base catalyst, His200, promotes the oxygen activation and ring opening reactions. Here, we utilize rapid freeze quench trapping and EPR spectroscopy to char- acterize novel intermediates using His200 mutants. Results are presented that support the central role played by the Fe(II) in juxta- posing the O2 and catecholic substrates and in transferring the electron density required to activate both for reaction (supported by NIH GM24689).

Fig. 1

P551 Activation and inhibition of the nuclease activity by metal ions: structural insights into metal-ion binding sites in CRN-4 Yu-Yuan Hsiao1, Woei-Chyn Chu2, Hanna S. Yuan1 1Institute of Molecular Biology, Academia Sinica, Taipe, Taiwan, 2Institutes of Biomedical Engineering, National Yang Ming University, Taipei, Taiwan, ROC. [email protected] Cell death related nuclease 4 (CRN-4) is one of the apoptotic nuc- References leases involved in DNA degradation in Caenorhabditis elegans. 1. Groce SL et al (2004) Biochemistry 43:15141–15153 CRN-4 contains a N-terminal DEDDh exonuclease domain and a C- 2. Gunderson WA et al (2008) J Am Chem Soc 130:14465–14467 terminal domain with unknown function. To reveal the molecular 3. Kovaleva EG, Lipscomb JD (2007) Science 316:453–457 basis of DNA fragmentation in apoptosis, we determined CRN-4’s crystal structures in apo-form, Mn2+-bound active form and Er3+- bound inactive form. Comparison of the Mn2+-bound and Er3+-bound CRN-4 structures revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain P550 bound to a structural zinc ion, termed the Zn-domain, and folded into Structure and function of a copper-containing nitrite a novel mixed a/b structure, containing basic surface residues ideal reductase from a marine thermophilic Gram-positive for RNA/DNA recognition. Site-directed mutagenesis further con- bacterium, Geobacillus kaustophilus HTA426 firmed the catalytic residues in the N-terminal DEDDh domain and the DNA-binding residues in the Zn-domain. The structural com- Yota Fukuda, Masaki Nojiri, Kazuya Yamaguchi, parison of CRN-4 to a number of dimeric DEDDh family nucleases, Shinichiro Suzuki further demonstrated that CRN-4 not only dimerizes but also interacts Department of Chemistry, Graduate School of Science Osaka with DNA in a unique way. Combining all these data, we suggest a University, Osaka, Japan. [email protected] structural model where DNA is bound at the Zn-domain and cleaved Geobacillus kaustophilus HTA426 is a marine thermophilic gram- at the DEDDh nuclease domain in CRN-4 when the cell is triggered positive bacterium isolated from deep-sea sediment of Mariana trench for apoptosis. and its genome contains a putative copper-containing nitrite reductase (CuNIR) gene. This protein was over-expressed in Escherichia coli. The purified recombinant protein was characterized. The UV-vis spectrum of GkNIR has a strong maximum at around 600 nm, which P552 is the characteristic of Type 1 Cu S(Cys) ? Cu ligand-to-metal XAS studies on the influence of weak interaction in blue charge transfer band (Figure). EPR spectroscopic analysis indicates that this protein has one type 1 Cu and one type 2 Cu, as previously copper proteins, fern plastocyanin and pseudoazurin reported on other known CuNIRs. The nitrite reduction assay dem- variants onstrates that GkNIR has a comparable catalytic rate constant to those Hiromi Togashi1, Junko Yano2, Vittal Yachandra2, 3 -1 1 of other CuNIRs at 25°C(kcat = 2.1 9 10 s ). And also, kinetic Takamitsu Kohzuma analysis of electron transfer (ET) between GkNIR and its possible 1Institute of Applied Beam Science, Ibaraki University, physiological electron donor, cytochrome c551, was carried out by Bunkyo 2-1-1, Mito, Japan, stopped-flow techniques at 25°C. The second-order ET rate constant 2Lawrence-Berkeley National Laboratory, Berkeley, CA 94720, USA (k = 3.9 9 105 M-1 s-1) is adequate for efficient electron transfer. Weak interaction is important to maintain structure and functions in However, this value is lower than that of CuNIR-Cyt c551 from protein molecules. Very recently, several second sphere effect around Alcaligenes xylosoxidans by an order of magnitude. the active site in metalloproteins have been reported. Pseudoazurin

123 S202 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224

(PAz) is a bacterial blue copper protein, and Met16 is located at the P554 second sphere of the active site. The Met16Phe PAz mutant suc- Subtle interaction between porphyrin and apoprotein cessfully reproduced the active site structure and properties of fern plastocyanin. Several amino acids were also introduced in the vicinity probed by a breathing Raman band 1 2 1 of the coordinated His81 residue in PAz, and the spectroscopic and Tomoko Miyazaki , Saburo Neya , Junji Teraoka 1 redox properties of PAz mutant proteins have been studied rationally Graduate School of Science, Osaka City University, to elucidate the effect of non-covalent weak interaction. Osaka 558-0022, Japan, 2 X-ray absorption spectra (XAS) of PAz variants and plastocyanins Graduate School of Pharmaceutical Sciences, Chiba University, from fern and sea-lettuce were measured to know the role of second Chiba 263-8522, Japan. [email protected] sphere interaction on the structure and functions of blue copper active There are two different kinds of interactions between iron porphyrins site. The XAS spectra of wild type PAz and Met16Phe PAz clearly and apo-proteins in hemoproteins controlling their functions, one is demonstrates the structural differences due to the weak perturbation through covalent bonds and the other is through space. It is difficult to through the non-covalent interaction at the active site. XAS mea- elucidate the latter interaction quantitatively because of the subtle surements were performed at SSRL, which is supported by the contact. To evaluate it, we focused on a vibration mode in the reso- National Institutes of HBRTP, DRR, and by the U.S. DOE, BES and nance Raman (rR) spectra and verified a few frequency differences in Office of OBER. reconstituted myoglobin (Mb) with an artificial iron porphyrin (DMDP) and the 13C-DMDP. A methin carbon substituted with 13Cis fixed on the opposite side to propionates in the porphyrin (see Fig.) and then potentially able to be a probe of investigating the subtle P553 contact in the deep inside of the heme pocket. 13 12 The catalytic mechanism of SoxAX cytochromes: RR difference spectrum of CMbN3– C is shown in the figure upon Soret band excitation (406.7 nm). The frequency difference is dis- insights from spectroscopy and site directed -1 tinctive (3 cm ) only for a m7 mode that is a Cm breathing mode. The mutagenesis same order of frequency shift is observed for the other derivatives. A 1 1 2 James Kilmartin , Mark Riley , Graeme Hanson , shift of 5 cm-1 is observed for the porphyrin itself, which is estimated 1 Ulrike Kappler as an amount of frequency shift inherent in the 13C labeling. Con- 1 School of Chemistry and Molecular Biosciences, University of sequently, it turns out that a subtle interaction reduces the frequency Queensland, Queensland, Australia, shift. It is noteworthy to mention that the shift for MbCN is different 2 Centre for Magnetic Resonance, University of Queensland, from other low-spin hemo-proteins and almost none. Queensland, Australia. [email protected] The SoxAX c-type cytochrome from Starkeya novella is a component of the multi-enzyme bacterial sulfur oxidation (Sox) pathway and is 13C ν7 thought to catalyse the covalent attachment of reduced sulfur com- pounds to a carrier protein, SoxYZ. The SoxAX protein has three N N redox centres; two low spin heme groups (His/Met and His/Cys Fe ligated), and a Cu(II) site with nitrogen ligands, which has been N N shown to be catalytically important [1]. We have investigated the Pr Pr influence of the axial cysteine ligand of the SoxA heme on SoxAX catalysis and on the spectroscopic properties of the redox centres present in SoxAX. The EPR spectrum of SoxAX displays a type I ‘‘large gmax’’ heme signal at g = 3.5 (SoxX heme with His/Met 13CMbN - 12CMbN ligation), multiple type II heme signals corresponding to the SoxA 3 3 heme with three different His/Cys ligation configurations and a Cu(II) signal consistent with 3 or 4N donors. The multiple SoxA related EPR signals are thought to arise from two different orientations of His/Cys 1000 850 700 550 and from a modification of the Cys ligand to His/Cys-persulfide, Raman Shift / cm-1 which may be a result of this heme ligand being involved in catalysis. Site directed mutagenesis was performed to replace the Cys ligand Fig. 1 with Met, and the SoxAXC199M protein was purified and charac- terised using EPR, MCD and a recently developed kinetic assay [1]. SoxAXC199M protein was found to bind copper in a 1:1 ratio like the P555 wild-type protein. The EPR spectra contained the Cu(II) signal and a A theoretical study on lipid oxidation of metal-binding low spin heme signal that should represent both heme groups of SoxAXC199M. Unexpectedly, a high spin iron signal was observed in peptides: implication in the neurotoxicity of b-amyloid the EPR spectrum but not in the MCD, this may indicate low con- (Ab) peptide centrations of a high spin heme in the SoxAXC199M sample. Kinetic Ren-jie Lin1, Soomin Jang2, Chen-chang Wu1, Feng-Yin Li1 assay data shows lower activity (37%) and a decrease in substrate 1Department of Chemistry, National Chung Hsing University, affinity for SoxAXC199M when compared to wild-type SoxAX. Taichung, Taiwan 402, Republic of China, These results demonstrate that catalysis takes place at the copper 2Department of Applied Chemistry, Sejong University, centre and show for the first time that the SoxA heme Cys ligand is Seoul 143-747, Korea. [email protected] not critically important for the enzymatic activity of SoxAX. Amyloid-b peptide (Ab) was the principal constituent of plaques and fibrils, commonly believed as the main source of the neurotoxicity Reference associated with Alzheimer’s disease (AD). Metal ions have been 1. Kappler U et al (2008) J Bio Chem 283:22206–22214 proposed to play a central role in the Ab neurotoxicity, which is

123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S203 believed by oxidizing the membrane of neuron cells. In this study, we Murata K, Nakamura N, Ohno H (2007) Biomacromolecules investigate the lipid oxidation by the Ab peptide and its possible 8:2080–2086 oligomers when binding with various metal ions, such as Cu and Zn, 2. Fujita K, Nakamura N, Igarashi K, Samejima M, Ohno H (2009) through ab initio calculation. Several models of copper-Ab complex Green Chem (in press) were proposed and investigated with ab initio calculation. All struc- tures of metal-Ab complexes were optimized at the M052X/ LANL2DZ level with the Gaussian 03 suit of program. Our results should provide some insight about the neurotoxicity caused by the P557 metal-Ab complexes and their associated aggregates. Modifications in the redox potential of the type I Cu References center of CueO by the mutations on the coordinating 1. Goate A, Chartier-Harlin M-C, Mullan M, Brown J, Crawford F, and non-coordinating amino acids Fidani L, Giuffra L, Haynes A, Irving N, James L, Mant R, Kunishige Kataoka1, Yasuo Maeda1, Shinji Kurose1, Newton P, Rooke K, Roques P, Talbot C, Pericak-Vance M, Madoka Sekimoto1, Naoya Shinohara1, Maiko Tsutsumi2, Roses A, Williamson R, Rossor M, Owen M, Hardy J (1991) Yuko Miura2, Seiya Tsujimura2, Kenji Kano2, Takeshi Sakurai1 Nature 349:704 1Graduate School of Natural Science and Technology, Kanazawa 2. Raffa D, Rauk A (2007) J Phys Chem B 111:3789 University, Kanazawa 920-1192, Japan, 2Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. [email protected] CueO is a multicopper oxidase involved in the Cu efflux system of P556 E. coli. CueO is promising best as the cathodic enzyme for biofuel Biocatalytic oxidation of cellobiose in hydrated ionic cells due to its efficient direct-electrochemistry between electrode and the type I Cu center, which transfers electron to the dioxygen- liquid reducing trinuclear Cu center. While the limiting currents obtained by Kyoko Fujita1, Nobuhumi Nakamura1, Kiyohiko Igarashi2, the four-electron reduction of dioxygen was very high, the overpo- Masahiro Samejima2, Hiroyuki Ohno1 tential of CueO is greater than those in fungal laccases because of 1Department of Biotechnology, Tokyo University of Agriculture and comparatively low redox potential of the type I Cu center. In the Technology, 2-24-16 Nakacho, Koganei, Tokyo, Japan, present study, we mutated Met510 axially coordinating the type I Cu 2Department of Biomaterials Sciences, Graduate School of center and Asp439 hydrogen-bonded with His443 coordinating to the Agriculture and Life Sciences, The University of Tokyo, 1-1-1 type I Cu center. We also mutated Pro444 to form a hydrogen bond Bunkyo-ku, Tokyo, Japan. [email protected] with the sulfur atom of Cys500 coordinating the type I Cu. Mutations Ionic liquids (ILs), typically possess almost no vapour pressure, are on Met510 with Gln gave the phytocyanin-like type I Cu center to recently being investigated for a variety of bio-applications. However, show an anisotropic character in the EPR spectrum and lowered in most cases, the proteins are not homogeneously dissolved and enzyme activities in accordance with the shift in the redox potential to retained the activity in ILs. We have been studying hydrated ILs as negative potential. The redox potential also shifted to negative solvents for proteins. Hydrated ILs maintain basic properties of pure potential by the mutations of Met510 with Ala and Thr, producing the ILs, but a small amount of water was effective enough to considerably phytocyanin-like type I Cu center, while the mutation with Leu made improve the protein solubility [1]. This work aims to investigate the the redox potential shift to positive potential, leading to an increase in biocatalysis of cellobiose dehydrogenase (CDH) in hydrated ILs. enzyme activity. The redox potential of the type I Cu center in CueO Hydrated choline dihydrogen phosphate ([ch][dhp]) is used as an IL was also shifted to positive potential by breaking down the hydrogen medium. bond between Asp439 and His443 and by forming a hydrogen bond CDH is successfully dissolved in hydrated [ch][dhp]. Furthermore, between Cys500 and an amino acid mutated for Pro444, also showing both inter- and intra-electron transfer of CDH were observed (Fig. 1) increases in enzyme activities. The present study shows that oxidizing [2]. It is suggested that the extraction of electrons from cellobiose has activity and efficiency as electrode catalyst of multicopper oxidases successfully carried out by biocatalysis in the hydrated ILs. are increased by the single mutations.

