Rhodium(III) and iridium(III) complexes with 1,2-naphthoquinone-1-oximate as a bidentate ligand: synthesis, structure, and biological activity.

J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 DOI 10.1007/s00775-009-0526-4 ORAL PRESENTATION D. Small Molecules (O2) Activation D-02 Ribonucleotide reductases—essential radical enzymes Britt-Marie Sjöberg Department of Molecular Biology and Functional Genomics, Stockholm University, 10691 Stockholm, Sweden. britt-marie.sjoberg@molbio.su.se Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to their corresponding deoxyribonucleotides, providing the sole biological means for de novo synthesis of the building blocks of DNA. Ribonucleotide reduction has evolved only once during evolution and all RNRs make use of a common thiyl radical-based mechanism for catalysis. The three currently known classes of RNR each operate under a distinct set of biochemical and environmental conditions [1]. Class I RNRs contain a diiron center that via oxygenmediated oxidation generates a protein-based tyrosyl radical that is a prerequisite to catalysis. Class II enzymes generate a radical via cleavage of vitamin B12 coenzyme (50 -deoxyadenosylcobalamin). Class III enzymes generate via S-adenosylmethionine cleavage (by action of an activase protein) a stable protein-based glycyl radical that is sensitive to oxygen. These differences form the basis for the classification of RNRs and result in distinct operational constraints (anaerobicity, iron/oxygen dependence and cobalamin dependence). While the propensity to synthesise deoxyribonucleotides is an essential function, the operational differences suggest that the type(s) of RNR(s) present in an organism will have an impact on the environmental conditions in which it can grow and reproduce. The effect of environmental parameters such as iron, cobalt and oxygen availability on the biochemistry of ribonucleotide reduction will impact our understanding of the adaptability of microorganisms to a range of environments. Reference 1. Torrents E, Sahlin M, Sjöberg B-M (2008) The ribonucleotide reductase family—genetics and genomics. In: Andersson KK, Uversky VN (eds) Molecular anatomy and physiology of proteins: ribonucleotide reductase. Nova Science Publishers. pp 17-776563659 (2007) D-03 Concerted iron and oxygen detoxification at the tri-nuclear Fe site of bacterial ferritin from Desulfovibrio vulgaris Hildenborough Alice S. Pereira1, Filipe Folgosa1, Márcia Guilherme, Américo G. Duarte, Cristina G. Timóteo1, Pedro Tavares1, Boi Hanh Huynh2 1 Centro de Quı́mica Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal; 2 Department of Physics, Emory University, Atlanta, GA 30322, USA. vhuynh@emory.edu Ferritins are ubiquitous and can be found in practically all known organisms ranging from bacteria, plants to vertebrates. They are 24-mers forming a hollow sphere with an inner cavity of *80 Å in diameter. The main function of ferritin is to concentrate and oxidize the cytotoxic Fe2+ ions and store the oxidized Fe in the inner cavity. In anaerobic bacteria, ferritin also serves an additional function of oxygen scavenging using molecular oxygen as the oxidant. It has been shown that rapid oxidation of Fe2+ (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a binuclear Fe site. In this paper, we report a spectroscopic (EPR and Mössbauer spectroscopy) and kinetic (stopped-flow absorption and rapid freeze quench technique) study of the Fe and O2 detoxification mechanism of the bacterial ferritin from Desullfovibrio vulgaris (Hildenborough). The results indicate that, distinct from H-type ferritins, the D. vulgaris ferritin employs a novel trinuclear Fe site for rapid oxidation of Fe2+. Also, in addition to the peroxodiferric intermediate, which was detected in the ferroxidation reaction of H-type ferritins, a mixed-valence FeIIFeIII intermediate and a transient tyrosyl radical are observed in the bacterial ferritin reaction. Involvement of a third Fe site is demonstrated unambiguously by the absence of the FeIIFeIII intermediate and tyrosyl radical, and by the stabilization of the peroxodiferric species in the E130A variant, in which the third Fe site has been removed. On the basis of these data, a minimal mechanistic scheme is proposed for the bacterial ferritin. D-04 Formation and function of the heterodinuclear Mn(IV)/ Fe(III) cofactor of Chlamydia trachomatis ribonucleotide reductase Wei Jiang, Jarod M. Younker, Paul Varano, Courtney M. Krest, Priti Singh, Jiajia Xie, Laura Dassama, Monique Maslak-Gardner, Michael T. Green, J. Martin Bollinger, Jr., Carsten Krebs Department of Chemistry and Biochemistry and Molecular Biology, The Pennsylvania State University, Pennsylvania 16802, USA. ckrebs@psu.edu Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the precursors for DNA synthesis and repair. This reaction is initiated by abstraction of the C30 -H bond of the substrate by a cysteine thiyl radical (C). In a class I RNR, the C is generated by a stable tyrosyl radical (Y), which is located in close proximity to a non-heme diiron(III/III) cluster, via a long-distance (*35 Å), conformationally gated, proton-coupled electron transfer (PCET) reaction that involves several conserved, redox-active amino acids along the PCET pathway. The recently discovered class I RNR from Chlamydia trachomatis (Ct) has all elements of the proposed PCET pathway conserved, except for the radical-harboring tyrosine, which is replaced with the non-oxidizable phenylalanine (F). This suggests that Ct RNR employs a different cofactor for generating the C. The Y-less RNRs have been termed class Ic RNR. We recently demonstrated that Ct RNR harbors a heterodinuclear Mn(IV)/Fe(III) cluster to generate the C, presumably via a PCET mechanism. Thus, the Y of a regular class I RNR is functionally replaced with a highvalent Mn(IV) site [1–3]. 