P558 Evidence for two electron entrances in bilirubin oxidase Cellobiose FAD 3+ 2+ 2 cyt c (Fe ) Kenichi Murata, Kazuki Kajiya, Nobuhumi Nakamura, - heme (Fe ) 2 e - e e- Hiroyuki Ohno Cellobiono 3+ heme (Fe ) lactone FADH 2+ Department of Biotechnology and Life Science, Tokyo University of 2 2 cyt c (Fe ) Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. [email protected] Bilirubin oxidase (BOD) catalyzes the one electron oxidation of Cellobiose substrates with the concomitant reduction of dioxygen to water at dehydrogenase neutral pH, and thus is a useful biocatalyst in the cathode compart- ment of a biofuel cell. In this study, we examined the oxidative activity of BOD toward 2,20-azinobis(3-ethylbenzthiazoline-6-sulfo- Fig. 1 Schematic representation of the pathway of electron transfer 2- 4- nate) (ABTS ) and hexacyanoferrate (Fe(CN)6 ) in water/alcohol from cellobiose, CDH to cyt c mixed solvents. These substrates are known as electron transfer mediators between BOD and an electrode. References The effect of alcohols (methanol, ethanol, and 1-propanol) on the 1. Fujita K, MacFarlane DR, Forsyth M, Yoshizawa-Fujita M, oxidation of the substrates was studied. Kinetic measurements of

123 S204 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 enzymatic activity were carried out using UV-vis spectrophotometer P560 at room temperature. The increase in absorbances of ABTS- at -1 -1 3- Zinc(II) binding to the HNH nuclease motif: 420 nm (e = 38,000 M cm ) and Fe(CN)6 at 420 nm (e = 1,010 M-1 cm-1) was monitored. what is the reason for its catalytic inactivity? 1 1 1 Alcohols showed competitive inhibition against the oxidation of Ida Noe´mi Jakab ,Be´la Gyurcsik , Hajnalka Bara´t-Jankovics , 2 3 3 ABTS2-, whereas K for oxidation of Fe(CN)4- was not affected by Zolta´n Kele , Krystyna Sklarska-Kiss , Antal Kiss , m 6 4 the alcohol content. Additionally, the apparent K for the oxidation of Kyosuke Nagata m 1 ABTS2- increased with increasing the hydrophobicity of alcohols. Department of Inorganic Analytical Chemistry, University of Alcohols competitively bind to the binding site for ABTS2- by Szeged, P.O. Box 440, 6701 Szeged, Hungary, 2 hydrophobic interaction. These results suggest that BOD has two Department of Medical Chemistry, University of Szeged, Do´mte´r 8., electron entrances. 6720 Szeged, Hungary, 3Biological Research Center of the Hungarian Academy of Sciences, Institute of Biochemistry, Temesva´ri krt. 62, 6726 Szeged, Hungary, 4Department of Infectious Biology, Graduate School of Comparative P559 Human Science and Institute of Basic Medical Science, University of Analysis of subunit interaction in human adult and fetal Tsukuba, 1-1-1 Tennohdai, Tsukuba 305-8575, Japan. hemoglobins through the characterization of the acid– [email protected] The HNH motif displays bba structure with a central metal ion alkaline transition binding site. It is abundant within the active centre of nuclease 1 1 1 1 2 T. Shibata , S. Nagao , H. Tai , S. Nagatomo , H. Hamada , enzymes. The metal ion binds to the scissile phosphate of the sub- 2 3 1 H. Yoshikawa , A. Suzuki , Y. Yamamoto strate. It is supposed to polarize the P–O bond, stabilize the transitions 1 Department of Chemistry, University of Tsukuba, 305-8571, Japan, state and the cleaved product [1]. Bacterial Colicin E7 functions in the 2 Institute of Clinical Medicine, University of Tsukuba, 305-8575, presence of zinc(II). It can be overexpressed in E. coli only in the Japan, presence of an inhibitory protein Im7. Recently, we elaborated a 3 Department of Materials Engineering, Nagaoka College of method to express the 43 amino acid containing C-terminal HNH Technology, 940-8532, Japan. [email protected] motif of this protein. Surprisingly, this protein did not exert cyto- Human adult hemoglobin (Hb A) has been recognized as an excellent toxicity, and the in vitro nuclease assays also proved to be negative. model for investigating the structure-function relationship in alloste- CD and fluorescence spectroscopies and mass spectrometry were ric proteins. Despite the detailed structural and spectroscopic applied for studying its zinc(II) ion binding. We have designed sev- information available for the protein, functional properties of the eral mutants of the nuclease domain of Colicin E7 revealing the protein have not been as fully exploited as expected. Major defect in essential amino acid residues. the functional characterization of Hb A is the lack of experimental techniques, which enable quantitative characterization of functional properties of the constituent subunits of the protein. In this study, we have developed techniques for determining the equilibrium constant (pKa) of the acid–alkaline transition in the individual subunits of metHb A and human fetal hemoglobin (metHb F). The pKa values of the constituent subunits of metHb A as well as metHb F provide novel and highly sensitive probes for characterizing the effects of structural changes of not only the interface between the subunits within the protein, but also the contact between heme and the protein in the heme pocket. The present study not only describes novel techniques for quantitative characterization of functional properties of the con- The nuclease domain of Coicinl E7 (HNH motif in blue) in complex stituent subunits of metHb A and metHb F, but also provides novel with DNA (PDB Id: 1ZNS) insight into the molecular mechanism through the subunit interaction.

Reference 1. Doudeva LG, Huang H, Hsia K-Ch, Shi Zh, Li Ch-L, Shen Y, Cheng Y-Sh, Yuan HS (2006) Protein Sci 15:269–280

P561 Structural characterization myoglobin peroxo intermediate Shusuke Kusama1, Masaki Unno1, Hui Chen2, Sason Shaik2, pKa Masao Ikeda-Saito1 1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan, 2Department of Organic Chemistry and the Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, Govat Ram Campus, 91904 Jerusalem, Israel. [email protected] Ferric peroxo form is one of important intermediates for mono-oxy- Scheme 1 Acid-alkaline transition of metHb genase heme enzymes. Crystal structural analysis of the reactive 123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S205 intermediate provides significant information to understand how the 0.40 enzymes control its reactivity. The instability of peroxo intermediate makes us difficult to trap it especially in crystal. The peroxo myo- globin (Mb) crystal was prepared by a cryo-reduction method to determine a crystal structure of the key intermediate. Oxy Mb crystal 0.30 was converted to the peroxo form at 100 K by irradiation of 1.0 A˚ wavelength X-ray at synchrotron. The generation of peroxo Mb was confirmed by the single-crystal absorption spectrum. The X-ray dif- fraction data for crystallography were collected with 0.6 A˚ wavelength X-ray to avoid radiation damage of the heme center. By 0.20 using the two distinct wavelengths of X-ray, we have successfully Absorbance determined the crystal structure of peroxo Mb at 1.20 A˚ resolution (Fig. 1). The crystal structure of peroxo Mb is very similar to that of oxy Mb including a double conformation of distal histidine (His 64) 0.10 and it is consistent with the result of QM/MM calculation.

0.00 300 400 500 600 Wavelength/ nm

Fig. 1 Absorption spectra of pMMO. pMMO (line), pMMO + dur- oquinol + O2 (dashed line), pMMO + duroquinol + O2 +CH4 (dotted line)

References 1. Lieverman RL et al (2005) Nature 434:177–182 2. Amanda SH et al (2008) Biochemistry 44:10954–10965 Fig. 1 The heme vicinity crystal structure of peroxo Mb

Reference 1. Unno M, Chen H, Kusama S, Shaik S, Ikeda-Saito M (2007) J P563 Am Chem Soc 129:13394–13395 Cooperative L-Trp binding to human tryptophan 2,3- dioxgenase revealed by Resonance Raman Spectroscopy Eiko Fukumura1,2, Hiroshi Sugimoto1, Yuko Misumi3, P562 Takashi Ogura3,4, Yoshitsugu Shiro1 1 EPR and UV-vis spectral changes of particulate Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, Hyogo 679-5148, Japan, methane monooxygenase from Methylosinus 2Department of Biological Science, Graduate School of Science, trichosporium OB3b Osaka University, Osaka, Japan, Kenji Tabata1, Masaru Saio1, Tomonori Yaguchi1, 3Department of Life Science and 4Picobiology Institute, Graduate Noriyuki Asakura1, Toshiaki Kamachi2, Ichiro Okura1 School of Life Science, University of Hyogo, Hyogo 678-1297, 1Graduate School of Bioscience and Bioengineering, Tokyo Institute Japan. [email protected] of Technology, Yokohama 226-8501, Japan, Tryptophan 2,3-dioxygenase (TDO) is a heme-containing enzyme 2 Graduate School of Engineering, Nagoya University, that catalyzes the oxidative cleavage of L-tryptophan (L-Trp) to N- Nagoya 464-8602, Japan. [email protected] formylkynurenine by the addition of O2 across the 2,3-bond of the Particulate methane monooxygenase (pMMO) is an integral mem- indole ring. This reaction is the first and rate-limiting step in the brane copper-containing enzyme that catalyses the conversion of kynurenine pathway in mammals. TDO is present as a homo-tetra- methane to methanol. The crystal structures of pMMO reveals pMMO meric enzyme and widely distributed across species, ranging from has two or three copper centers [1, 2]. In this study, EPR and UV-vis bacteria to mammals. Here we report the conformational changes in spectral changes of pMMO in enzymatic reaction were investigated. the heme pocket of recombinant human TDO (rhTDO) in ferric form EPR spectra of pMMO were measured by addition of duroquinol, O2, that are induced by L-Trp binding using both resonance Raman and methane. The changes in super hyperfine structure of EPR spectra optical absorption spectroscopies. The deconvolution analysis of the suggested pMMO has three types EPR active copper ions. Figure 1 heme Raman bands at various concentrations of L-Trp revealed that shows spectral changes of pMMO. Absorption around 320 nm was the wild-type enzyme exhibits homotropic cooperativity in L-Trp increased by adding O2 to reduced pMMO. The addition of O2 and binding. The spectra of Y42A mutant and the inter-subunit contacts methane did not cause the increase of absorption around 320 nm. reported in the bacterial TDO structures suggest that the Y42 of These results suggest that copper–dioxygen complex was formed by rhTDO is responsible for the cooperative binding of L-Trp by par- reaction with reduced pMMO and dioxygen, and reacts with methane. ticipating in the active site of the adjacent subunit [1].

123 S206 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224

Above results clearly indicate that the bis(DMED)Mo complex mimics both of reactivity and structure of the oxotransferases. Detailed reaction mechanism for the PCET and OAT reactions and ligand effects on the reactions are presented.

P565 Characterization of cytochrome c-554 from Methylosinus trichosporium OB3b, a heme protein exhibiting a HALS EPR signal E. Harbitz, K. K. Andersson 1Department of Molecular Biosciences, University of Oslo, Oslo, Norway. [email protected] Reference A novel low-spin cytochrome c-554 has been purified from the 1. Fukumura E, Sugimoto H, Misumi Y, Ogura T, Shiro Y (2009) J methane oxidizing bacteria Methylosinus trichosporium OB3b. This Biochem (in press) cytochrome exhibits a ferric HALS (highly anisotropic low-spin) low-temperature EPR signal, with a typical HALS lineshape and a gmax value of 3.41 [1–4]. MALDI-TOF-MS has shown that this cytochrome has a molecular weight of 12,230 Da, and that it only P564 contains one heme group. Light absorption and CD spectroscopy indicates a His/Met coordination of the heme iron. The sequence of Proton-coupled electron transfer and oxygen atom the first 39 N-terminal amino acids and 27 C-terminal amino acids transfer reactivities of MoO/Mo system has been determined and sequence alignments performed. The most Hideki Sugimoto1, Hiroyuki Tano1, Hiroyuki Miyake1, similar hits represent c-type cytochromes from bacteria belonging to Shinobu Itoh2 the phylum Bradyrhizobiaceae, in which Methylosinus trichosporium 1Department of Chemistry, Graduate School of Science, Osaka City OB3b is included. C-type cytochromes have similar His/Met coor- University, Osaka 558-8585, Japan, dination of the heme iron they can exhibit different EPR signals. 2Department of Material and Life Science, Graduate School of The correlation between different EPR signals and structure is not Engineering, Osaka University, Osaka 565-0871, Japan. clearly known for His/Met coordinated hemes. Characterization of [email protected] this protein aims to provide a broader basis for understanding this Molybdenum-containing oxotransferases utilize water as the ultimate correlation. source or sink of oxygen in the overall catalytic reaction, coupled with proton-coupled electron transfer (PCET) and oxygen atom References transfer (OAT). OAT reactions by Mo complexes have been inves- 1. Zoppellaro G, Harbitz E, Kaur R, Ensign AA, Bren KL, An- tigated extensively, however, little is known on PCET reactions of dersson KK (2008) J Am Chem Soc 130:15348–15360 Mo complexes. We have report herein PCET/OAT reactions of 2. Zoppellaro G, Bren KL, Ensign AA, Harbitz E, Kaur R, Hersleth IV 2- bis(dithiolene)Mo complexes. [Mo O(DMED)2] (Chart 1) shows H-P, Ryde U, Hederstedt L, Andersson KK (2009) Biopolymers reversible MoV/MoIV redox couple in acetonitile (Fig. 1a red line). In (submitted) the presence of hydroxide ion, the redox couple becomes irreversible. 3. Teschner T, Benda R, Schu¨nemann V, Trautwein AX, Arciero This CV change completed by adding 2 equiv. of OH– (Fig. 1a blue DM, Hooper AB, Andersson KK (2002) Hyperfine Interactions C line, 1b), and its MoO2 form was generated stoichiometrically after 2 5:249–252 electron oxidation by electrochemical technique. Additionally, the 4. Zoppellaro G, Teschner T, Harbitz E, Karlsen S, Schu¨nemann V, MoO2 complex transferred its one oxygen atom some substrates. Trautwein AX, Arciero DM, Hooper AB, Ciurli S, Andersson KK (2006) Chem Phys Chem 7:1258–1267 O Cl - - - - S R S OMe S S = OMe -S R -S -S -S O Cl P566 DMED bdt bdtCl2 Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway Chart 1 in Staphylococcus aureus Michael T. Tiedemann1, Naomi Muryoi2, David E. Heinrichs2, 1 (a)− (b) Martin J. Stillman + 2 OH 1Department of Chemistry, 2Department of Immunology and Microbiology, The University of pa

I Western Ontario, London, ON N6A 5B7, Canada. ∆ [email protected]. Staphylococcus aureus, an antibiotic resistant bacterium, is a signif- icant problem in hospitals and communities worldwide. Bacterial 0123− -0.2 0 0.2 [OH ] / [Mo] survival is largely dependent on iron scavenging and Staphylococcus E / V vs. SCE aureus has adopted specialized mechanisms for scavenging iron from the host. The cell wall and membrane-associated iron regulated sur- Fig. 1 face determinant (Isd) proteins allow Staphylococcus aureus to

123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S207

the electron transfer pathway, but are still controversial regarding the rate determining step and the enzyme’s active form [2, 3]. In this work, we present the first non-catalytic voltammetric signals of a cd1NiR exemplar, using the protein purified from Pseudomonas nautica strain 617. This was achieved by anchoring the enzyme molecules onto self-assembled monolayers of hydrophilic thiols, as characterized by the AFM technique. Using this electrochemical approach, it was possible to assess the reduction potential and the dynamic behavior of both redox centers upon reduction, and was helping to understand the intra-molecular electron transfer steps, along with the proton-coupled reactions.

References 1. Besson et al (1995) Anaerobe 1:219–226 2. Oganesyan et al (2007) Inorg Chem 46:10950–10952 3. Wherland et al (2005) Chem Phys Chem 6:805–812 Acknowledgments: FCT (POCI/QUI/58026/2004, SFRH/BD/28921/ 2006).