123 S34 References 1. Jiang W, Yun D, Saleh L, Barr EW, Xing G, Hoffart LM, Maslak M-A, Krebs C, Bollinger JM Jr (2007) Science 316:1188–1191 2. Jiang W, Yun D, Saleh L, Bollinger JM Jr, Krebs C (2008) Biochemistry 47:13736–13744 3. Bollinger JM Jr, Jiang W, Green MT, Krebs C (2008) Current opinion in structural biology 18:650–657 D-05 Two-site competitive inhibition in hemoglobindehaloperoxidase A from Amphitrite ornata Stefan Franzen, Vesna de Serrano, Michael F. Davis, Matt Thompson Department of Chemistry, North Carolina State University, Raleigh, NC 27695 USA. Stefan_Franzen@ncsu.edu The enzyme dehaloperoxidase A (DHP A) from the marine worm Amphitrite ornata is a unique hemoglobin that functions as a peroxidase, capable of converting 2,4,6-trihalophenols (2,4,6-TXP, X = B, C, F) into the corresponding 2,6-dihalogenated quinines as well as other products. We have determined that 4-halophenols are inhibitors that bind in the distal pocket and displace the distal histidine (H55) based on X-ray crystal structures with the series of inhibitors (4-XP, X = I, Br, Cl, and F) in a unique binding interaction for a hemoglobin will be presented. The published X-ray crystal structures [1] of DHP A reveal that H55 is flexible and has two major conformations as observed in the closed (Fig. 1a, PDB 2QFK) and open (Fig. 1b, PDB 3DR9) forms of Sperm Whale myoglobin (SWMb). Figure 1c shows that binding of 4-XP inhibitor is consistent with the open conformation of H55. The 2,4,6-TXP substrates substrate affects H55 and none of the internal residues. The distal histidine acts to stabilize bound O2 in hemoglobins and as an acid–base catalyst in peroxidases. Thus, in a dual-function protein the regulation of these functions is effected by changes in the geometry of the distal histidine. Bound J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 phenols are simultaneously substrate and inhibitor of an enzyme and allosteric effectors of O2 binding. Reference 1. Chen Z, de Serrano VS, Betts L, Franzen S (2009) Distal histidine conformational flexibility in dehaloperoxidase from Amphitrite ornata. Acta Cryst D 65:34–40 D-06 Methane hydroxylation at the iron and copper sites of methane monooxygenase Kazunari Yoshizawa Institute for Materials Chemistry, Kyushu University, Fukuoka 819-0395, Japan. kazunari@ms.ifoc.kyushu-u.ac.jp DFT studies on methane hydroxylation by enzymes and related metaloxo species are presented. We propose a non-radical mechanism for methane hydroxylation by soluble and particulate methane monooxygenase having the reaction by the bare FeO+ and CuO+ complexes in mind. This mechanism is applicable when a metal-oxo species is coordinatively unsaturated. Direct interaction between methane and a metal active center can form a weakly bound methane complex in the initial stages of this reaction. Subsequent C–H bond cleavage to form an intermediate with an HO–Fe–CH3 moiety in a non-radical manner and recombination of the resultant OH and CH3 ligands take place at a metal active center to form a final methanol complex. Thus, this is a non-radical, two-step reaction. The fact that methyl radical is 10– 20 kcal/mol less stable than secondary and tertiary carbon radicals and benzyl radicals leads us to propose this mechanism. References Yoshizawa K (2006) Acc Chem Res 39:375–382 Yoshizawa K, Yumura T (2003) Chem Eur J 9:2347–2358 Yoshizawa K, Shiota Y (2006) J Am Chem Soc 128:9873–9881 D-08 The oxoiron(IV) reaction landscape Lawrence Que Jr Department of Chemistry, Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN 55455, USA. larryque@umn.edu Oxoiron(IV) centers are involved in the oxygen activation chemistry of nonheme iron enzymes. Experimental evidence for the generation of oxoiron(IV) species from many synthetic iron(II) precursors has been obtained. Such biomimetic complexes serve as useful models for the proposed enzymatic oxidants. However, almost all of these synthetic complexes have an S = 1 spin state, while the iron(IV) centers in oxidizing intermediates of nonheme iron enzymes have an S = 2 spin state. Design strategies to achieve the latter will be discussed. Of particular interest is a functional model for the a-ketoglutaratedependent iron enzymes. This iron(II) complex (1) activates O2 to generate an oxidant that can oxidize hydrocarbon substrates shape selectively. Fig. 1 X-ray structures of DHP 123 J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 D-09 Non-heme iron model complexes as models of superoxide reductase: FeIII-OOR formation, characterization and dependence on ligand environment David P. Goldberg, Frances Namuswe, Gary D. Kasper, Yunbo Jiang Department of Chemistry, The Johns Hopkins University, Baltimore, MA 21218, USA. dpg@jhu.edu The synthesis, spectroscopic characterization and reactivity of nonheme iron peroxo, oxo, and related species is an area of intense interest. This talk will describe our recent efforts in the synthesis of small-molecule models of such species. The active site of the iron enzyme superoxide reductase (SOR) is our specific target. A new series of non-heme iron(II) complexes with N4S(thiolate) ligation will be described, approximating the His4Cys coordination observed in the reduced, iron(II) form of SOR. These complexes have been shown to react with alkylhydroperoxides (e.g. tBuOOH) to give metastable iron(III)-alkylperoxo species that can be trapped at low-temperature. The spectroscopic characterization (UV–Vis, EPR, RR, EXAFS) of these FeIII–OOR complexes, combined with a systematic variation of the S and N donors, has allowed us to correlate important spectroscopic features with the properties of the ligand environment at iron. One trend that arises from these data suggests a role for the unusual thiolate donor found in the active site of SOR. References 1. Namuswe F, Kasper GD, Sarjeant AAN, Hayashi T, Krest CM, Green MT, Moënne-Loccoz P, Goldberg DP (2008) J Am Chem Soc 130:14189–14200 2. Krishnamurthy D, Kasper GD, Namuswe F, Kerber WD, Sarjeant AAN, Moënne-Loccoz P, Goldberg DP (2006) J Am Chem Soc 128:14222–14223 S35 References 1. Kovacs JA, Brines LM (2007) Acc Chem Res 40:501–509 2. Shearer J, Scarrow RC, Kovacs JA (2002) J Am Chem Soc 124:11709–11717 3. Kitagawa T, Dey A, Lugo-Mas P, Solomon EI, Kovacs JA (2006) J Am Chem Soc 128:14448–14449 D-11 New pathways of nonheme-iron-catalyzed oxidation processes Peter Comba Anorganisch-Chemisches Institut, Universität Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany. peter.comba@aci.uni-heidelberg.de Bispidine ligands are extremely rigid, easy to synthesize and available in a large variety. They enforce coordination geometries derived from cis-octahedral, and the two vacant coordination sites with the tetradentate ligand systems are sterically and electronically distinct. Reasons are thoroughly analyzed on the basis of computational work as well as experimental structural data, thermodynamics and reactivities, specifically for the corresponding iron complexes. As a consequence, we are able to directly influence the electronic properties and reactivities of the corresponding catalyst systems, and this has lead to interesting and novel observations with respect to the oxidation power, reaction mechanisms, spin states of the high-valent complexes and catalytic efficiencies. D-10 Understanding how thiolates contribute to the function of non-heme iron enzymes Julie A. Kovacs, Santiago Toledo, Elaine Nam, Jessica Pikul, Rodney D. Swartz, Pauline Alokolaro Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195-1700, USA. kovacs@chem.washington.edu The cysteinate-ligated non-heme iron enzyme superoxide reductase (SOR) has been shown to selectively reduce superoxide to afford hydrogen peroxide via an iron–peroxo intermediate that undergoes preferential Fe–O, as opposed to O–O, bond cleavage [1]. The orientation of the sulfur has been proposed to play an important role in promoting the catalytic reaction [1]. Biomimetic analogues [2, 3] of this site will be described which provide insight as to why nature utilizes a trans thiolate [1] to promote SOR function. A new series of coordinatively unsaturated pyridine/thiolate-ligated synthetic models will be described which reduce O2- via metastable intermediates that, in some cases, can be reversibly protonated affording an equilibrium acidity constant (Ka) for the exchangeable proton. By tuning the electronic properties of the metal ion, we will show that we can alter the preferred reaction pathways. D-12 Oxidation reactivities of peroxo-diiron(III) and -dicopper(II) complexes: functional models for dioxygen activating dimetalloenzymes Masatatsu Suzuki Department of Chemistry, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 9201192, Japan. suzuki@cacheibm.s.kanazawa-u.ac.jp Reactivities of peroxo-diiron(III) and -dicopper(II) complexes are of particular interest as functional models for the dioxygen activating diiron and dicopper metalloenzymes such as methane monooxygenase, toluene monooxygenase, and tyrosinase. We have synthesized two types of peroxodiiron(III) complexes, [Fe2(LPh4)(RCO2)(O2)]2+ (R = Ph3C (oxy-1) and Ph (oxy-2)), the former performs regioselective hydroxylation of a phenyl group of LPh4 and the latter exhibits reversible dioxygen binding (LPh4 = N,N,N0 ,N0 -tetrakis-[(1-methyl-2phenyl-4-imidazolyl)methyl]-1,3-diamino-2-propanolate) [1]. The reactions mimic toluene monooxygenase and hemerythrin reactivity, respectively. We have also succeeded in preparation of (l-g2:g2peroxo)dicopper(II)2 complexes, [Cu2(O2)(R-L)]2+ (oxy-R-1: R = H, MeO, t-Bu, and NO2), where R-L = 1,3-bis[bis(6-methyl-2-pyridylmethyl)aminomethyl]-5-R-benzene, which can perform not only 123 S36 J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 hydroxylation of the m-xylyl linker of R-L, but also epoxidation of styrene via an electrophilic addition of the peroxide to the C=C bond [2]. In addition, oxy-H-1 can oxidize a variety of aliphatic C–H bonds which have the bond dissociation energies (BDE) * 75 *92 kcal mol-1 via H-atom abstraction. A good correlation between the second order rate constants of oxidation of the C–H bonds of the substrates and their BDEs was observed. References 1. Yamashita M, Suzuki M et al. (2007) J Am Chem Soc 129:2 2. Matsumoto T, Suzuki T et al. (2006) J Am Chem Soc 128:3874 D-13 Push effect in mononuclear nonheme iron complex Yutaka Hitomi, Kengo Arakawa, Takuzo Funabiki, Masahito Kodera Department of Molecular Chemistry and Biochemistry, Doshisha University, Kyotanabe 610-0321, Japan. yhitomi@mail.doshisha.ac.jp It has been well accepted that an axial thiolate ligand to heme iron induces the O–O heterolysis of iron(III)-OOH species in the reaction cycle of cytochrome P450s, which is further assisted by a hydrogen bonding network to the distal oxygen atom of iron(III)-OOH species. A similar mechanism is operative in heme-dependent peroxidases, such as horseradish peroxidase, which is known as the push–pull mechanism. In the case of peroxidases, both the imidazolate ligand to the heme iron (push effect) and a protonated His residue in the distal heme pocket (pull effect) polarizes the O–O bond to promote heterolysis of the O–O bond. Recently, we started a research program to develop bio-inspired oxidation catalysts based on the abovementioned reaction mechanisms of heme-dependent oxygenases. As a part of this program, we have synthesized a series of mononuclear nonheme iron(III) complexes supported by amidate-based ligands [FeIII(dpaqR)(H2O)]+ (1R), where R = NO2, Cl, H, and Me. The amidate coordination was introduced to mimic ‘‘push effect’’. We found that its methoxide complex [Fe(dpaq)OMe]+ (2R) reacts with hydrogen peroxide to afford the corresponding iron(III)-OOH species (3R) at low temperatures. Complex 3R showed an amidate-to-iron(III) LMCT band at around 500 nm, which gradually decayed even at 233 K, Such instability cannot be observed with other iron complexes supported by neutral ligands, i.e., iron-N4Py complex. The order of the instability of 3R, R = Me [ H [ Cl [ NO2, clearly demonstrates that the amide anion coordination facilitates the decomposition of iron(III)-OOH species via ‘‘push effect’’. + OH O N III N Fe N N O N R R = Me, H, Cl and NO2 [FeIII(dpaqR)OOH]+ (3R) D-15 The effects of the secondary coordination sphere on metal-mediated oxidations A. S. Borovik Department of Chemistry, University of California-Irivne, Irvine, CA 92697, USA. aborovik@uci.edu Hydrogen bonds stabilize and direct chemistry performed by metalloenzymes. With inspiration from enzymes, we have utilized an approach that incorporates intramolecular hydrogen bond donors to 123 determine their effects on the stability and reactivity of metal complexes. Our premise is that control of secondary coordination sphere interactions will promote new function in synthetic metal complexes. For instance, we have developed a series of complexes with terminal oxo and hydroxo ligands, which are surrounded by intramolecular hydrogen bond networks. Our work has established that hydrogen bonds can compete with pi-bonds to stabilized monomeric oxometal complexes. This talk will discuss our recent efforts in understanding dioxygen activation by metal complexes and the reactivity of resultant oxometal complexes, in which the oxo ligands are highly basic. Our results indicated that mechanistic changes are correlated with the basicity of the oxo ligands. D-16 Striking lessons from nickel-superoxides and related systems Shenglai Yao1, Matthias Driess1, Carsten Milsmann2, Eckhard Bill2, Karl Wieghardt2 1 Institute of Chemistry, Metalorganics and Inorganic Materials, Sekr. C2, Technische Universität Berlin, Strasse des 17. Juni 135, 10623, Germany; 2 Max Planck Institute of Bioinorganic Chemistry, Mülheim, Germany. matthias.driess@tu-berlin.de Diatomic chalcogen molecules X2 in the coordination spheres of transition-metals can play an eminent role in oxidation processes in biological and catalytic systems. While peroxo and persulfido ligands and their suitability for X-atom transfer reactions have been studied in great detail, much less is known about paramagnetic superoxo (O2 radical anion) and supersulfido (S2 radical anion) complexes. Moreover, the heavier analogues (Se2 and Te2 anions) are hitherto unknown. We now learned that using a b-diketiminate nickel(+1) precursor complex gives facile access to the isolable and surprisingly stable superchalcogenido complexes 1 (X=O) and 2 (X=S) [1, 2] (Fig. 1). LNi II .O LNi .S II S O 1 2 . S S . S S LNi II Ni II L 22 Unexpectedly, complex 2 exists as a diamagnetic dimer 22 with a four-sulfur two-electron bond. The peculiar structure–reactivity relationships of 1 and 2 and related