P568 Fig. 1 Noncooperative cadmium(II) binding to human metallothionein 1a scavenge iron from the heme in hemoglobin. There are six Isd pro- D. E. K. Sutherland, M. J. Stillman teins (IsdH, IsdB, IsdA, IsdC, IsdE and IsdF) located at different Department of Chemistry, The University of Western Ontario, depths in the cell wall and membrane (Fig.). Magnetic circular London, ON N6A 5B7, Canada. [email protected] dichroism spectroscopy, together with electrospray ionization mass Metallothionein (MT) is a ubiquitous metalloprotein found in all spectrometry were used to determine that the heme in IsdA-N bacteria, mammals and invertebrates. Mammalian MTs contain two transfers through IsdC-N, to IsdE. Heme transfer was demonstrated to independent metal binding domains with a total of 20 cysteines. In occur in a unidirectional fashion and propagate down the pathway in the overall structure, the a and b domains each encapsulate a metal the sequence IsdB-N 2 ? IsdA-N ? IsdC-N ? IsdE or, alterna- thiolate core. The mechanistic and structural properties of the tively, initiating from IsdH-N3 ? IsdA-N ? IsdC-N ? IsdE. Only individual metal binding domains, and the protein as a whole, are NEAT-H3 and NEAT-B2 could transfer bidirectionally that is in the illustrated. Through the use of ESI-MS, it has now been shown that reverse direction as well, and only with each other. metallothionein binds cadmium in a noncooperative manner. Upon addition of substoichiometric amounts of cadmium, all partially metallated intermediates were observed. Additionally, novel References supermetallated structures, both Cd -b-MT and Cd -a-MT, were 1. Muryoi N, Tiedemann MT, Pluym M, Cheung J, Heinrichs DE, 4 5 observed at eight equivalents of cadmium. Together these structures Stillman MJ (2008) J Biol Chem 283:28125–28136 may be critically important to the role of MT in heavy metal- 2. Tiedemann MT, Naomi M, Heinrichs DE, Stillman MJ (2008) detoxification as well as metal-exchange through protein–protein Biochem Soc Trans 36:1138–1143 interactions.

P567

Probing the regulation mechanism of cytochrome cd1 from Pseudomonas nautica: an electrochemical approach Alexandra S. Serra1, Ste´phane Besson1, Ana S. Viana2, Isabel Moura1, Jose´ J. G. Moura1, M. Gabriela Almeida1,3 1REQUIMTE, Department of Quı´mica, CQFB, Fac. Cieˆncias Tecnologia, University Nova Lisboa, 2829-516 Caparica, Portugal, 2CQB, Fac. Cieˆncias Univ. Lisboa, Campo Grande, 1749-016 Lisboa, Portugal, 3Escola Sup. Sau´de Egas Moniz, 2829-511 Caparica, Portugal. [email protected] Cytochrome cd1 nitrite reductase (cd1NiR) is a homodimeric enzyme that catalyzes the one electron reduction of nitrite to nitric oxide. Each References monomer contains a c-type and d1-type hemes, the latter being the 1. Sutherland DEK, Stillman MJ (2008) Biochem Biophys Res catalytic center [1]. Previous studies on the catalytic mechanism Commun 372:840–844 performed with enzymes from other bacterial sources mostly agree on 2. Rigby Duncan KE, Stillman MJ (2007) FEBS J 274:2253–2261

123 S208 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224

P569 tightest binding, in mutant. The bimolecular rate constant increases 7 -1 -1 10 -1 -1 The active site of particulate methane monooxygenase from 10 M s in the wild type to *10 M s . Stephen M. Smith1, Swati Rawat2, Timothy L. Stemmler2, Amy C. Rosenzweig1 1Departments of Biochemistry, Molecular Biology, and Cell Biology and of Chemistry, Northwestern University, Evanston, IL 60208, USA, 2Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, MI 48201 USA. [email protected] Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that converts methane to methanol in methanotrophic bac- teria. In contrast with industrial catalysts, this biological oxidation of methane takes place at ambient temperature and pressure. The active site of pMMO is highly debated, with many groups suggesting a Reference functional role for copper and/or iron. The recent crystal structures of 1. Liang Z-X, Nocek JM, Huang K, Hayes RT, Kurnikov IV, pMMO from Methylococcus capsulatus (Bath) [1] and Methylosinus Beratan DN, Hoffman BM (2002) J Am Chem Soc 124(24): trichosporium OB3b [2] revealed a conserved dinuclear copper center 6849–6859 located in the pmoB subunit. A second conserved site occupied by zinc in M. capsulatus (Bath) pMMO and copper in M. trichosporium OB3b pMMO was recently hypothesized to contain a diiron active site based on Mo¨ssbauer spectroscopic and activity data [3]. While the active site location is still debatable, here we show new activity and P571 spectroscopic data indicating that copper, not iron, is the metal in the Spectroscopic and functional characterization active site of pMMO. of the human enteric ferric reductase (Dcytb) incorporated into Nanodiscs References Shin-ichi J. Takayama,1 Tomoko S. Misono,1 Stephen G. Sligar,2 1. Lieberman RL, Rosenzweig AC (2005) Nature 434:177–182 A. Grant Mauk1 2. Hakemian AS, Kondapalli KC, Telser J, Hoffman BM, Stemmler 1Department of Biochemistry and Molecular Biology and the Centre TL, Rosenzweig AC (2008) Biochemistry 47:6793–6801 for Blood Research, University of British Columbia, Vancouver, BC 3. Martinho M, Choi DW, DiSpirito AA, Antholine WE, Semrau V6T 1Z3, Canada, JD, Mu¨nck E (2007) J Am Chem Soc 1129:15783–15785 2Departments of Biochemistry and Chemistry, College of Medicine, and Beckman Institute, University of Illinois, Urbana, IL, USA. [email protected] P570 Duodenal cytochrome b (Dcytb) is an iron-regulated heme protein Use of electrostatic surface residue modifications to that is highly expressed in the duodenal brush border membrane and that has been proposed to catalyze reduction of elemental dietary Fe3+ stabilize reactive configurations in the myoglobin/ to Fe2+ by ascorbate to permit iron uptake by the Dmt1 transporter. cytochrome b5 complex Recently, Dcytb has been expressed in good yield from transformed Amanda K. K. Griffin, Peng Xiong, Judith M. Nocek, E. coli [1]. The resulting detergent-solubilized Dcytb exhibited the Brian M. Hoffman expected electronic spectrum but was not reduced by ascorbate, Department of Chemistry, Northwestern University, Evanston, IL, presumably as the result of detergent-induced destabilization of the USA. amandagriffi[email protected] heme binding sites. To eliminate detergent, we have now reconsti- Macromolecular interactions are at the heart of biology, and inter- tuted Dcytb into Nanodiscs, stabilized lipid bilayer discs (*7nm protein electron transfer (ET) measurements give a unique probe of diam.) each of which can bind one Dcytb molecule [2]. In this study, these interactions. For ET proteins, these interactions are generally Dcytb was reconstituted into Nanodisc-stabilized lipid bilayers controlled by the interfacial dynamics and not the ET process itself, as comprised of DMPC. Dcytb reconstituted into these Nanodiscs is we have illustrated through the study of interprotein ET within the soluble in the absence of detergents, is reduced by ascorbate, and physiological complex myoglobin (Mb)/cytochrome b5 (b5) [1]. We exhibits an EPR spectrum consistent with a greater component of here report dramatic progress in taking a pair of proteins that interact native structure than is the case for Dcytb in detergent. Nanodisc- weakly and altering them to form a well defined, functional complex stabilized Dcytb is being used for kinetic and spectroscopic charac- between Mb and b5. terization and for identification of inhibitors and activators of Dcytb To change the affinity of this electrostatically bound complex, we for use in development of therapeutic agents. have made a suite of Mb charge-reversal mutants consisting of D44K, D60K, E85K, and neutralization of the heme propionates. These References progressively strengthen the affinity between the two proteins from a 1. Ludwiczek S, Rosell FI, Ludwiczek ML, Mauk AG (2008) binding constant of 103 M-1 in the wild type to over 106 M-1, Biochemistry 47:753–761

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2. Nath A, Atkins WM, Sligar SG (2007) Biochemistry 46:2059– nitrogenase-like enzymes, dark-operative protochlorophyllide oxido- 2069 reductase (DPOR) and chlorophyllide oxidoreductase (COR). Both DPOR and COR consist of two separable components, the ATP- dependent reductase components (L-protein and X-protein) and the catalytic components (NB-protein and YZ-protein), respectively. P572 Metallocenters of these components have been largely uncharacter- Affinity of structurally modified metalothionein ized. Here, we report comparative biochemical analysis of the catalytic components, NB-protein and YZ-protein from Rhodobacter to heavy metals capsulatus. Dithionite-reduced YZ-protein showed an EPR signal Shin-ichi Takeuchi1, Miwa Sugiura1,2, Hidenori Hayashi1,2 indicative of a [4Fe-4S] cluster. Though the Fe–S cluster of NB- 1Graduate School of Science and Technology, Ehime University, protein was EPR-silent, we found that NB-protein has a unique [4Fe- Matsuyama, Japan, 4S] cluster (NB-cluster) coordinated by three Cys residues and one 2Cell-Free Science and Technology Research Center, Ehime Asp residue from the crystallographic structure. To address the University, Matsuyama 790-8577, Japan. functional significance of these residues, we constructed and analyzed [email protected] a series of NB-protein mutants in which, the Cys and Asp residues Cyanobacterial metallothionein, for example, SmtA from Synecho- were altered to Ala and Ala/Ser/Cys, respectively. We will also dis- coccus PCC 7942 traps Zn2+ in cells to detoxify metal ions as well as cuss the involvement of these residues in the complex formation of to retain Zn2+ homeostasis. SmtA consists of 56 amino acids and is BchN and BchB. enriched in Cys and His residues that ligate the metal ions. Compared with eukaryotic metallothioneins, rather simple structure of SmtA References makes the genetic modification easy, and is useful to create a metal- 1. Nomata J, Mizoguchi T, Tamiaki H, Fujita Y (2006) J Biol Chem binding protein that would be applicable to the phytoremediation of 281:15021–15028 the metal-contaminated soils by genetically modified higher plants. In 2. Nomata J, Ogawa T, Kitashima M, Inoue K, Fujita Y (2008) this study, we structurally modified the metal-binding site by inserting FEBS Lett 582:1346–1350 additional amino acids in the C-terminal region of the SmtA homo- logue from Synechococcus PCC 7002, and examined the ability of binding metal ions other than Zn2+. To create larger space for binding the heavy metal ions, we genetically inserted one to three amino acid P574 residues between H49 and G50 (site A), and G46 and C47 (site B) as shown in Figure. Non FokI-type zinc finger nuclease Insertion of two residues into ‘‘site A’’ increased binding affinity to Shigeru Negi1, Saeko Masuyama1, Koji Kano2, Yukio Sugiura1 Cd2+ and decreased the affinity to Zn2+. In contrast, any insertions 1Faculty of Pharmaceutical Sceince, Doshisha Women’s University, into ‘‘site B’’ were ineffective against affinities to both Cd2+ and Zn2+. Kyotanabe, Kyoto 610-0395, Japan, We will present about relationship between modified structure and 2Department of Molecular Chemistry and Biochemistry, affinity of SmtA homologue to the heavy metal ions. Doshisha University, Kyotanabe, Kyoto 610-0321, Japan. [email protected] The design of functional protein is one of the most attractive issues in the post-genomic era. In particular, the artificial nucleases that pro- B A mote efficient cleavage of DNA are valuable as gene therapeutic PCC 7942 MTSTTLVKCACEPCLCIVDPSKAIDRNGLYYCSEACADGHTGGSKGCGHGGCNCHG PCC 7002 MVTVTQMKCACESCLCIVDLNSAIQKEGKSYCSQACADGHPAGSEGCGHGGCTCHQ regents. A zinc finger protein provides the ideal framework for cre- ating the artificial nuclease. We previously reported the construction Fig. 1 Amino acid sequences of SmtA of Synechococcus PCC 7942 of artificial zinc finger nuclease in a metal dependent manner by and PCC 7002. A and B are positions of insertion of additional amino mutating the Cys2His2-type to the His4-type (Sp1(F2H4)) in the acids second finger domain of the transcription factor Sp1 zinc finger protein (Sp1(zf(123)) without adding the DNA cleavage domain [1]. However, the low DNA cleavage activity of the His4-type zinc finger P573 nuclease is still an important problem to be solved. In order to Characterization of metallocenters of two nitrogenase- improve the low catalytic activity, we created the three His4-type zinc like enzymes, dark-operative protochlorophyllide finger domains (Sp1(F1H4), Sp1(F2H4) and Sp1(F3H4)) for each finger domain of Sp1(zf123), and tested their cleavage activities for oxidoreductase and chlorophyllide oxidoreductase, phosphate ester and plasmid DNA. Furthermore, we designed a new in bacteriochlorophyll biosynthesis type of zinc finger nuclease (ZWH4) possessing four zinc finger Jiro Nomata1, Kozue Ebata1, Norifumi Muraki2, Masaharu domains (Fig. 1). ZWH4 was created by combining the Sp1(zf123) Kitashima3, Kazuhito Inoue3,4, Genji Kurisu2, Yuichi Fujita1,5 and His4 zinc finger domains. This chimera protein is expected to 1Graduate School of Bioagricultural Science, Nagoya University, function as a novel artificial nuclease with high sequence specificity Nagoya 464-8601, Japan, for DNA duplexes. 2Department of Life Science, University of Tokyo, Tokyo 153-8902, Japan, 3Department of Biology, Kanagawa University, Kanagawa 259-1293, Japan, 4Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan, 5Presto, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan. [email protected] In the late steps of bacteriochlorophyll biosynthesis, porphyrin ring is converted to bacteriochlorin ring by the sequential reductions of two Fig. 1 Schematic representation of ZWH4 123 S210 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224