studies on the corresponding Se and Te systems [3] as potential building blocks for metalloenzyme models will be discussed in my talk. References 1. Yao S, Bill E, Milsmann C, Wieghardt K, Driess M (2008) Angew Chem Int Ed 47:7110 2. Yao S, Milsmann C, Bill E, Wieghardt K, Driess M (2008) J Am Chem Soc 130:13536 3. Driess M et al. submitted D-17 Bio-inspired alkane oxidation catalysts based on nickel complexes Shiro Hikichi, Hideho Okuda, Naoki Imamura, Akiyoshi Ishii, Jun Nakazawa Department of Material and Life Chemistry, Faculty of Engineering, Kanagawa University, Yokohama 221-8686, Japan. hikichi@kanagawa-u.ac.jp Development of the efficient oxygenation processes for hydrocarbons (especially alkanes) is still one of the challenging subjects from the J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 S37 1 Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea, 2 Department of Chemistry, Stanford University, Stanford, CA 94305, USA, 3 Picobiology Institute, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan. jaeheung@ewha.ac.kr The binding and activation of dioxygen at transition metal centers are of great importance for understanding the reaction mechanisms of metalloenzymes and utilizing metal complexes as oxidation catalysts. NiO2 intermediates have been developed and investigated to elucidate highly active species in the oxidative functionalization of organic substrates [1]. The synthesis and spectroscopic characterization of a NiO2 complex with a 14-membered macrocyclic ligand were reported previously [2]. Herein, we report the formation, characterization, and reactivity of a novel NiO2 complex having a 12-membered macrocyclic ligand, [Ni(12-TMC)(O2)]+ (12-TMC = 1,4,7,10-tetramethyl1,4,7,10-tetraazacyclododecane). PR3 electrophile O=PR3 RCOO nucleophile radical RCHO Aliphatic C-H oxidation Fig. 1 Rectivity of 1 II Ni SR N N N N N N N N O2 III N N SR Ni Ni = H B N N Ni O O III Ni O2 O OH Ni OOH SR 14-TMC Ph Ph Me C OH + C O Me H Ph Me C O O H II SR NO2 N N Ph Me C H H Ph Me C H R= Ni I CH2Cl2 D-19 Biomimetic and bioinspired oxidation by ruthenium and osmium oxo complexes OOH viewpoints of green and sustainable chemistry. In this work, oxidizing catalyses of TpR-supported nickel complexes with peroxides as well as O2 were investigated. An alkylperoxo complex of nickel(II), [NiII(OOtBu)TpiPr2] (1) exhibited the substrates-depending reactivity as shown Fig. 1. The thermal decomposition of 1 might proceed through O–O and Ni–O homolysis, and the resulting radical species would contribute to the catalytic cyclohexane oxygenation with TBHP [1]. In contrast, in situ generated acylperoxonickel analogues 2 exhibited high selectivity for the cyclohexane oxygenation to the corresponding alcohol. We also investigated O2 oxidizing capability of a thiolato complex, [NiII(SC6H4NO2)TpMe2] (3). Complex 3 reacted with O2 and abstracted a benzylic H atom of ethylbenzen (Fig. 2). Reference 1. Hikichi S, Okuda H, Ohzu Y, Akita M (2009) Angew Chem Int Ed 48:188–191 D-18 Contribution of the supporting ligands to NiO2 intermediates 2 References 1. Kimura E, Machida R (1984) J Chem Soc Chem Commun 499–500 2. Kieber-Emmons MT, Annaraj J, Seo MS, Van Heuvelen KM, Tosha T, Kitagawa T, Brunold TC, Nam W, Riordan CG (2006) J Am Chem Soc 128:14230–14231 II Ni Cl Fig. 2 Alkane oxygenation mediated by 3 1 12-TMC 1 Jaeheung Cho , Ritimukta Sarangi , Jamespondi Annaraj , Sung Yeon Kim1, Minoru Kubo3, Takashi Ogura3, Edward I. Solomon2, Wonwoo Nam1 Tai-Chu Lau Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Hong Kong. bhtclau@cityu.edu.hk Nature makes use of iron oxo species to carry out various oxidation reactions. In this presentation the kinetics and mechanisms of the oxidation of various inorganic and organic substrates by ruthenium and osmium oxo complexes will be discussed. These substrates include alkanes, arenes, NO2-, I-, NCS- etc. The formation of metal-oxo species is an important step in many enzymatic and chemical oxidation processes. In this presentation the kinetics of the oxidation of trans[RuIV(tmc)(O)(solv)]2+ to trans-[RuVI(tmc)(O)2]2+ (solv = H2O or CH3CN) by MnO4- in aqueous solutions and acetonitrile will also be described. We provide evidence that the initial rate-limiting step in water occurs by hydrogen atom transfer, while that in acetonitrile occurs by oxygen atom transfer. References 1. Man WL, Lam WWY, Wong WY, Lau TC (2006) J Am Chem Soc 128:14669–14675 2. Lam WWY, Man WL, Lau TC (2008) Inorg Chem 6771–6778 3. Lam WWY, Man WL, Leung CF, Wong CY, Lau TC (2007) J Am Chem Soc 129:13646–13652 D-21 Human P450 heme–oxygen intermediates Stephen G. Sligar1, James R. Kincaid2, Ilia G. Denisov1, Piotr J. Mak2, Stephanie L. Gantt1, Aditi Das1, Yelena V. Grinova1 123 S38 J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 1 School of Molecular and Cellular Biology, University of Illinois, Urbana, IL 61801, USA; 2 Department of Chemistry, Marquette University, Milwaukee, WI 53233, USA. s-sligar@uiuc.edu We have examined the electronic, vibrational and magnetic properties of the intermediate states of cytochrome P450 using a combination of cyrogenic optical, EPR and Raman spectroscopy and documented the channels leading to productive and uncoupled pathways. We extended our initial studies using the bacterial P450 CYP101 to the human enzymes involved in drug metabolism (CYP3A4) and steroid hormone biosynthesis (CYP19, CYP17 and CYP11A1) by using self-assembled nanoscale lipid bilayer ‘‘Nanodiscs’’. In these membrane-bound systems we document redox potentials, autoxidation rates, heme–oxygen intermediates and the existence of a substantial ‘‘oxidase’’ activity represented by water production from a putative ‘‘Compound I’’ state and will discuss these results in terms of the chemical reactivity of peroxo and iron– oxo intermediates. O2 + S NADPH FAD FMN H 2 O + S-O NADP + References 1. Mak PJ, Denisov IG, Victoria D, Makris TM, Deng T, Sligar SG, Kincaid JR (2007) J Am Chem Soc 129:6382–6383 2. Denisov IG, Mak PJ, Makris TM, Sligar SG, Kincaid JR (2008) J Phys Chem A 112:13172–13179 3. Grinkova YV, Denisov IG, Waterman MR, Arase M, Kagawa N, Sligar SG (2008) Biochem Biophys Res Comm 372:379–382 D-22 Substrate recognition and conformational stability of the active site in cytochrome P450cam Rabindra K. Behera, Soumen K. Manna, Shyamalava Mazumdar Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400005, India. shyamal@tifr.res.in The cytochrome P450cam from P. putida is studied as a model of the heme mono-oxygenases that are involved several vital biochemical processes such as drug metabolism, detoxification against xenobiotics and biosynthesis of steroid hormones. The active site of these enzymes consists of a deeply buried heme center anchored through a cysteine residue of the enzyme and the intervening amino acid residues control the entry and binding of the substrate at the active site of the enzyme. Site-specific mutation at the surface near the substrate entry channel of the enzyme showed distinct variations in the substrate association rate constants, suggesting the existence of a recognition site for the substrate at the enzyme surface. The structure of the enzyme indicated presence of a unique potassium ion binding site near the substrate access channel of the enzyme. Binding of the potassium ion not only facilitated substrate binding but also stabilized the active form of the enzyme. Moreover, substitution of the potassium ion by calcium ion was found to cause inactivation of the enzyme forming a P420-type species that was reversibly converted to the active P450 form on addition of potassium ion. The present talk will describe some of these results in the light of understanding the conformational properties of the heme pocket and the effects of substrate binding to the active site in cytochrome P450cam. 123 D-23 How nature uses oxygen—lessons from enzymes and model compounds John T. Groves Department of Chemistry, Princeton University, Princeton NJ 08544, USA. jtgroves@princeton.edu The central paradigms of aerobic life processes derive from our understanding of how the redox chemistry of oxygen is manipulated and controlled by iron- and manganese-containing proteins. Nature has exploited the large, intrinsic driving force for the reduction of oxygen to water in a variety of intriguing ways. Now that the core chemistry of these processes has been revealed, they can be seen as a set of remarkably similar themes involving oxo- and peroxo-transition metal complexes. In this lecture we compare and contrast those mechanistic strategies looking alternately at heme proteins such as cytochrome P450, non-heme iron proteins such as AlkB and NDO and models metalloporphyin complexes containing iron and manganese. The oxygen activation and transfer reactions catalyzed by the heme and non-heme families of iron-containing oxygenase enzymes produce reactive metal–oxo intermediates. We find that a full understanding of the nature of these reactions requires consideration of metal ligation and peripheral substitution effects [1], spin-state crossing effects [2] and the stochastic effects of in-cage recombination and cage escape [3]. The lecture will describe recent experimental results and some new insights regarding these fascinating and important reactions. References 1. Jin N, Ibrahim M, Spiro TG, Groves JTJ (2007) Am Chem Soc 129:12416–12417 2. De Angelis F, Jin N, Car R, Groves JT (2006) Inorg Chem 45(10):4268–4276 3. Austin RN, Luddy K, Erickson K, Pender-Cudlip M, Bertrand E, Deng D, Buzdygon RS, van Beilen JB, Groves JT (2008) Angew Chem 47:5232–5234 Acknowledgments We thank the NIH (R37 GM36298, MERIT award), the NSF (CHE0316301; Special Creativity Award) and the NSF-EMSI, CEBIC, at Princeton University, (CHE-9814301) for generous support of this research. D-24 Human indoleamine 2,3-dioxygenase: new inhibitors and their functional consequences A. Grant Mauk,1 Federico I. Rosell,1 Gavin Carr2 Raymond J. Andersen2 1 Department of Biochemistry and Molecular Biology, The Centre for Blood Research, University of British Columbia, Vancouver BC V6T 1Z3, Canada; 2 Departments of Chemistry and Earth and Ocean Sciences, University of British Columbia, Vancouver, BC V6T 1Z? Canada. mauk@interchange.ubc.ca The role played by indoleamine 2,3-dioxygenase (IDO) in the immune escape mechanism of solid tumors has made it an appealing target for therapeutic intervention [1]. Using high throughput screening assays, we have identified several new and potent inhibitors of human IDO in extracts prepared from marine invertebrates and from microbes cultured from marine sediments. The structures of several of these new inhibitors have been determined. Their mechanism of inhibition and of some synthetic analogues related to exiguamine A [2] are uncompetitive with respect to Trp and exhibit submicromolar inhibitory constants. To evaluate the effects of these inhibitors on the interaction of the enzyme with diatomic gaseous J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 S39 ligands, we have begun to study their effect on the affinity of the enzyme for dioxygen and on the binding of CO. The results indicate that these new inhibitors may be useful mechanistic probes as well as potential therapeutic leads. Acknowledgments This study was supported by grants from the National Cancer Institute of Canada and the Canadian Cancer Society (to A.G.M. and R.J.A.). References 1. Muller AJ, Scherle PA (2006) Nat Rev Cancer 6:613–625 2. Carr G, Chung MK, Mauk AG, Andersen RJ (2008) J Med Chem 51:2634–2647 D-25 Crystal structures of the substrate-free and the decoy molecule-bound forms of cytochrome P450BSb 1 1 2 Osami Shoji , Takashi Fujishiro , Shingo Nagano , Yoshitsugu Shiro2, Yoshihito Watanabe3 1 Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan; 2 RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan; 3 Research Center for Material Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. shoji.osami@a.mbox.nagoya-u.ac.jp In contrast to most P450s, P450BSb (CYP152A1) isolated from Bacillus subtilis utilizes hydrogen peroxide to catalyze the hydroxylation of long-alkyl-chain fatty acid. Recently, we have reported that P450BSb can catalyze oxidation of a variety of non-natural substrates, such as hydroxylation of ethylbenzene, by employing a simple substrate trick: a series of short alkyl-chain carboxylic acids are misrecognised as substrates [1]. In order to examine possible structural changes induced by the substrate and decoy molecule binding, the crystal structural studies of the substrate-free form and a decoy molecule-bound form were carried out and compared those structures with that of the palmitic acid bound form reported (1IZO). Reference 1. Shoji O, Fujishiro T, Nakajima H, Kim M, Nagano S, Shiro Y, Watanabe Y (2007) Angew Chem Int Ed 46:3656–3659 D-27 The oxidation of L-tryptophan in biology Nishma Chauhan,1 Sarah J. Thackray,2 Sara A. Rafice,2 Igor Efimov,1 Jaswir Basran,3 Christopher G Mowat,2 Stephen K. Chapman,2 Emma Raven 1 Department of Chemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK; 2 EaStCHEM, School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK; 3 Department of Biochemistry, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK. emma.raven@le.ac.uk The L-kynurenine pathway—which leads to the formation of NAD—is the major catabolic route of L-tryptophan metabolism in biology. The initial step in this pathway is oxidation of L-tryptophan to N-formylkynurenine (Scheme 1). In all biological systems examined to date, this is catalysed by one of two heme enzymes, tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO), in a reaction mechanism that involves binding of O2 to ferrous