Reference unusual P450 intermediate. We also propose that VioE traps X within 1. Nomurai A, Sugiura Y (2004) J Am Chem Soc 126:15374–15375 the surface cavity to prevent from spontaneous conversion into 1 and promote construction of 3 by proximity and orientation effect. These finding should help to construct combinatorial biosynthesis system, which can produce novel bisindole compounds. P575 Chemical function of tyrosinase from Aspergillus oryzae Nobutaka Fujieda, Michiaki Murata, Shinobu Itoh Pioneering Research Unit for Next Generation, Kyoto University, Uji 611-0011, Japan, Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan. [email protected] Tyrosinase is a copper containing metalloprotein monooxygenase, known as type 3 copper proteins and a bifunctional enzyme, cata- lyzing the o-hydroxylation of phenols and subsequent oxidation of catechols to quinones. Tyrosinase is able to oxidize various phenolic compounds and also peptide and protein bound tyrosine, and thus is of great interest for different biotechnological applications. Knowledge of fungal tyrosinases is still limited, and the work has been hampered by relatively low production yields of the enzymes. Tyrosinase from References Aspergillus oryzae was over-expressed from E.coli (BL21(DE3)) by 1. Makino M, Sugimoto H, Shiro Y, Asamizu S, Onaka H, Nagano using tyrosinase-encoring gene (melB) [1]. In this study, production, S (2007) Proc Natl Acad Sci USA 104:11591–11596 purification, chemically characterization of recombinant tyrosinase 2. Hirano S, Asamizu S, Onaka H, Shiro Y, Nagano S (2008) J Biol and mutational study are reported. Chme 283:6459–6466 Recombinant wild type tyrosinase has been successfully expressed 3. Wang Y, Hirao H, Chen H, Onaka H, Nagano S, Shaik S (2008) J and purified by the GST-tag affinity chromatography. It has been Am Chem Soc 130:7170–7171 found that as-isolated tyrosinase was inactive but can be activated by the pre-treatment with trypsin or an acid (pH 3.0). Detailed steady- state kinetic analysis on the phenolase activity has been performed by monitoring the O2-consumption rate using a Clark-type oxygen P577 electrode in a borate buffer solution. The results have clearly showed Investigation of metal ions effect on the flavonoid– that the reaction involves an electrophilic aromatic substitution mechanism as in the case of mushroom tyrosinase. In addition, human serum albumin interaction by capillary C104A mutant has been successfully expressed by using pGEX-KG- electrophoresis frontal analysis C104A in order to examine the function of Cys–His thioether bond in Tatjana Knjazeva, Mihkel Kaljurand the enzyme active site. Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia. Reference [email protected] 1. Obata H, Ishida H, Hata Y, Kawato A, Abe Y, Akao T, Akita O, The flavonoids constitute a large class of naturally occurring poly- Ichishima E (2004) J Biosci Bioeng 97:400–405 phenols with many beneficial health effects and wide-ranging biological activities. Human serum albumin is a primary carrier of flavonoids in blood and binding can occur both non-covalently and covalently depending on conditions. Some trace metal ions exist in P576 the plasma and can affect the binding properties of serum albumin with pharmaceutical or biologically active molecules. For this reason, Structure and mechanism of enzymes involved investigation of flavonoids and serum albumin interaction in the in indolocarbazole biosynthesis presence of various metal ions is important for better understanding Shingo Nagano1, Hiroyasu Onaka2, Sason Shaik3, mechanism of their actions. Shiro Yoshitsugu3 The interaction of structurally different flavonoids with human serum 1RIKEN SPring-8 Center, Sayo, Hyogo 679-5148 Japan, albumin was investigated under near-physiological conditions— 2Toyama Prefectural University, Toyama 939-0398, Japan, 67 mM phosphate buffer (pH 7.4) at temperature 37°C. Using a fixed 3Hebrew University of Jerusalem, Givat Ram Campus, 91904 HSA concentration and varying concentrations of flavonoid, the Jerusalem, Israel. [email protected] binding constants and the number of binding sites were determined by Staurosporine and violacein are members of indolocarbazole com- CE-FA method. At the same experimental conditions, the effect of pounds with various biological activities, including antitumor various metal ions on the binding parameters was studied. The results activity. Both biosynthesis pathways share a common short-lived showed a decreasing of the binding constants, supposedly, due to the intermediate (X), which spontaneously converted into chromopyrrolic competition between the flavonoids and metal ions when both of them acid (1). Cytochrome P450 StaP constructs skeleton of staurosporine existed in the serum albumin solution. (2) from 1 and VioE is proposed to trap X to convert into protode- Investigation of binding processes and influence of different factors is oxyviolaceinic acid (3). Based on the crystal structures of StaP and important for storage of bioactive compounds in the plasma and VioE, and together with mutagenesis, substrate docking simulation, evaluation of medicine effects. The CE-FA proved to be a simple, and theoretical calculation, we propose that StaP makes the core rapid, specific and repeatable method, which could be implemented in structure via indole cation radical as a catalytic intermediate, which is pharmacokinetic studies and new drug development process.

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P578 1Fukui Institute for Fundamental Chemistry, Kyoto University, Relationship between oxygen affinity of myoglobin Kyoto 606-8103, Japan, 2Institute of Applied Radiation Chemistry, Technical University and the equilibrium constant of acid–alkaline transition of Lodz, Zeromskiego 116, 90-924 Lodz, Poland. in metmyoglobin [email protected] T. Shibata1, K. Mizuseki1, S. Nagao1, H. Tai1, S. Nagatomo1, Considerable protein effects on the homolytic Co–C bond cleavage to A. Suzuki2, K. Imai3, Y. Yamamoto1 form 50-deoxyadenosyl (Ado) radical and cob(II)alamin and on the 1Department of Chemistry, University of Tsukuba, subsequent H-transfer from the methylmalonyl-CoA substrate to the Tsukuba 305-8571, Japan, Ado radical in MMCM have been extensively studied by DFT and 2Department of Materials Engineering, Nagaoka College of ONIOM(DFT:MM) methods. Several models have been used to Technology, Nagaoka 940-8532, Japan 3Department of Frontier systematically study the protein effect. The calculations have shown Bioscience, Hosei University, Koganei 184-8584, Japan. that the Co–C bond dissociation energy is very much reduced in the [email protected] protein, compared to that in the gas phase. The large protein effect Functional regulation of oxygen binding hemoprotein myoglobin can be decomposed into the cage effect, the effect of coenzyme (Mb) is thought to be achieved through the heme environment fur- geometrical distortion, and the protein MM effect. The initial Co–C nished by nearby amino acid residues, and subtle tuning of the bond cleavage and the subsequent hydrogen transfer were found to intrinsic heme Fe reactivity. Although the heme environmental effects occur in a stepwise manner in the protein, although the concerted on the O2 affinity of the protein have been elucidated in some detail, pathway for the Co–C bond cleavage coupled with the hydrogen the relationship between the heme electronic structure and Fe reac- transfer is more favored in the gas phase. The entire catalytic cycle in tivity has remained to be clarified. In this study, we have performed MMCM will also be discussed. the substitution of strongly electron-withdrawing perfluoromethyl (CF3) group(s) as heme side chains(s) (Fig. 1), which affords great variation in the electronic structure of the porphyrin moiety of the heme, and hence allows detailed characterization of the relationship between the O2 affinity of Mb and the heme electronic structure. Additionally, in order to quantitatively assess the effects of the CF3 substitution(s) on the electronic properties of the heme Fe atom in the protein, we used the equilibrium constant (pKa) of the acid–alkaline transition in metmyoglobin (metMb).

Fig. 1 The structures and R3 H R7 numbering system for mesoheme (R2 = R7 = SˇCH3, 3 5 7 R Reference R2 2 8 8 R3 = R8 = SˇCH2CH3), 7-PF 1. Li X, Chung LW, Paneth P, Morokuma K (2009) J Am Chem Soc N N (R2 = SˇCH3, (in press) 3+ R3 = R8 = SˇCH2CH3, H 20 Fe 10 H R7 = SˇCF3), and 2,8-DPF ˇ N N (R2 = R8 = SCF3, 18 12 R3 = R7 = SˇCH3) CH3 17 15 13 CH3 P580

CH2 H CH2 A New l-oxo trinuclear iron(III) chain built up from the basic l-oxo trinuclear iron(III) pivalate complex: CH2 CH2 possible building blocks in ferritin core formation COC - COC - 2 2 Mara Johann, Eva Rentschler Johannes-Gutenberg University Mainz, Institute of Inorganic Chemistry, Duesbergweg 10-14, 55128 Mainz, Germany. [email protected]

Using the pKa value as a quantitative measure of the effect of the Ferritin (Fr) contains up to 4,500 high-spin iron(III) centers. It plays heme modification on the electron density of the heme Fe atom, we an important role in storage and recycling of iron in most life forms. have confirmed that the introduction of electron-withdrawing group(s) Many high nuclear metal compounds that model the storage of iron in t to the heme as peripheral side chain(s) leads to a decrease in the Fr, like [Fe6O2(OH)2(O2CBu )12] [1] have been synthesized. Various electron density of the heme Fe atom, which in turn decreases the O2 approaches to build up iron clusters with high nuclearity can be found affinity of Mb. in literature [2-5]. One procedure is the oligomerization of trinuclear l-oxo bridged iron(III) units under hydrolytic conditions that proba- bly also proceed during the core formation of Fr. The here presented t l-oxo iron(III) chain, {Fe3O(O2CBu )6(OPhCHO)(HOPhCHO)}n, P579 was prepared by reaction of the well-known trinuclear iron(III) core, t + DFT and ONIOM(DFT:MM) studies on enzymatic [Fe3O(O2CBu )6(H2O)3] with an excess of 4-hydroxybenzaldehyde + mechanism in B12-dependent methylmalonyl-CoA (HOPhCHO). The {Fe3O7} cores of the chain are connected through a deprotonated OPhCHO, the third open coordination position of the mutase core is occupied by a protonated HOPhCHO. The compound is 1 1 1 2 Xin Li , Lung Wa Chung , Keiji Morokuma , Piotr Paneth characterized by X-ray, i.r. and moessbauer spectroscopy. Magnetic

123 S212 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 susceptibility data show antiferromagnetic coupling within the 1Department of Biochemistry, Molecular Biology, Nippon Medical + {Fe3O7} core and a weak interaction within the chains given by the School, Tokyo 113-8602, Japan, 2 hWeiß temperature of -0.6 K. The presented 1D chain compound is an Department of Chemistry, Juntendo University, Chiba 270-1695, + interesting new example for the oligomerization of {Fe3O7} units Japan, where the trinuclear core is retained. 3Institute of Environmental Microbiology, Kyowa Kako Co., Tokyo 194-0035, Japan, 4 References Department of Veterinary Clinical Medicine, University of Illinois, 1. Micklitz W, Lippard SJ (1988) Inorg Chem 27:3067 Urbana, IL 61801, USA, 5 2. Christou G et al (2000) J Chem Soc Dalton Trans 4446 Department of Chemistry, University of Georgia, Athens, GA 3. Lippard SJ et al (1997) J Am Chem Soc 119:1037 30602-2556, USA, 6 4. Gorun SM, Lippard SJ et al (1987) J Am Chem Soc 109:3337 SPring-8/JASRI/RIKEN, Hyogo 679-5198, Japan. 5. Heath SL, Powell AK (1992) Angew Chem Int Ed 31:191 [email protected] MitoNEET (a mammalian mitochondrial outer membrane protein) is a potential pharmacological/clinical target of the insulin-sensitizer pioglitazone for the treatment of type II diabetes. In Thermus ther- P581 mophilus HB8, two hypothetical genes coding for mitoNEET homologs were found. Constitutive transcription of one of the two Spectroscopic characterization of evolutionary old genes was confirmed by expression profile analysis by microarray. ferredoxins This gene was heterologously overexpressed in Escherichia coli, and Hanne Nørgaard1, Signe Smedegaard Helt1, Bee Lean Ooi1, the recombinant protein, Tth-NEET0026, was characterized as a 2 1 Wilfred R. Hagen , Hans E. M. Christensen soluble protein, containing two mitoNEET-like [2Fe–2S](Cys)3(His)1 1Department of Chemistry, Bldg. 207, Technical University of clusters per dimer. The crystal structure of Tth-NEET0026 at 1.80-A˚ Denmark, 2800 Kgs. Lyngby, Denmark, resolution reveals a unique topology compared with the human 2Department of Biotechnology, Delft University of Technology, mitoNEET domain structure, although both proteins accommodate a 2628 BC Delft, The Netherlands. [email protected] [2Fe-2S] cluster in a structurally conserved CDGSH-type zinc-finger- Life on Earth may have evolved near hydrothermal vents on the like domain. Our first structure of the prokaryotic mitoNEET homo- ocean floor more than 3.6 billion years ago. On the surface of log suggests a putative redox function as a result of evolutionary these formations, early catalytic activity is believed to have taken redesign of the CDGSH-type metal-binding module. place while iron-sulfur proteins—the oldest known proteins—may have been functional on these surfaces. By phylogenetic analysis of Reference 82 ferredoxins (8Fe and 7Fe) from archaea and bacteria, a phy- 1. Kounosu A et al (2008) Acta Cryst Sect F 64:1146–1148 logenetic tree was constructed [1]. The amino acid sequence of the Last Common Ancestor (LCA) contains 55 amino acids and within the sequence, there is an internal repeat of 23 amino acids. LCA contains eight cysteines (four in each 23 amino acid repeat) and is P583 likely to coordinate two [4Fe–4S] clusters. The internal repeat is A molecular mechanism for entropic control of redox believed to be a result of a duplication of an even older peptide— the Origin of Iron–Sulfur Proteins (Ori-ISP). Ori-ISP consists of 23 potential of cytochrome c amino acids and within them four cysteines. We have expressed Shin-ichi Mikami, Kiyofumi Irie, Hulin Tai, Shigenori Nagatomo, and purified both proteins and investigated them using EPR spec- Yasuhiko Yamamoto troscopy. EPR titration of LCA shows that the protein contains two Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, clusters. LCA was found to coordinate either two [4Fe–4S] clusters Japan. [email protected] or one [4Fe-4S] cluster and one [3Fe–4S] cluster. Ori-ISP was also Redox potential (Em) of cytochrome c (cyt c) is determined through investigated by EPR and the results imply that Ori-ISP forms both enthalpy (DH) and entropy (DS) contributions. Although struc- dimers with magnetically interacting [4Fe–4S] clusters. tural factors such as Fe–Met coordination bond and hydrophobic environment around heme have been shown to regulate DH, molec- Reference ular mechanism that controls DS has been largely remained to be 1. Davis BK (2002) Prog Biophys Mol Biol 79:77–113 explored. In this study, a molecular mechanism responsible for DS control of Em of cyt c has been revealed. The DH and DS values for the reduction of Pseudomonas aeruginosa cytochrome c551 (PA) and its selected mutants were obtained from the P582 analysis of temperature dependence of the Em value at pH 6.0. Four Thermophile ‘‘NEET’’: structure toward functional mutants, F34Y, E43Y, R47L, and W56F (Fig. 1), in which, electro- understanding static interaction between heme 17-propionate side chain and nearby amino acid residues was affected, exhibited the DS values largely Toshio Iwasaki1, Asako Kounosu1, Daijiro Ohmori2, different from that of PA (Table 1). 1H NMR study of these mutants Yoko Hayashi-Iwasaki3, Tairo Oshima3, Sergei A. Dikanov4, revealed that the DS value was controlled through the redox-depen- Darin M. Cowart5, Uma Mahendra Kumar Koppolu5, dent change of the ionization state of heme 17-propionic acid side Robert A. Scott5, Takashi Kumasaka6 chain. This finding provides not only novel insights into the molecular

123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S213

mechanisms of the Em regulation of cyt c, but also new strategy for tuning the redox function of the protein through protein engineering.

80 nA P584

Fig. 1 Tertiary structure of PA. Residues substituted in the study are indicated by a space 0.3 0.2 0.1 0 -0.1 -0.2 -0.3 filling model E / V vs. Ag / AgCl

Fig. 1 Cyclic voltammogram using the E31R/R99A-Ami-immobi- lized electrode

P585 Functional conversion of nickel-containing urease by the addition of copper ion Yasuhito Mori1, Shinnichiro Suzuki1, Kazuya Yamagichi1 1Department of Chemistry, Graduate School of Science, Osaka Table 1 Em, DH, and DS values of PA and its mutants University, Osaka 560-0043, Japan. [email protected]

-1 Urease catalyzes the hydrolysis of urea to form ammonia and car- Protein E (mV) (25 ) ∆ (kJmol ) ∆ (JK-1mol-1) m H S bamate, possesses a dinuclear nickel active site with the protein F34Y 260 5 -38 2 -42 3 providing a bridging carbamylated lysine residue as well as an E43Y 285 5 -55 2 -92 3 aspartyl and four histidyl ligands. Meanwhile some oxidases having dinuclear copper ions at the active site have been known, for example, PA306 5 -48 2 -69 3 catechol oxidase and tyrosinase. In this study, we have found that the R47L 265 5 -39 2 -45 3 addition of copper ions to urease decreases hydrolase activity, and the W56F 280 5 -38 2 -36 3 oxidase activity of the protein has appeared. We have investigated the veffect of the amount of added copper ion on the enzyme activ- ities and the metal contents. The relationship between the oxidase activity and the copper contents of the protein can be analyzed by ‘‘the distinct model’’, not the identical or the consecutive models. Kd1 and Kd2 were calculated to be 3.4 ± 0.6 and 518 ± 60 mM, Electron transfer reactions of wild-type and mutant respectively. forms of amicyanin immobilized on an electrode Kd1 Ni1M2 urease þ Cucu1M2 urease þ Ni via an arginine residue K ðM ¼ Ni or CuÞ 2 d2 Nobuhumi Nakamura, Ayako Kaneko, Hiroyuki Ohno M1Ni urease þ CuM1Cu2 urease þ Ni

Department of Biotechnology and Life Science, Tokyo University ½Cuf ½Cuf of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. r ¼ þ Kd1 þ½Cuf Kd2 þ½Cuf [email protected] The orientation of the redox center toward electrodes seriously affects the electron transfer process, in particular, the electron transfer rate. As a keto aldehyde group of phenylglyoxal selectively reacts with a r copper contents at the active site, guanidino group of an arginine residue, in this study, amicyanin [Cu]f concentration of free copper ions, (Ami) from Paracoccus denitrificans was immobilized on the elec- Kd1, Kd2 dissociation constants for copper ions. trode via an arginine residue by using p-carboxyethylphenylglyoxal We concluded that the nickel ions at the active site of urease were (CEPG). The redox response for a wild type form of amicyanin replaced with copper ions by the addition of copper ion to urease (WT-Ami) immobilized on the 6-mercapto-1-hexanol (MH)/CEPG solution and the function of the enzyme was converted from hydro- modified electrode was observed. Because the redox response of lase to oxidase activities by the replacement of the metal ions form R99A-Ami was not observed, we could say the electron transfer nickel to copper ions. reaction for the WT-Ami occurred through the R99 residue. To achieve the fast electron transfer reaction between Ami and the MH/ CEPG electrode, four Ami mutants (E31R/R99A, M51R/R99A, H56R/R99A, and T93R/R99A) were constructed. The redox respon- ses of E31R/R99A and M51R/R99A were observed. The value of the separation (DE) between anodic and cathodic peaks for the E31R/ R99A-Ami- immobilized electrode was smaller than that for the WT-Ami- immobilized electrode (Fig. 1). The fast electron transfer could be achieved when the 31st residue was used for the immobi- lization of Ami on the electrode.

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P586 form (2), most stable spin state was a broken-symmetry state (BS2), The NMR structure of a novel protein containing a new which was found out to be more stable than a non-collinear state (Tilt). A model Hamiltonian investigation showed that these BS2 and heterometallic molybdenum–copper cluster Tilt states are quite close in energy for their conflicting two inter- 1 1 1 1 Sofia R. Pauleta , M. S. Carepo , A. G. Duarte , A. S. Pereira , actions, the exchange and double exchange interactions. Inherent 2 2 1 1 P. Turano , I. Bertini , J. J. G. Moura , I. Moura electronic and spin structures in [3Fe–4S] clusters are discussed in 1 REQUIMTE, CQFB, Dep. Quı´mica, FCT-UNL, 2829-516 Caparica, this study. Portugal, 2CERM, Via Sacconi 6, 50019 Sesto Fiorentino (FI), Italy. [email protected] The ORange Protein (ORP) from Desulfovibrio gigas is an orange coloured 11.8 kDa protein that contains a mixed-metal sulphide 3- cluster, of the type [S2MoS2CuS2MoS2] , non-covalently bound to the polypeptide chain [1, 2]. A blast search revealed that this protein has sequence homology of around 30–50% with conserved proteins from eubacteria and hyperthermophilic archaea with unknown func- tion. They all contain a conserved domain common to the nitrogenase accessory factor (NifB C-terminal domain, NifX and NafY). The ORP was produced for NMR studies by heterologous expression in E. coli as the apo-form [3]. The holo form was reconstituted by the in situ synthesis of the metal cluster upon the addition of copper sulphate and thiomolybdate or thiotungstate. The polypeptide chain of ORP is composed of 117 residues of which 13 Fig. 1 Calculated non-collinear spin state of an oxidized [3Fe–4S] 5 are prolines. C detection experiments enabled the extension and 3- cluster ([3F(III)-4S](SH)4 ). Arrows indicate the atomic spin confirmation of the sequential assignment for both apo and recon- densities stituted-forms of ORP3. The over-all solution structures of the apo and reconstituted ORP are similar, with an a/b motif, characteristic of the members of the ribonuclease H family. The two structures are Reference similar and the mapping of the chemical shift differences between 1. Shoji M, Takeda R, Kitagawa Y, Yamanaka S, Kawakami T, them was used to elucidate which region of the polypeptide chain is Okumura M, Yamaguchi K (2009) Int J Quantum Chem involved in the binding of the metal cluster. These results give insights into the metal binding mode of chaperons involved in the synthesis of the nitrogenase metal cofactor. P589 References Hydroxylation of aromatic rings by myoglobin mutants 1. George GN et al (2000) JACS, 122:8321–8323 (2000) Chikako Shirataki1, Christian Wiese2, Osami Shoji1, 2. Bursakov SA et al (2004) JIB 98:833–837 Yoshihito Watanabe3 3. Pauleta SR et al (2007) Biomol NMR Assignm 1:81–83 1Department of Chemistry, Graduate School of Science, This work was supported by the project POCI/QUI/55350/2004 Nagoya University, Nagoya 464-8602, Japan, 2 (Fundac¸a˜o para a Cieˆncia e Tecnologia) and by the EU-NMR Department of Pharmacology and Medical Chemistry, University of Research Infrastructure (Contract no. 026145). Mu¨nster, Munich, Germany, 3Research Center of Material Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. [email protected] Myoglobin (Mb) is a small heme protein responsible for the oxygen P588 storage. Even though Mb does not show any enzymatic activities, it Theory of chemical bonds in metalloenzymes: a GSO- can be converted into a heme enzyme by substitution of amino acid DFT study of electronic and spin structures in [3Fe–4S] residues around the heme. In fact, we have proved that the aromatic clusters ring of Trp-43 in F43W/H64D/V68I mutant is hydroxylated upon the 1 2 2 addition of 1 eq. hydrogen peroxide [1]. Mitsuo Shoji , Yasutaka Kitagawa , Takashi Kawakami , In this study, we found that the hydroxylation of 1-methoxynaph- Shusuke Yamanaka3, Mitsutaka Okumura2, Kizashi Yamaguchi3 1 thalene is catalyzed by a series of H64D mutants. Furthermore, Graduate School of Engineering, Nagoya University, progress of the hydroxylation can be simply monitored by observing a Nagoya 466-8603, Japan, 0 0 2 color change due to the formation of 4, 4 -dimethoxy-[2,2 ]-binaph- Department of Chemistry, Graduate School of Science, thalenylidene-1,10-dione, so-called ‘‘russig’s blue’’. We here report Osaka University, Toyonaka Osaka 560-0043, Japan, 3 the hydroxylation reaction of the aromatic ring by the Mb mutants and Center of Quantum Science and Technology Under Extreme offer a useful colorimetric assay for detecting hydroxylation activity. Conditions (KYOKUGEN), Osaka University, Osaka 560-8531, Japan. [email protected] The electronic and spin structures of [3Fe-4S]+/0 clusters were investigated by using generalized spin orbital approach based on the density functional theory (GSO-DFT). In the oxidized form + ([Fe(III)3S4] ), a non-collinear spin (NC) state was found out to be lower in energy than a conventional broken-symmetry state, i.e. antiferromagnetically coupled (AF) state. These electronic structures were characterized by using the natural orbital analysis. In reduced

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Reference environment, and symmetry of the complex. In a dielectric environ- 1. Pfister TD, Ohki T, Ueno T, Hara I, Adachi S, Makino Y, Uey- ment corresponding to proteins (e = 20), the shift of redox potential ama N, Lu L, Watanabe Y (2005) J Biol Chem 280:12858–12866 is calculated to be about 64 mV for Fe–SH–N H-bonds, correlating well with data estimated from experiments on iron-sulfur proteins [2]. In aqueous solution (e = 80), a shift of 58 mV was computed for Fe– SH–O H-bonds in agreement with measured values in iron sulfur P590 proteins with partially exposed H-bonds. This demonstrates the Sulfido–, and Selenido–bis(dithiolene)Mo complexes dependence of the iron–sulfur complex redox potentials on the environment (solvent or protein). related to 6 group metal enzyme reaction centers Hiroyuki Tano1, Hideki Sugimoto1, Hiroyuki Miyake1, Shinobu Itoh2 1Department of Chemistry, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan, 2Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan. [email protected] Mo=S and Mo=Se complexes with one or two dithiolene ligands are of great importance due to their relevance to reaction centers of molybdenum hydroxylases and dehydrogenases. However, mononu- clear Mo=S/Mo=Se complexes are rare. Here, we report bis(dithiolene)MoS/MoSe complexes containing identical dithiolene ligands. Treatment of Mo(CO)2(L)2 (L = cyclohexene-1,2-dithiolate) with excess amount of Na2SorNa2Se and 2 equiv. of Ph4PBr in CH3CN afforded a blue suspension. Repeated recrystallizations References from CH3CN/diethyl ether gave green–brown crystals of 1. Galstyan AS, Knapp EW (2009) J Comput Chem 30:203–211 IV IV (Ph4P)2[Mo S(L)2](1) and brown crystals of (Ph4P)2[Mo Se(L)2] 2. Hunsicker-Wang LM et al (2003) Biochemistry 42:7303–7317 (2). Each molybdenum center (Mo1) is coordinated with a respective terminal chalcogenido and four sulfur atoms from two dithiolene ligands. Both Mo1 atoms are raised above the basal plane comprising four S atoms by 0.78 A˚ and adopt a distorted square–pyramidal P592 structure with the dihedral angle between S1Mo1S2 and S3Mo1S4 Highly efficient isomerization of endoperoxides planes as 129°. The two complexes exhibit one reversible Mo(V)/ catalyzed by synthetic heme thiolate as a model Mo(IV) redox couple at the same redox potential (E1/2 = –0.74 for 1 of cytochrome P450 and –0.74 V vs. SCE for 2)inCH3CN. This suggests that the HOMOs Hiroshi Suzuki, Takehiro Yamane, Naoki Umezawa, are hybridized with Mo dxy and dithiolene orbitals. By a treatment Nobuki Kato, Tsunehiko Higuchi with an equivalent ferrocenium hexafluoro-phosphate (FcPF6), the Graduate School of Pharmacy, Nagoya City University, CH3CN solution of 1 changed to gray colour. The gray species was V Nagoya 467-8603, Japan. [email protected] characterized to be (Ph4P)[Mo S(L)2] by UV–vis spectroscopy, ESI– V Prostaglandin H2 (PGH2), a natural endoperoxide, is catalytically MS, and EPR. Similarly, (Ph4P)[Mo Se(L)2] was generated and characterized. isomerized to prostacyclin or thromboxane A2 by cytochrome P450s. Hydroperoxides are widely used as oxidants by heme-containing peroxidases and P450. However, no metalloenzyme other than the P450-type one is known to catalyze reactions involving endoperox- ides, which do not have active hydrogen. We investigated the isomerization mechanism of PGH2 using SR complex, which is a model of P450 having heme-thiolate structure [1]. Isomerization of endoperoxide proceeded very rapidly with SR, whereas imidazole or chloride-ligated heme had slight or no catalytic activity [2]. The redox potential (FeIII/FeIV)ofSR was lower than those of the other com- plexes. This ready accessibility of the high-valent iron form of heme P591 thiolate is considered critical for O–O bond cleavage of endoperoxide, Redox potentials of iron sulfur proteins which has no active hydrogen. with Fe–SH–N and Fe–SH–O hydrogen bonds Next, spin-trap experiments using 3,3,5,5-tetramethylpyrroline Ana P. Ga´miz-Herna´ndez, Artur S. Galstyan, E. Walter Knapp -N-oxide were carried out to examine the formation of radical species. Department of Biology, Chemistry and Pharmacy, Institute of The ESR spectrum of the reaction mixture clearly showed a sextet Chemistry and Biochemistry, Freie Universita¨t Berlin, Fabeckstr. 36a, signal assignable to an alkoxyl radical-derived product. 14195 Berlin, Germany. [email protected] CHO The energetics of redox states for several iron sulfur protein cofactors O Fe(por)L (1.0 mol%) CH2 O + + R O CH2 CHO CDCl3, 25 C CHO were computed using a combination of electrostatic and density R N N Endoperoxide part Fe N N R functional theory [1]. Accordingly, H-bonds in iron–sulfur complexes of PGH2 Reactivity: L = R SŠ >> >> ClŠ shift the redox potentials positively independent of each other. The R N N R Turnover L : -1 -2 -1 no reaction magnitude of the shift depends on the type of H-bond, dielectric Frequency 10.5 sec 1.1 x 10 sec Fe(por)L