heme. We have examined the activity of three heme dioxygenases (human IDO, human TDO and X. campestris TDO) with 1-Me-L-Trp. In contrast to previous work, we find that 1-Me-L-Trp is a slow substrate. These observations are inconsistent with current proposals in the literature for the mechanism of substrate oxidation, and we propose an alternative. References 1. Sono M, Roach MP, Coulter ED, Dawson JH (1996) Chem Rev 96:2841 2. Chauhan N et al. (2009) J Am Chem Soc (in press) D-28 How do novel functions evolve from existing protein scaffolds? C. S. Raman Department of Biochemistry and Molecular Biology, University of Texas Medical School, MSB 6.128, 6431 Fannin Houston, TX 77030, USA. c.s.raman@uth.tmc.edu Understanding the relationship between protein structure and function is one of the greatest challenges in structural biology. In this lecture, I will discuss our recent efforts to gain molecular insights into how new biosynthetic pathways evolve. Specifically, my talk will focus on the generation of oxygenated lipid mediators by the cytochrome P450 superfamily. Lipid mediators constitute a broad spectrum of molecules, including vasoactive substances in mammals and volatile organic compounds that confer characteristic flavors to fruits and vegetables. Plant oxylipins (such as jasmonates) and animal prostaglandins are short-lived but potent peroxide-derived lipid mediators that share strikingly similar biological activities, including metabolic regulation, reproduction, and host defense. Their biosynthesis involves extraordinary rearrangements of labile organic peroxides by a novel group of heme thiolate enzymes belonging to the cytochrome P450 superfamily. In spite of three decades of intense research, it has been difficult to unravel how some of these molecules are produced. Equally unclear is the evolutionary origin of the enzymes that synthesize these diverse groups of signaling molecules. Here, I will offer an atomic description of the enzymes involved in oxylipin biosynthesis. I will also elaborate on how our structural efforts led to the discovery of new oxylipin signaling pathways in both bacteria and marine invertebrates [1]. References 1. Lee DS, Nioche P, Hamberg M, Raman CS (2008) Nature 455:363–368 Acknowledgments This work is supported by The Robert A. Welch Foundation, Pew Scholars Program in Biomedical Sciences, The American Heart Association, and The NIH. 123 S40 D-29 Mechanisms, kinetics and thermodynamics of halide oxidation by human heme peroxidases Christian Obinger, Johanna Stampler, Markus Auer, Paul G. Furtmüller Department of Chemistry, Division of Biochemistry, BOKU, University of Natural Resources and Applied Sciences, Muthgasse 18, 1190 Vienna, Austria. christian.obinger@boku.ac.at The recently proposed peroxidase-cyclooxygenase superfamily includes enzymes from all living kingdoms with a broad spectrum of enzymatic features and physiological roles [1]. These proteins have differentiated very early in the evolutionary history as cornerstone of the innate immune defence system. Before organisms have developed an acquired immunity, their antimicrobial defence depended on enzymes that were recruited upon pathogen invasion and produced microbicidal reactive oxidants and diffusible radical species. A unique activity of these enzymes is their ability to use hydrogen peroxide and halides (Cl-, Br-, I-) and/or thiocyanate as two-electron donors to generate halogenating oxidants such as hypohalous acids [2]. Here, we discuss the mechanisms, kinetics and thermodynamics of halide oxidation by mammalian peroxidases. It is presented how structural differences in their active sites (e.g. heme to protein linkages, heme asymmetry and distortion) are reflected by distinct thermodynamic and kinetic features [3]. References 1. Zamocky M, Jakopitsch C, Furtmüller PG, Dunand C, Obinger C (2008) Proteins 71:589–605 2. Arnhold J, Monzani E, Furtmüller PG, Zederbauer M, Casella L, Obinger C (2006) Eur J Inorg Chem 3801–3811 3. Zederbauer M, Furtmüller PG, Brogioni S, Jakopitsch C, Smulevich G, Obinger C (2007) Nat Prod Res 24:571–584 D-30 Conformational dynamics in the reductase domain of nitric oxide synthase Huiying Li1, Aditi Das2, Hiruy Sibhatu1, Joumana Jamal1, Stephen G. Sligar2, Thomas L. Poulos1* 1 Departments of Molecular Biology and Biochemistry, Chemistry and Pharmaceutical Sciences, University of California, Irvine, CA 92697, USA; 2 Departments of Biochemistry, Chemistry, The Center for Biophysics and Computational Biology, The College of Medicine, University of Illinois, Urbana, IL 61801, USA. poulos@uci.edu As in P450s electrons in nitric oxide synthase (NOS) are delivered from a FAD/NADPH/FMN reductase domain to the heme, a required reaction for proper O2 activation and substrate hydroxylation. In NOS, however, the heme and reductase domains are linked together as a single polypeptide chain and calmodulin binding to NOS regulates electron flow from the reductase to heme. It is generally thought that the reductase domain is quite flexible and that large movements must occur for the FMN domain to pick up an electron from FAD and then undergo a large repositioning required for proper alignment with the heme domain. Calmodulin binding favors the ‘‘output state’’ wherein the FMN is in position to reduce the heme. The crystal structure of the reductase domain [1] shows that, like in P450 reductase, the FMN and FAD are quite close. We now have shown that mutations at the FMN/FAD interface are critical in controlling the FMN redox potential and the ability of calmodulin to promote the ‘‘output state’’. The changes are subtle indicating that the dramatic effects observed when calmodulin binds are due to small changes in free energy. 123 J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 Reference 1. Garcin ED, Bruns CM, Lloyd SJ, Hosfield DJ, Tiso M, Gachhui R, Stuehr DJ, Tainer JA, Getzoff ED (2004) J Biol Chem 279(36):37918–37927 D-31 Theoretical studies on short-lived intermediates in the catalytic cycle of cytochrome P450 and their reactivity patterns Sam P. de Visser1 1 Manchester Interdisciplinary Biocenter, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK. sam.devisser@manchester.ac.uk The cytochromes P450 are important heme enzymes in biosystems involved in drug metabolism and detoxification processes in the body. in spite of extensive experimental and theoretical studies there are still controversies regarding the nature of the active oxidant that performs the substrate monoxygenation. We have done a series of density functional theory and quantum mechanics/molecular mechanics calculations on cytochrome P450 enzymes and active site mimics to elucidate the properties of potential oxidants and their reactivity patterns [1, 2]. In this presentation we will highlight the latest achievements of our group using DFT and