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References ICP-MS, XPS, DLS, AFM, CV, DPV, CD, FM) and molecular 1. Higuchi T, Uzu S, Hirobe M (1990) J Am Chem Soc 112:7051– dynamics simulation. First, it was found that La(III) could clearly 7053 change the peroxidase activity in horseradish and show the ‘‘hormesis 2. Yamane T, Makino K, Umezawa N, Kato N, Higuchi T (2008) effect’’ with increasing the concentration of La(III). Second, when Angew Chem Int Ed 47:3656–3659 horseradish is treated with the low concentration of La(III), La(III) is distributed on the plasma membrane in horseradish cell, thus La(III) cannot penetrate through the cell membrane. Meanwhile, La(III) located on the plasma membrane probably promotes or accelerates P593 substance exchange in the plant. It was found that when horseradish is The effects of mutation of the axial ligand treated with the low concentration of La(III), the HRP activity is increased about 101.5%. Whereas, the Rz value or the enzymatic on the conformational stability of the heme active site activity index and structure of HRP in horseradish are not obviously of thermostable cytochrome c changed. Therefore, La(III) located on the outer surface of cyto- Rabindra Kumar Behera1, Hiroshi Nakajima2, plasmatic membrane may regulate the substance exchange occurring Yoshihito Watanabe2, Shyamalava Mazumdar1 in both, intra and extra-cellular, and then large amounts of HRP 1Tata Institute of Fundamental Research, Mumbai 400005, India, protein in plant may be synthesized, leading to the increase in the 2Department of Chemistry, Graduate School of Science, Nagoya bioactivity of HRP in horseradish. Third, when horseradish is treated University, Nagoya 464-8602, Japan. [email protected] with the high concentration of La(III), a large amount of La(III) is distributed on cell wall, some of La(III) has been entered into the Cytochrome C552 is a highly thermostable c-type heme protein from T. thermophilus involved in the terminal step of the bacterial respi- protoplast of horseradish, leading to the distortion of the protoplast in horseradish. Meanwhile, the bioactivity of HRP in horseradish are ratory electron transfer process. The active site of cytochrome C552 consists of a low spin iron(III) in a saddle shaped heme group axially clearly inhibited by La(III). A new peroxidase complex containing coordinated to His15 and Met69. La(III) (La-HRP) was obtained from horseradish treated with the high Mutagenic replacements of the axial Met69 with His and with Ala concentration of La(III), whereas, the Rz value of La-HRP is about have been carried out to investigate the role of the axial ligation on 2.4. Fourth, the catalytic activity of the La-HRP complex is much the conformational properties and thermostability of the active site of lower than that of HRP obtained from the horseradish without the the protein. Detailed circular dichroism and other spectroscopic treatment of La(III). The decrease in the activity of the La-HRP studies have been carried out to determine the thermal and chemical complex is due to the conformation destruction comparing with native induced unfolding properties of the wild type and the mutant proteins. HRP in horseradish, in which, La(III) can interact with O and/or N The analyses of the results suggested that the tertiary structure around atoms in the polypeptide backbone of the peroxidase in the cell and the heme of the M69H mutant had very similar unfolding free energy formed the La-HRP complex, thereby changing the structure of native (DGAq) but had slightly higher thermostability compared to the wild HRP in horseradish, in turn, affecting the geometry and electron type protein, albeit the stability of the secondary structure was not density of the active site, and leading to the inhibition of the catalytic affected by this mutation. Analogous studies on the M69A mutant, activity of the peroxidase in horseradish. however, showed that the replacement of the methionine by alanine at this site leads to higher thermodynamic (DGAq) as well as thermal stability (Tm) to the heme center of the protein. A ligand exchange process forming a low-spin heme species was P595 observed during the unfolding of the wild type as well as of the Catalytic mechanism of nitrile hydratase: role of serine mutant proteins. The M69A that consists of a high-spin ferric heme in ligand and the conserved tyrosine residue the folded form showed a clear isosbestic point corresponding to the Yasuaki Yamanaka1, Koichi Hashimoto1, Akashi Ohtaki1, spin transition associated with the ligand exchange at high tempera- Keiichi Noguchi2, Masafumi Yohda1, Masafumi Odaka1 ture and in presence of GdnHCl. 1 Department of Life Science and Technology, Graduate School of Engineering, 2Instrumentation Analysis Center, Tokyo University of Agriculture P594 and Technology, Kogenei, Japan. [email protected] Effects of La(III) on the activity, structure and electron Nitrile hydratase (NHase) catalyzes the hydration of nitriles to the corresponding amides. It consists of a and b subunits with the non- transfer of horseradish peroxidase (HRP) in plant heme iron(III) catalytic center, in which two Cys ligands, aCys112 Lihong Wang1,2, Shaofen Guo2, Xiaolan Ding3, Qing Zhou2, and aCys114, are post-translationally modified to Cys–sulfinic acid 1 Xiaohua Huang (Cys–SO2H) and –sulfenic acid (Cys–SOH), respectively. Here, we 1Jiangsu Key Laboratory of Biofunctional Materials, College of replaced the Ser ligand (aSer113) and the neighbor conserved Tyr Chemistry and Environment Science, Nanjing Normal University, residue (bTyr72) of Fe-type NHase from Rhodococcus sp. N771 Nanjing 210097, China, (ReNHase) by Ala and Phe, respectively, and studied their functions. 2 The Key Laboratory of Industrial Biotechnology, Ministry of In aS113A, the Km value is comparable to that of wild-type NHase Education, Jiangnan University, Wuxi 214122, China, while the kcat value decreased to approximately 6%. In contrast, the 3State Key Laboratory of Biomembrane and Membrane bY72F mutant exhibited no NHase activity under the experimental Biotechnology, Department of Biological Science and Biotechnology, condition. The structure of the aS113A mutant was similar to that of Tsinghua University, Beijing 100084, China. the wild-type NHase, but significant changes were observed in the [email protected] hydrogen bond networks around the metallocenter. We also crystal- Effects of La(III) on the activity, structure and electron transfer of lized the aS113A mutant in the nitrosylated inactive state with tert- horseradish peroxidase (HRP) in plant were investigated for the first butylacetonitrile (tBuCN) and activated the mutant NHase in crystals time using the optimum combination of transmission electron by light. Before light illumination, tBuCN was observed at the cata- microscopic autoradiography (EMARG) technology, the methods of lytic cavity but it was transformed to trimetylacetamide after 60-min biophysical chemistry (such as SDS-PAGE, IEF, MALDE-TOF/MS, reaction, confirming that the aS113A mutant has the nitrile hydration

123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S217 activity. Crystal structure of the bY72F mutant was similar to that of alaskensis will be presented. Direct electrochemistry (cyclic vol- the wild-type NHase too. Moreover, Cys114-SOH was oxygenated to tammetry and differential pulse voltammetry) will be used in order to Cys–SO2H. The loss of NHase activity is attributed to the replace- detect all the redox transitions attainable by the core and EPR spec- ment of bTyr72 to Phe or to the oxidation of aCys114–SOH to Cys– troscopy will be used in complementary way. A comparative study SO2H. Based on the obtained results, the functions of aSer113 and will be performed with the previously characterized ferredoxins from bTyr72 will be discussed. Desulfovibrio gigas. Physiological role and relation to metal growth conditions will be discussed. Acknowledgements: Fundac¸a˜o para a Cieˆncia e a Tecnologia for financial support (BPD/14938/2004). P596 Role of surfactants as promoters of direct Reference electrochemistry of the dinuclear CuA site 1. Moura JJG et al (1994) Ferredoxins. In: PecK JHD, Le Gall J of cytochrome c oxidase from Thermus thermophilus (eds) Methods in enzymology, vol 234 Jitumani Rajbongshi1,2, Diganta Kumar Das2, Shyamalava Mazumdar1 1Tata Institute of Fundamental Research, Mumbai 400005, India, 2Department of Chemistry, Gauhati University, Guwahati 14, India. P598 [email protected] NMR analysis of the histidine–iron interaction Cytochrome c oxidase is the terminal enzyme in the electron transport chain of aerobic respiration. The CuA centre in the subunit II of and heme conformation in cytochromes c cytochrome c oxidase is a unique dinuclear copper center containing Kara L. Bren, Sarah E. J. Bowman, Matthew D. Liptak bis-thiolato bridge in a valance delocalized [Cu+1.5–Cu+1.5] state of Department of Chemistry, University of Rochester, Rochester, NY the metal center. This metal center acts as the electron entry site of the 14627-0216, USA. [email protected] enzyme and accepts electrons from cytochrome c. Direct electro- Cytochromes c (cyts c) are characterized by covalent attachment of chemistry of the soluble CuA domain obtained by genetically heme to the polypeptide, typically to a Cys–X–X–Cys–His motif. In truncating the membrane bound part of the enzyme from Thermus comparison with cyts b, which bind heme noncovalently, low-spin thermophillus has been examined on glassy carbon electrode using cyts c display a greater range of reduction potentials. Notably, cyts c various surfactants as promoters. The bare glassy carbon electrode reaches potentials below -400 mV versus NHE, whereas cyts b with gives very weak electrochemical response for the protein, while potentials below -200 mV versus NHE are not known. To test the surfactants were found to promote the direct electrochemistry, pos- hypothesis that the properties of the heme attachment peptide influ- sibly by decreasing the propensity of irreversible adsorption of the ence reduction potential of cyts c, we are preparing mutants of cyts c protein on the electrode and thereby increasing the active surface area targeting the variable residues in the Cys–X–X–Cys–His motif as well of the electrode. The results showed that addition of cationic sur- as residues that interact with the Cys. We have identified mutants that factant cetyl trimethyl ammonium bromide (CTAB) significantly we propose to display (1) changes in the amount of ruffling of the enhances the electrochemical response of CuA on the glassy carbon heme and (2) changes in hydrogen bonding to the heme axial His. 1 13 electrode. The results have been used to understand the mechanism of Analysis of the hyperfine chemical shifts of H and C nuclei in these electron transfer from cytochrome c to the copper center during the variants of paramagnetic Fe(III)cyts c reveals how hydrogen bonding enzymatic reaction. to the axial His and heme conformation influence heme electronic structure. The goals of this work are: (1) to develop relationships between heme 1H and 13C chemical shifts and heme conformation to facilitate NMR analysis of heme conformation for low-spin ferric P597 hemes, and (2) to learn how heme conformation and the properties of Biochemical and spectroscopic/electrochemical the axial His as modulated by the heme attachment peptide influence characterization of a new ferredoxin isolated reduction potential. from Desulfovibrio alaskensis Raquel Grazina, Patrı´cia P. Sousa, Marta Carepo, Isabel Moura, Jose´ J. G. Moura REQUIMTE, Departamento de Quı´mica, Centro de Quı´mica Fina e Biotecnologia, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. [email protected] Ferredoxins are simple iron–sulphur proteins that contain prosthetic groups composed of iron and sulphur atoms and they play a functional role in electron transfer processes relevant for sulphate-reducing bacteria (SRB) metabolism. Four distinct types of ferredoxins are found in SRB, containing [3Fe–4S], [4Fe–4S], [3Fe–4S] plus [4Fe– 4S] and 2 9 [4Fe–4S] clusters. Previous characterizations of other ferredoxins from other Desulfovibrio species, such as ferredoxin II from Desulfovibrio gigas (DgFdII), demonstrate that the Fe–S clusters of these proteins can achieve several redox processes. The DgFdII presents two distinct electrochemical signals correspondent to the following transitions of the cluster: [3Fe–4S]+1/0 and [3Fe–4S]0/-2. Reference Herein the characterization of this new ferredoxin from Desulfovibrio 1. Bowman SEJ, Bren KL (2008) Nat Prod Rep 25:1118–1130

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P599 mechanisms have not been understood. We noticed the difference in Contribution of the N-terminal domain of Haloarcula the environments around the substrate binding pockets. In the present study, we replaced bArg90 and cArg136 of SCNase, which were japonica ferredoxin to haloadaptation substituted by hydrophobic residues in all known NHases, by Trp and Rie Yatsunami, Hiroki Hada, Naoki Hirota, Takatoshi Matsuo, Phe, respectively (R90W, R136F, R90W, R136F). All mutant SCN- Akiko Ikeda, Toshiaki Fukui, Satoshi Nakamura ases were expressed in E. coli and purified like wild-type enzyme. Department of Bioengineering, Tokyo Institute of Technology, Each mutant SCNase had no SCNase activity. But, when each mutant 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. was incubated with methacrylonitrile, a small fraction of meth- [email protected] acrylamide was produced. All mutant SCNase converted several other Extremely halophilic archaea (haloarchaea) are extremophiles that nitriles to the corresponding amides, indicating that the mutants can grow in hypersaline environments even in the NaCl-saturated possessed NHase activity. These results indicate that the catalytic concentration. Since intracellular salt concentrations are also very mechanisms of SCNase and NHase were basically conserved, and that high in these microorganisms, their entire biochemical systems adapt their substrate-specificities were determined by the environments to function at such high salt concentrations. Indeed, some proteins around the substrate-binding pockets including bArg90 and cArg136 from haloarchaea have been investigated to be functionally active or the corresponding residues of NHases. only under high salt conditions. Halophilic proteins, in general, have a higher molar excess of acidic amino acid residues, fewer basic resi- Reference dues, and a higher number of small hydrophobic residues than the 1. Arakawa T, Kawano Y, Kataoka S, Katayama Y, Kamiya N, nonhalophilic counterparts. Yohda M, Odaka M (2007) J Mol Biol 366:1497–1509 The Ferredoxin (Fd) from Haloarcula japonica possesses a plant-type [2Fe–2S] cluster and is stable at high salt concentrations. Ha. japonica Fd (HjFd) includes an N-terminal additional domain rich acidic amide acid, as well as a common core domain that contains the Fe–S cluster. P601 To investigate the function of the N-terminal domain of HjFd, a Catalase activity of cytochrome P450BSb in the presence modified Fds (Fig. 1) were prepared and characterized. The data of the decoy molecule presented here show that the N-terminal domain contributes to halo- Shota Tanaka1, Takashi Fujishiro1, Osami Shoji1, adaptation of HjFd. Yoshitsugu Shiro2, Yoshihito Watanabe1,3 1Department of Chemistry, Graduate School Science Nagoya University, Nagoya 464-8602, Japan, 2RIKEN SPring-8 Cent. Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan, 3Research Center for Material Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. [email protected] u.ac.jp Cytochrome P450BSb(P450BSb) is a peroxygenase with catalytic activity for the hydroxylation of long-alkyl-chain fatty acids. Recently, we have demonstrated that P450BSb catalyzes oxidations of several non-natural substrates in the presence of a short-alkyl-chain carboxylic acid (decoy molecule) [1]. Catalase activity of P450BSb has not yet been examined, whereas the hydroxylation activity has been well investigated. In this study, catalase activity of P450BSb in the presence and in the Fig. 1 Schematic structure of Ha. japonica Fd (HjFd), spinach Fd absence of the decoy molecules was investigated. and chimeric Fds

P600 Effect of amino acid residues around the substrate- binding pocket on the substrate specificity of thiocyanate hydrolase Satoshi Namima, Shota Hori, Takatoshi Arakawa, Masafumi Yohda, Masafumi Odaka Department of Biotechnology, Tokyo University of Agriculture and Technology, Kogenei, Japan. [email protected] Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 catalyzes is the first enzyme of bacterial degradation of thiocyanate - - (SCN ) catalyzing the hydrolysis of SCN to calbonylsulfide and Fig. 1 The proposed reaction mechanism of the substrate-misrecog- ammonia. SCNase shares high amino acid sequence homology with nition system Co-type NHase. Recently, we determined the crystal structure of SCNase, which highly conserved with that of NHase including the Co active center with two cysteine modifications [1]. Although these structural similarities suggest the conservation of their reaction Reference mechanisms, SCNase exhibits no NHase activity, and NHase has no 1. Shoji O, Fujishiro T, Nakajima H, Kim M, Nagano S, Shiro Y, SCNase activity. Also, in both enzymes, the detailed catalytic Watanabe Y (2007) Angew Chem Int Ed 46:3656–3659