valence bond methods and thermodynamic cycles to explain the fundamental differences between heme and nonheme monoxygenases [3]. References 1. Shaik S, Kumar D, de Visser SP (2008) J Am Chem Soc 130:10128–10140 2. Tahsini L, Bagherzadeh M, Nam W, de Visser SP (2009) Chem Eur J (submitted) 3. de Visser SP (2006) J Am Chem Soc 128:9813–9824 D-32 Structural studies of the intermediates in the reaction between myoglobin and peroxides: effects of cryoradiolytic reduction K. Kristoffer Andersson1, Hans-Petter Hersleth1 1 Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway. k.k.andersson@imbv.uio.no The intermediates generated in the reaction between myoglobin and peroxides mimic the intermediates found in many peroxidases, oxygenases and catalases [1, 2]. These myoglobin intermediates are also relevant because myoglobin is proposed to take part as scavenger of reactive oxygen species during oxidative stress. We have in this study combined crystallography and single-crystal light absorption spectroscopy (microspectrophotometry). Radiation-induced changes of the different intermediates in this reaction cycle have been observed and followed by microspectrophotometry [2–5]. We have been able by cryoradiolytic reduction of an oxymyoglobin equivalent (compound III) to generate and trap the so-called peroxymyoglobin intermediate, J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 S41 References 1. Thomas KE, Wasbotten IH, Ghosh A (2008) Inorg Chem 47:10469–10478 2. Roos BO, Veryazov V, Conradie J, Taylor PR, Ghosh A (2008) J Phys Chem B 112:14099–14102 Scheme 1 Reaction catalysed by IDO and TDO a Fe(II)-superoxide form indicated by quantum refinement analysis [2, 4]. By annealing of this compound the oxygen–oxygen bond is broken and the reaction propagates to the compound II intermediate [3–5]. The structures have further been refined with quantum refinement [1, 4, 5] together with Ulf Ryde. References 1. Hersleth H-P, Ryde U, Rydberg P, Görbitz CH, Andersson KK (2006) J Inorg Biochem 100:460–476 2. Hersleth H-P et al. (2008) Inorg Chim Acta 361:831–843 3. Hersleth H-P, Uchida T, Røhr ÅK, Teschner T, Schünemann V, Kitagawa T, Trautwein AX, Görbitz CH, Andersson KK (2007) J Biol Chem 282:23372–23386 4. Hersleth H-P, Hsiao Y-W, Ryde U, Görbitz CH, Andersson KK (2008) Biochem J 412:257–264 5. Hersleth H-P, Hsiao Y-W, Ryde U, Görbitz CH, Andersson KK (2008) Chem Biodiv 5:2067–2089 D-33 Metallocorroles as models of high-valent heme protein intermediates Abhik Ghosh, Abraham Alemayehu, Kolle E. Thomas, Can Capar, Emmanuel Gonzalez Department of Chemistry, University of Tromsø, 9037 Tromsø, Norway. abhik@chem.uit.no Metallocorroles are intriguing as stable models of heme protein compound I and compound II intermediates. They exhibit an amazing range of electronic structures, spectroscopic and electrochemical properties. Here we will present our findings on b-octakis(trifluoromethyl)corrole complexes (see diagram below) recently prepared in our laboratory [1], focusing on copper, manganese, and iron derivatives. Although chloroiron corrole derivatives have long been recognized as noninnocent [2], copper corroles have until now been thought of as true Cu(III) complexes. Combined crystallographic, spectroscopic, electrochemical and high-level theoretical (CASPT2) studies now indicate that even copper corroles are best viewed as antiferromagnetically coupled CuII-corrole2-. This antiferromagnetic coupling, mediated by a specific Cu(dx2-y2)-corrole(p) orbital interaction, is so strong that it results in a buckling of the corrole macrocycle, i.e. even sterically unhindered copper corroles are inherently buckled, a situation that is very different from cobalt corroles, which are planar even for highly hindered corrole ligands. We will attempt to find parallels between these and other intriguing aspects of corrole chemistry and spectroscopic features of metalloenzyme intermediates such as chloroperoxidase compound II and the ox1 state of methylcoenzyme M reductase. D-35 Exploring the role of the active site methionine residue in PHM catalysis Brenda A. Broers1, Corinna Hess2, Andrew T. Bauman1, Judith P. Klinman2, Ninian J. Blackburn1 1 Department of Science and Engineering, Oregon Health and Sciences University, Beaverton, OR 97006, USA; 2 Department of Chemistry, University of California, Berkely, CA 94720, USA. ninian@ebs.ogi.edu Methionine 314 plays a critical role in PHM catalysis. No clear role for this residue has emerged but a number of proposals suggest that it may (i) stabilize the Cu(II)-superoxo intermediate (ii) stabilize the reduced form of the enzyme and (iii) provide a fluxional coordinate for H tunneling. For WT PHM the intensity of the Cu–S interaction in the Cu(I) EXAFS data is inversely proportional to catalytic activity over the pH range 3–8. At pH 8, the reduced Cu(I) spectrum can be simulated by replacing the CuM Cu–S(Met) interaction with a Cu–N/ O. However, the pH 3.5 data still show the presence of a strong Cu–S interaction, and establish that the form observed at low pH in WT cannot be due to a M314 ‘‘on’’ conformer, but must arise from a different Cu–S interaction. These data are confirmed by studies of CO binding which show that M314H does not bind CO at pH 8, but that both WT and mutant exhibit a new FTIR band at 2,111 cm-1 assignable to the low-pH S-containing species. Therefore, lowering the pH causes a conformational change at one of the Cu centers which brings a new Cys or Met residue into a favorable orientation for coordination to the metal center generating an inactive form. These data, together with parallel results on tyramine b monooxygenase, are discussed in relation to the PHM catalytic mechanism (Fig. 1). Fig. 1 Structure of the PHM Active Site. The YVG substrate is shown in red. M314 is shown coordinated to CuM as a yellow side chain 123 S42 J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 D-36 Peptidylglycine a-hydroxylating monooxygenase (PHM): oxygen activation and small molecule binding by a copper center D-38 Copper-dioxygen and cupric-hydroperoxo mononuclear complexes: formation, characterization and substrate reactivity Eduardo E. Chufán1, Sean T. Prigge2, Betty A. Eipper3, Richard E. Mains3, L. Mario Amzel1 1 Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; 2 Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; 3Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030, USA. mamzel@jhmi.edu Peptidylglycine a-hydroxylating monooxygenase (PHM) catalyzes the first step in the activation of peptide hormones and neurotransmitters: the stereospecific hydroxylation of the Ca of a C-terminal peptidylglycine. The enzyme uses for catalysis two separate Cu sites (CuM and CuH) located *11 Å from each other; CuM is the metal involved directly in catalysis while CuH is associated with electron transfer. Using X-ray