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P602 2. Brondino CD, Rivas MG, Roma˜o MJ, Moura JJG, Moura I Functional studies of the x-hydroxylases and its redox (2006) Acc Chem Res 39(10):788–796 3. Moura JJG, Brondino CD, Trinca˜o J, Roma˜o MJ (2004) J Biol partners from Pseudomonas oleovorans Inorg Chem 9:791–799 Chun-Wei Chang, Ho-Hsuan Chou, Chien-Hung Lai, Steve S.-F. 4. Brondino CD, Passeggi MC, Caldeira J, Almendra MJ, Feio MJ, Yu Moura JJG, Moura I (2004) J Biol Inorg Chem 9:145–151 Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan, ROC. 5. Boyington JC, Gladyshev VN, Khangulov SV, Stadtman TC, Sun [email protected] PD (1997) Science 275(5304):1305–1308 The integral-membrane bound non-heme di-iron x-hydroxylases (AlkB) comprising evolutionarily histidine-rich motifs facilitate the hydroxylation at the terminal carbon of medium-chain-length alkanes (C5–C12)inPseudomonas oleovorans. From the protein primary P604 sequence analysis, these motifs presumably are acted as part of active site to coordinate with the irons with the formation of di-iron core Chronometric method for the kinetic characterization complex for carrying out the C–H activation chemistry of hydrocar- of phenolic substrates of laccase bons. Its redox partners, rubredoxin-2 and rubredoxin reductase in the Jesus Martinez-Ruiz1, Magdalena Parra1, Virginia Tomas2, cytoplasm, are responsible for the supplement of electrons from Ramiro Martinez-Gutierrez3, Francisco Garcia-Canovas1, NADH towards x-hydroxylase. Jose Tudela1 We successfully expressed and purified the recombinant His-tagged 1GENZ-Grupo de Investigacion Enzimologia, Departamento de alkane hydroxylase and rubredoxin-2 from E. coli. On the basis of Bioquimica y Biologia Molecular-A, Universidad de Murcia, Murcia, spectroscopic data, the isolated His-tagged rubredoxin-2 containing Spain, only one iron atom locates at the C-terminal domain. The conse- 2Departamento de Quimica Analitica, Facultad de Quimica, quent iron-sulfur complex could be reduced by NADPH-dependent Universidad de Murcia, 30071 Murcia, Spain, spinach ferredoxin reductase and subsequently results in promising 3NOVOZYMES SPAIN S.A., Madrid, Spain. [email protected] conformational change observed from its secondary structural Laccase (EC 1.10.3.1) catalyzes the oxidation for molecular oxygen studies. During the purification of the recombinant His6-tagged x- of phenols up to phenoxyl radicals. The broad substrate selectivity hydroxylase, it required more competing imidazole to be eluted out and the high oxidation potential of LAC, have lead to be used in 2+ from the Ni column chromatography than the regular His6-tag biotechnological applications. The phenoxyl radicals originate containing proteins. The activity studies for both the purified unstable quinones that can evolve up to polymers, which difficult their recombinant His-tagged and wild type alkane hydroxylase indicated reliable kinetic characterization. These radicals are reduced by that, after the addition of FeSO4, these two variants are able to ascorbic acid, which oxidizes to dehydroascorbic acid. The spectro- convert 1,7-octadiene to 1,2-epoxy-7-octene in the presence of photometric assays of laccase oxidizing several phenolic substrates in recombinant His-tagged rubredoxin-2 (one Fe), NADPH and spin- the presence of ascorbic acid, they show a lag period inversely pro- ach ferredoxin reductase. The overall study supports that histidine- portional to the steady state rate of the enzymatic activity on each rich motifs at the active site for iron coordination in x-hydroxylase substrate. A kinetic equation is proposed to explain the experimental are presumably lay in the aqueous exterior of the protein. data and to predict the kinetic behaviour of the enzymatic system. Thus, the affinity (1/Km) and the rapidity (Vmax) of the oxidation of several phenolic substrates yielding unstable products have been properly determined. The obtained Km (140–970 lM) were: 4- P603 hydroxyanisole \ propylgallate \ methylgallate \ 4-tertbuthylphe- Influence of Mo or W in formate oxidation by formate nol \ gallic acid \ 4-cresole. That is due to electronic, steric and dehydrogenases hydrophobic properties of the phenolic substrates. This work has been partially supported by grants from several Spanish Cristiano S. Mota1, Maria G. Rivas1, Pablo J. Gonzalez1, organizations. Projects BIO2006-15363 (MICINN, Madrid), BIO- Carlos D. Brondino2, Jose J. G. Moura1, Isabel Moura1 BMC 06/01-0004 (BioCARM, Murcia) and 08856/PI/08 (Fundacion 1REQUIMTE, CQFB, Department of Quı´mica, Faculdade de Cieˆncias Seneca, CARM, Murcia). Predoctoral fellowships JMR BES-2007- e Tecnologia, University Nova de Lisboa, 2829-516 Caparica, 16208 (FPI, MICINN, Madrid) and MPC 09378/FPI/08 (Fundacion Portugal, Seneca, CARM, Murcia). 2Department of Fisica, Universidad Nacional del Litoral, Santa Fe, Argentina. [email protected] The biological oxidation of formate to carbon dioxide can be per- formed by formate dehydrogenases that can contain either Mo or W P605 in their active sites [1–5]. The reason for Mo and/or W preference is Optimization of the horseradish peroxidase catalysed still unknown. In order to evaluate how the metal affects the catalysis, several Mo and W containing Fdhs were isolated from biodegradation of 2,4,6-trichlorophenol Desulfovibrio species (also obtained under metal control growth Magdalena Parra1, Jesus Martinez-Ruiz1, Virginia Tomas2, conditions) and kinetic and spectroscopic studies were performed. In Ramiro Martinez-Gutierrez3, Francisco Garcia-Canovas1, addition, the isotopic effect of the alpha proton of the substrate was Jose Tudela1 evaluated using deutero-formate. Azide, nitrate and cyanide were 1GENZ-Grupo de Investigacion Enzimologia, Departamento de tested as inhibitors. Bioquimica y Biologia Molecular-A, Universidad de Murcia, Murcia, Acknowledgements: We thank Fundac¸a˜o para a Cieˆncia e a Tecno- Spain, logia (MCTES) for financial support (POCI/QUI/57701/2004). 2Departamento de Quimica Analitica, Facultad de Quimica, Universidad de Murcia, 30071 Murcia, Spain, 3 References NOVOZYMES SPAIN S.A., Madrid, Spain. [email protected] 1. Rivas MG, Gonza´lez PJ, Brondino CD, Moura JJG, Moura I Horseradish peroxidase (HRP, EC 1.11.1.7) is an enzyme with Fe(III) (2007) J Inorg Biochem 101:1617–1622 in the hemin group of its active site. 2,4,6-Trichlorophenol (TCP) is a

123 S220 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 pesticide, antiseptic and wood preservative, classified as environ- 1Department of Chemistry, Oakland University, Rochester, MI, USA, mental pollutant and probable human carcinogen by the USEPA. The 2Department of Chemistry, University of Rochester, Rochester, NY, airborne fungi can convert TCP into 2,4,6-trichloroanisole (TCA), USA. [email protected] chief cause of cork taint from polluted cork stoppers to wines. There Elevated oxalate concentrations in humans (hyperoxaluria) has been are few studies about the enzymatic biodegradation of TCP by HRP, implicated in a number of pathological conditions such as formation mainly focused on the identification of the oxidation product, 2,6- of calcium oxalate stones in the kidney, renal failure, cardiomyopa- dichloro-1,4-benzoquinone (DCBQ), by MS and NMR. The aim of thy, and cardiac conductance disorders. Oxalate oxidase (OxOx) this work is the kinetic optimization of the assay conditions for this catalyzes the manganese-dependent oxidative decarboxylation of enzymatic reaction, with separation and determination of TCP and oxalate to carbon dioxide and hydrogen peroxide. Oxalate decar- DCBQ by HPLC-DAD. The enzymatic activity and stability, lead to boxylase (OxDC) is also a manganese-dependent enzyme and the best assay conditions for the biodegradation in 15 min of catalyzes the conversion of oxalate to formate and carbon dioxide. 1.05 mM TCP, which are 22.50 nM HRP, 1.18 mM hydrogen per- Despite the detailed structural knowledge for both OxOx and OxDC, oxide (H2O2) and 50 mM pH 5.0 sodium citrate buffer at 25°C. The very little is known about the detailed catalytic mechanism for these stoichiometry TCP:H2O2 = 1:1 was established. HRP generates in enzymes. In the work, we will discuss the synthesis, characterization, each catalytic cycle two molecules of TCP-radical, which react and reactivity of small molecule manganese compounds and extrap- between them to originate only one molecule of DCBQ, with olate their properties and reactivity to the manganese enzymatic regeneration of one molecule of TCP. systems. We will also discuss the implications for our findings and This work has been partially supported by grants from several Spanish describe other applications for these catalysts. organizations. Projects BIO2006-15363 (MICINN, Madrid), BIO- BMC 06/01-0004 (BioCARM, Murcia) and 08856/PI/08 (Fundacion Seneca, CARM, Murcia). Predoctoral fellowships JMR BES-2007- P608 16208 (FPI, MICINN, Madrid) and MPC 09378/FPI/08 (Fundacion Indigo carmine biodegradation catalysed Seneca, CARM, Murcia). by horseradish peroxidase Lidia Jimenez1, Magdalena Parra1, Virginia Tomas2, Ramiro Martinez-Gutierrez3, Francisco Garcia-Canovas1, Jose Tudela1 P606 1GENZ-Grupo de Investigacion Enzimologia, Departamento de Expression and purification of mutated hpn protein, Bioquimica y Biologia Molecular-A, Universidad de Murcia, Murcia, and studies of function of cysteine residues in hpn Spain, 2Departamento de Quimica Analitica, Facultad de Quimica, Shuang QI, Hongzhe SUN, Jian-Dong HUANG Universidad de Murcia, 30071 Murcia, Spain, Department of Biochemistry, The University of Hong Kong, 3NOVOZYMES SPAIN S.A., Madrid, Spain. [email protected] Pokfulam, Hong Kong, People’s Republic of China, Department of Horseradish peroxidase (HRP, EC 1.11.1.7) is an enzyme with Fe(III) Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, in the hemin group of its active site. Indigo carmine (IC) is a pH and People’s Republic of China. [email protected] redox indicator used as dye in the manufacturing of capsules. IC is Hpn is a small nickel-storage protein in Helicobacter pylori. harmful to the respiratory tract if swallowed, and irritant to the skin Consisting of 60 amino acids, it is rich in histidine (28 histidine and eyes. There are few studies about the biodegradation of IC by residues), glutamate, glycine and serine residues. There are also oxidative enzymes, mainly focused on the identification of the oxi- four cysteine residues that may play important roles in its structure dation product, isatin-5-sulfonic acid (ISA), by MS and NMR. The and function. Our previous study shows that Hpn exists in equi- aim of this work is the kinetic optimization of the assay conditions for librium among different multimeric forms in solution, which may the enzymatic oxidation of IC by hydrogen peroxide with HRP as be due to intermolecular disulfide bonds formation via cysteine biocatalyst, with separation and determination of IC and ISA by residues. HPLC-DAD and by UV-visible spectrophotometry. The enzymatic In the present study, we replaced the four-cysteine residues with activity and stability, lead to the best assay conditions for the uni- alanine to abolish disulfide bond formation. Growth assay showed exponential biodegradation in 30 min of 85 lM IC, which are 20 nM Escherichia coli hpn that BL21(DE3) transformed with mutant gene HRP, 100 lM hydrogen peroxide and 50 mM pH 3.0 sodium citrate hpn can grow similarly as the cells transformed with wild type gene buffer at 25°C. The stoichiometry IC:H2O2 = 1:1 was established. under high nickel concentration, suggesting that the mutant hpn HRP generates in each catalytic cycle two molecules of IC-radical, protein can still protect the cells against nickel toxicity. This result which react between them with regeneration of one molecule of IC, to implies that the mutated protein has similar nickel-binding capacity as originate only one molecule of a transient oxidation product that is the wild type hpn in vivo. The mutant protein was over-expressed and hydrolyzed yielding two molecules of ISA. purified. I show that the mutated protein still aggregates, and aggre- This work has been partially supported by grants from several Spanish gation state is in equilibrium. Therefore, the underlying mechanism of organizations. Projects BIO2006-15363 (MICINN, Madrid), BIO- hpn protein aggregation may not only result from intermolecular BMC 06/01-0004 (BioCARM, Murcia) and 08856/PI/08 (Fundacion disulfide bond formation, but also from the interactions among other Seneca, CARM, Murcia). Predoctoral fellowships JMR BES-2007- amino acids, such as histidines. 16208 (FPI, MICINN, Madrid) and MPC 09378/FPI/08 (Fundacion Seneca, CARM, Murcia).

P607 P609 Model studies for oxalate-degrading manganese Interaction of pseudopeptidic disulfides enzymes and disulfide-oxides towards metal cations: relation Ferman A. Chavez1, Piotr L. Pawlak,1 Manashi Panda,1 to biological systems Derek J. Averill,1 William W. Brennessel2 Isabelle Artaud

123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S221

Laboratoire de Chimie et Biochimie, Pharmacologique et Toxicolo- gique, UMR8601 CNRS Universite´ Paris Descartes, 45 rue des Sts Pe`res, 75270 Paris Cedex 06, France. [email protected] All the sulfur oxidized species derived from thiols or disulfides have been clearly identified in biological systems and are now supposed to play a key role in enzyme activity or as signaling agents. For example, the reduction of sulfinates involves the intermediate formation of an interprotein thiosulfinate [1]. Disulfides are found in Hepcidin, a 25 aa peptide responsible for the regulatory of iron homeostasis [2]. This system is supposed to bind iron. The only systems, which contain oxidized cysteines, including the highly unstable sulfenate, ligated to a metal center, are nitrile hydratases [3] and thiocyanate hydrolases. The processus of oxidation of these cysteines is, however, not clearly elucidated. The chemical approach using cyclic pseudopeptidic thiosulfinates or thiosulfonates [4, 5], as well as cyclic or acyclic pseudopeptidic bis- disulfides allows proposing some new hypotheses to explain the formation of the nitrile hydratase metal active site and the possible reversible metallation of hepcidine.

References 1. Vivancos AP, Castillo EA, Biteau B, Nicot C, Ayte J, Toledano MB, Hidalgo E (2005) Proc Natl Acad Sci USA 102:8875 2. Hunter HN, Fultan DB, Ganz T, Vogel HJ (2002) J Biol Chem 277:37597–37603 3. Miyanaga A, Fushinobu S, Ito K, Wakagi T (2001) Biochem Reference Biophys Res Commun 288:1169–1174 1. Feducia J, Dumarieh R, Gilvey LBG, Smirnova T, Franzen S, 4. Galardon E, Bourle`s E, Artaud I, Daran J-C, Roussel P, Tomas A Ghiladi RA (2009) Biochemistry 48:995–1005 (2007) Inorg Chem 46:4515–4522 5. Bourle`s E, Alves de Sousa R, Galardon E, Giorgi M, Artaud I (2005) Angew Chem Int Ed 44:6162–6165 P611 The search for the hydrocarbon substrate binding site in the particulate methane monooxygenase (pMMO) Kok Yaoh Ng, Steve S.-F. Yu, Sunney I. Chan P610 Institute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan. [email protected] Mechanism and function of dehaloperoxidase Compelling evidence that the catalytic site of the particulate methane isoenzymes A and B monooxygenase (pMMO) is located at Site D in the protein structure Reza Ghiladi, Rania Dumarieh, Jennifer D’Antonio has recently been reported by the Chan laboratory [1]. A hydrophobic Department of Chemistry, North Carolina State University, Raleigh, pocket capable of binding the hydrocarbon substrates known to be NC 27695-8204, USA. [email protected] hydroxylated or epoxidated by pMMO has now been identified Dehaloperoxidase (DHP), the oxygen transport hemoglobin from adjacent to Site D by Global Protein Surface Survey (GPSS) analysis the marine worm Amphitrite ornata, is the first globin identified to of the published crystal structure [2] on the GPSS Website, http:// possess a biologically relevant peroxidase activity. Of the two DHP gpss.mcsg.anl.gov as well as Dockligand (LigandFit) on Discovery isoenzymes known to exist, DHP A has been extensively studied Studio 1.7 (Accelrys Software Inc.) [3]. In this analysis, pentane was and shown to oxidize trihalophenols to dihaloquinones via a selected as the probing hydrocarbon because it represented the sub- Compound ES intermediate in a dehalogenation reaction that uses strate with the largest surface area and volume that could be oxidized hydrogen peroxide as a substrate [1]. Here, we report the first by the enzyme. The predicted hydrophobic pocket was sufficiently kinetic and spectroscopic characterization of DHP isoenzyme B, long to bind only C1–C5 hydrocarbons, and it was wide enough to demonstrate its ability to oxidize trihalophenols, and characterize accommodate only straight-chain alkanes. The hydrophobic ‘‘chan- the Compound ES intermediate of this isoenzyme using both nel’’ was lined by the aromatic residues Trp48, Phe50, Trp51, and Trp stopped-flow UV-visible and rapid-freeze-quench electron para- 54 of PmoA, the 28-kDa subunit, and was ‘‘closed’’ at one end. magnetic resonance spectroscopies. Furthermore, we describe the Located at the open substrate entrance to the pocket is Gly46. With reaction of oxyferrous DHP A and B with hydrogen peroxide the ‘‘probing’’ hydrocarbon substrate fully inserted into the pocket, leading to the first characterization of authentic DHP Compound II. the putative tricopper cluster was directed at the secondary carbon of In the presence of trihalophenol substrate, this reaction leads to the substrate near the depth of the pocket, perfectly poised for O-atom catalytic turnover forming dihaloquinone product, representing transfer to the secondary carbon when the tricopper cluster was the first known peroxidase cycle capable of commencing from activated by dioxygen, in agreement with experiment. an oxyferrous starting oxidation state in vivo. The mechanis- tic implication is that dehaloperoxidase does not require a References functional switch to perform both of its biologically relevant 1. Chan SI, Yu SS-F (2008) Accounts of Chemical Research activities. 41:969–979

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2. Lieberman RL, Rosenzweig AC (2005) Nature 434:177–182 inhibitors. Additional studies aim to elucidate the origin of this unique 3. Ng KY, Tu L-C, Wang Y-S, Chan SI, Yu SS-F (2008) substrate specificity. Chembiochem 9:1116–1123

P614 P612 Structure-dependent ion interaction and free radical Cloning and characterization of nitric oxide synthase generation of Ab/Cu(II) complexes 1 3 3 4 5 from Rhodococcus erythropolis N771 C. A. Yang ,S.C.Ke, Y. H. Chen , T. H. Lin , H. B. Huang , Y. C. Chen1,2 Yuki Maeda, Satoshi Shimizu, Akashi Ohtaki, Taishin Kaneshiro, 1Institute of Life Science, Tzu Chi University, Hualien 970, Taiwan, Masafumi Yohda, Masafumi Odaka 2Institute of Medical Biotechnology, Tzu Chi University, Hualien Department of Biotechnology, Graduate School of Engineering, 970, Taiwan, Tokyo University of Agriculture and Technology, Kogenei, Japan. 3Department of Physics, National Dong Hwa University, Hualien 974, [email protected] Taiwan, Nitric oxide (NO) is synthesized by arginine-oxidizing NO synthase 4Department of Medical Research and Education, Taipei Veterans (NOS) in mammals and used for protection against pathogens, blood General Hospital, Shihpai, Taipei 112, Taiwan, pressure regulation and nerve cell transmission. Recently, a number 5Institute of Molecular Biology, National Chung-Cheng University, of bacterial protein sequences with substantial similarity to those of Chiayi 621, Taiwan. [email protected] the mammalian NOSs have been identified [1]. Although their According to the amyloid cascade hypothesis, the b-amyloid (Ab) biological function has not been understood well, they are catego- aggregates are the toxic species to neuron and the main cause of rized as bacterial NOS proteins. Previously, we found that the Alzheimer’s disease. The neurotoxicity of Ab has been associated activity of Fe-containing nitrile hydratase is regulated by with the formation of free radical by a mechanism that is still being NO-binding and its photo-release in Rhodococcus erythropolis N771 studied and possibly dependent on the coordination of Ab with redox [2]. Since the bacterial strain did not have nitrite-reducing activity, active metals such as Fe3+ and Cu2+. Our previous study has char- the presence of a bacterial NOS (ReNOS) was suggested in R. acterized the conformation of Ab in the presence or absence of erythropolis N771. In this study, we isolated the bacterial NOS gene transition metals into three categories, random coil, unstable helix and of R. erythropolis N771 (ReNOS). PCR primers were designed stable helix by chemical approach. In the present study, we applied based on amino acid sequence homologies between known bacterial similar conditions to correlate the Ab conformation with ion inter- NOSs. A 543bp DNA fragment was amplified by using the R. action and free radical generation. Unlike the general concepts, it is erythropolis N771 genome as the template. The amplified DNA the first time to demonstrate that, on a structure-dependent manner, fragment exhibited 73.5% (in amino acid sequence) and 74.3% 2+ not only can monomeric Ab40 coordinate with Cu but also induce (in DNA sequence) identities with the NOS of Rhodococcus sp. 2+ hydrogen peroxide. The Cu affinity is stronger for monomeric Ab40 RHA1. We are now attempting to clone the whole gene of ReNOS. in random coil than that in stable helix. On the other hand, monomeric The latest results will be presented. 2+ Ab40/Cu in stable helical conformation can induce the generation of H2O2, whereas, in contrast, monomeric Ab40 in random coil structure References shows to inhibit the redox reaction of Cu2+. With the increase of 1. Crane BR (2008) Biochem Soc Trans 36:1149–1154 incubation time via. the formation of Ab40 aggregates, the generation 2. Odaka M, Fujii K, Hoshino M, Noguchi T, Tsujimura M, of H2O2 can induced for both random coil and stable helix and Nagashima S, Yohda M, Nagamune T, Inoue Y, Endo I (1997) J Ò 2+ show to be dependent on the molar ratio of A40/Cu . In conclusion, Am Chem Soc 119:3785–3791 the observations implicate that, unlikely the amyloid hypothesis, the role of Cu2+ in free radical generation may involve at monomeric stage even far early before Ab aggregates into oligomer or large assemblies. P613 Understanding the structure and substrate specificities of the hydroquinone dioxygenases P615 1 Timothy E. Machonkin Syntheses, characterizations, and catecholase activities 1Department of Chemistry, Whitman College, Walla Walla, WA 99362, USA. [email protected] of some mono and tetranuclear copper complexes 2,6-Dichlorohydroquinone 1,2-dioxygenase (PcpA) from Sphingo- Shuranjan Sarkar1, Chang-Seop Hong2, Hong-In Lee1 monas chlorophenolica is a member of a small class of poorly 1Department of Chemistry, Kyungpook National University, characterized Fe(II)-containing ring-cleaving dioxygenases that have Daegu 702-701, Republic of Korea, limited sequence homology to the extradiol catechol dioxygenases. 2Department of Chemistry, Korea University, Seoul 136-704, Recently, a crystal structure was reported of a putative bacterial Republic of Korea. [email protected] glyoxalase I (PDB number, 1ZSW) with a higher sequence homology Some copper proteins exhibit different chemical reactivity toward to PcpA than any extradiol dioxygenase. From this structure, we external substrates, notwithstanding sharing the same active inter- generated a homology-based model of the structure of PcpA that mediate. For example, tyrosinase, which involves a common side-on predicted it would have significant differences in the tertiary structure peroxo species at its dinuclear copper reaction center, catalyzes and the location of the active site compare to the extradiol dioxy- the o-hydroxylation of phenols to catechols as well as the oxidation genases. Site-directed mutagenesis of the putative ligands confirmed of catechols to o-quinones (catecholase activity). Here, we explore the active site location predicted by the structural model. Steady-state catechol oxidation reaction catalyzed by mono and tetranuclear kinetic studies indicated that PcpA exhibits a high degree of substrate copper complexes. We synthesized three copper complexes, CuL1R1 specificity. Only hydroquinones with substituents at the 2- and [L1 = benzenamine, 2,20-{1,2-ethanediyl)bis (N-(2-pyr idinylmethy 6-positions are substrates, and monosubstituted hydroquinones are lene)}, R1 = OCH3], CuL2R2 [L2 = tris(2-phenylmethylimino 123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S223

ethyl)amine, R2 = OOCCH3] and Cu4(L3)4 [L3 = benzenamine, P618 0 2,2 -{1,2-ethanediyl) bis(N-(2-hydroxylphenylmethylene)}] with N4 Structural and functional models for the active site and N2O2 donor set ligands, respectively. Those complexes were structurally characterized to show one or more open coordination of Quercetin 2,3-Dioxygenase: electronic effects sites, which are appropriate for catechol oxidation. In this poster, the of the ligands catalytic action of the complexes were investigated by using the Ying-Ji Sun1, Xiao-Hong Zheng1, Shinobu Itoh2 oxidizing agent 3,5-di-tert-butylcatechol and the reactions were 1Department of Chemistry, The School of Chemical Engineering, monitored with EPR and absorption spectroscopy. Dalian University of Technology, 116024 Dalian, China, 2Department of Chemistry, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan. [email protected] Quercetin 2,3-dioxygenase (2,3-QD) is a mononuclear copper(II) P616 enzyme that activates molecular dioxygen to catalyze the oxidative Synthesis, crystal structure, and reactivity of degration of polyphenolic flavonols to the corresponding depside and Ni(II)cyclam-TTF [TTF 5 Tetrathiafulvalene] carbon monoxide. The active site of 2,3-QD exhibits two different Ju-Eun Lee1, Myoung Soo Lah2, Hong-In Lee1 forms, one of which consisting of three histidine imidazoles (His66, 1Department of Chemistry, Kyungpook National University, His68, and His112), one water molecule, and one carboxylate group of Daegu 702-701, Republic of Korea, glutamate (Glu 73) which coordinated to Cu(II) directly, whereas no 2Department of Applied Chemistry, Hanyang University, such coordination in the other form [1]. Such a coordination environ- Ansan 426-791, Republic of Korea. [email protected] ment of the enzyme active site may play an important role in controlling Charge-transfer complexes, composed of TTF and metal ions, exhibit the reactivity of 2,3-QD. However, little is known about the mecha- interesting electric and magnetic properties. We have synthesized new nistic details of the enzymatic reaction. In order to get insights into the type of metal-TTF complex in which TTF is functionalized with catalytic role of Glu73 and electronic effects on the structure and carboxylate groups and Ni(II) is activated with polyazamacrocyclic reactivity, we herein designed and synthesized a serials of bis(2-pyr- cyclam. The X-ray crystallographic study shows the complex is idylalkyl)amine model ligands carrying a para or meta-X substituted the 1-D chain framework of TTF-Ni(cyclam)-TTF-Ni(cyclam). This ortho-benzoic acid derivative as the ligand sidearm (Fig. 1). The cor- electroactive complex can work as integrated amperometric biosen- responding M(II)-model complexes [M(II): Cu, Fe and Co] have been sors for glucose and fructose. We also present the characteristic synthesized, and their structures and spectroscopic features as well as differences when nickel is replaced with copper. the reactivity towards flavonol have been investigated in details.

X N N COOH

N P617

Synthesis, crystal structure, and peroxides-like activity X: OMe, Me, H, Br, NO2 of a novel manganese complex: Mn(II)imtren [imtren 5 Tris {(2-(4-imidazolyl) Fig. 1 Structure of the model ligands methyliminoethyl)amine)}] Su-Yeon Sim1, Shuranjan Sarkar1, Myoung Soo Lah2, Hong-In Lee1 Reference 1Department of Chemistry, Kyungpook National University, 1. Fusetti F, Schroter KH, Dijkstra BW (2002) Structure 10:259–268 Daegu 702-701, Republic of Korea, 2Department of Applied Chemistry, Hanyang University, Ansan 426-791, Republic of Korea. [email protected] P619 Manganese-containing enzymes are involved in detoxification of reactive oxygen species, oxygen evolution, and non-redox catalysis. Synthesis and activity of artificial metalloprotein This brings enormous interest in the chemistry of manganese com- Seah Ling KUAN, Tanja WEIL plexes in aspect of structures and reaction mechanisms. In present Department of Chemistry, National University of Singapore, study, we synthesized a mononuclear manganese complex, Kent Ridge, Singapore 119260. [email protected] [MnHL](ClO4)2 [HL = Tris(2-(4-imidazolyl)methyl iminoeth- The burgeoning interest in artificial metalloprotein design stems, in yl)amine], to understand the peroxidase and catalase activities of the part, from their applications in enantioselective catalysis [1–6]. Our complex. The HL ligand is imidazolyl donor pendants. This ligand group is interested in the design of protein hybrids carrying transition can chelate a metal ion to form highly symmetric octahedral geometry metals within the protein scaffold, at the periphery of the active site. as well as contain labile donor atoms. Here, we illustrate the prepa- We present here the synthesis of protein hybrids via anchoring of ration, characteristics, and catalytic activities of [MnHL](ClO4)2. ruthenium complexes containing phosphate group selectively to the The catalytic activities were investigated by the volumetric methods, active site of the protein (Fig. 1). In this context, a natural binding site UV-kinetics, and EPR spectroscopy in the reaction of the complex is converted into an artificial catalytic centre by combining the cat- with ABTS and hydrogen peroxide. alytically active enzyme RNase and a metal complex. The synthesised

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metalloprotein complexes are characterised to determine how the incorporation of the transition metal complex affects the protein structure and their catalytic activity in the Heck reaction will also be investigated.

References 1. Steinreiber J, Ward TR (2008) Coord Chem Rev 252(5–7):751–766 2. Pordea A, Creus M, Panek J, Duboc C, Mathis D, Novic M, Ward O His TR (2008) J Am Chem Soc 130(25):8085–8088 Protein Scaffold 3. Pierron J, Malan C, Creus M, Gradinaru J, Hafner I, Ivanova A, P O- Sardo A, Ward TR (2008) Angew Chem Int Ed 47(4):701–705 Ln Ru Spacer - O 4. Pordea A, Ward TR (2008) Chem Commun (36):4239–4249 His 5. Rasmussen BS, Pedersen JM, Soerensen J, Egebjerg J, Schioett B, Ln = Auxillary ligand Mortensen KK, Skrydstrup T (2007) ChemBioChem 8(16):1974– e.g. diphos, diammine 1980 6. Ueno T, Abe S, Yokoi N, Watanabe Y (2007) Coord Chem Rev 251(21–24):2717–2731

Fig. 1 Protein hybrid carrying ruthenium complexes

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