diffraction, sitedirected mutagenesis and kinetic characterization, we identify important intermediates in the reaction mechanism. In addition, we proposed a path for the transfer of an electron from CuH to the site of CuM. To determine whether only oxygen does not bind to CuH or this lack of binding is a general feature of CuH, we determined the structures of the complexes of PHM with other small molecules such as nitrite (NO2-) and azide (N3-). Nitrite anion coordinates CuM but, surprisingly, not CuH despite the high concentration of nitrite used in the experiments (nitrite/protein [ 1,000). Similarly, azide binds CuM but not CuH. The lack of binding of small molecules to CuH may be correlated with its functional role as an electron-transfer site. Kenneth D. Karlin Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA. karlin@jhu.edu For a number of copper metalloenzymes, copper(I)-dioxygen adducts or derived species are oxygenating (oxygenase) or oxidant (oxidase) active species. Mononuclear CuI/O2 activation chemistry is considered to be important and of considerable recent interest. Here, we will describe: (i) CuI/dioxygen chemistry involving tripodal tetradentate ligands which lead to end-on bound superoxo-copper(II) complexes. Their kinetics of formation, spectroscopic and structural characterization along with reactivity traits will be presented. (ii) The chemistry of copper(II) or dicopper(II) hydroperoxo complexes, i.e., CuIIn (-OOH) (n = 1 or 2), will also be described and we show that these can effect substrate C–H activation/oxygenation chemistries, such as N-alkyl hydroxylation or oxidative N-dealkylation. (iii) Chemistry of a new system where two-electron reduction of O2 is effected at a single copper ion ligand complex. D-37 Identifying the active site of particulate methane monooxygenase Amy C. Rosenzweig 1 Departments of Biochemistry, Molecular Biology, and Cell Biology and Chemistry, Northwestern University, Evanston, IL, USA. amyr@northwestern.edu Particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol in methanotrophic bacteria. The 300 kDa pMMO enzyme is a trimeric integral membrane protein, comprising three copies each of three subunits: pmoB, pmoA, and pmoC. In spite of extensive spectroscopic characterization and the availability of two crystal structures [1, 2], the location and metal composition of the pMMO active site remain unknown. We have detected three different metal centers crystallographically: a highly conserved dinuclear copper center, a nonconserved mononuclear copper center, and a site that can be occupied by zinc or copper. On the basis of spectroscopic data, other researchers have reported the presence of trinuclear copper center or a dinuclear iron center, of which each was proposed to be the active site. We present here new activity, biochemical, and spectroscopic data that directly address the validity of these models. References 1. Hakemian AS, Kondapalli KC, Telser J, Hoffman BM, Stemmler TL, Rosenzweig AC (2008) Biochemistry 47:6793 2. Rosenzweig AC (2008) Biochem Soc Trans 36:1134 123 References 1. Maiti D, Lee D-H, Gaoutchenova K, Würtele C, Holthausen MC, Sarjeant AAN, Sundermeyer J, Schindler S, Karlin KD (2008) Angew Chem Int Ed 47:82–85 2. Maiti D, Narducci Sarjeant AA, Karlin KD (2008) Inorg Chem 47:8736–8747 D-39 Copper chemistry within biomimetic chambers Olivia Reinaud Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université Paris Descartes, CNRS UMR 8601, 45 rue des Saints-Pères, 75006 Paris, France. Olivia.Reinaud@parisdescartes.fr We are interested in modeling mono-nuclear active sites of copperenzymes with ligands reproducing not only the first coordination sphere (a poly-histidine core by an aza-site), but also the hydrophobic funnel controlling the substrate binding. For this purpose, we have designed a ligand family, the calix[6]aza-cryptands, based on the calix[6]arene core onto which a poly-aza cap has been grafted at the small rim. The resulting capped structures provide a strong chelate effect, preclude any bimetallic interaction and enforce exogenous ligation exclusively through the funnel. The corresponding Cu(I) and Cu(II) complexes, their coordination properties and supramolecular behavior will be presented, together with recent insights into their ability to activate dioxygen. N NH NHHN N N NN P NH HNHN Calix[6]tren Calix[6]tmpa Calix[6]PN3 J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 References 1. Izzet G, Douziech B, Prangé T, Tomas A, Jabin I, Le Mest Y, Reinaud O (2005) Proc Natl Acad Sci USA 102:6831–6836 2. Izzet G, Rager M-N, Reinaud O (2007) Dalton Trans 771–780 3. Izzet G, Zeng X, Akdas H, Marrot J, Reinaud O (2007) Chem Commun 810–812 4. Izzet G, Zeng X, Over D, Douziech B, Zeitouny J, Giorgi M, Jabin I, Le Mest Y, Reinaud O (2007) Inorg Chem 46:375–377 5. Izzet G, Zeitouny J, Akdas-Killig H, Frapart Y, Ménage S, Douziech B, Jabin I, Le Mest Y, Reinaud O (2008) J Am Chem Soc 130:15226–15227 D-41 Activation and catalysis of inactive phenoloxidase and hemocyanins Heinz Decker Institute for Molecular Biophysics, University of Mainz, 55099 Mainz, Germany. hdecker@uni-mainz.de Among type 3 copper proteins hemocyanins (Hc) serve as oxygen carrier, phenoloxidase (PO) comprising tyrosinase (Ty) and catecholoxidase (CO) are enzymes starting the biosynthesis of melanin being involved in innate immunity, wound healing, coloring of hair and eyes, browning of fruit and plants. We suggest molecular mechanisms for the activation and catalysis [1–5]. Our model explains why oxidation process turns hair grey [6]. Acknowledgment This work was granted by the DFG and EC. References 1. Decker H, Schweikardt T, Tuczek F (2006) Angew Chem Engl Ed 45:4546–4550 2. Baird S, Kelly SM, Price NC, Jaenicke E, Meesters C, Nillius D, Decker H, Nairn J (2007) Biochem Biophys Acta 1774:1380–1394 3. Chong Y, Ludtke SJ, Woolford DSA, Khant HA, Chiu W, Schweikardt T, Decker H (2009) submitted 4. Decker H, Schweikardt T, Nillius D, Salzbrunn U, Jaenicke E, Tuczek F (2007) Gene 398:183–191 5. Nillius D, Jaenicke E, Decker H (2008) FEBS Lett 582:749–754 6. Wood JM, Decker H, Hartmann H, Chavan B, Rokos H, Spencer JD, Hasse S, Thornton J, Shalbaf M, Paus R, Schallreuter KU (2009) FASEB J. doi:10.1096/fj.08-125435 D-42 Tyrosinase: mechanistic studies and new reactivity Luigi Casella Dip. Chimica Generale, Univ. Pavia, Via Taramelli 12, 27100 Pavia, Italy. bioinorg@unipv.it Tyrosinase is a ubiquitous dinuclear copper enzyme catalyzing the efficient oxidation of phenolic and catecholic substrates to quinones, and is therefore classified as an internal monooxygenase. The activity and mechanism of tyrosinase have been the focus of enzymatic and model studies in our laboratory for almost two decades [1]. A recent X-ray structure determination of the enzyme from S. castaneoglobisporus clarified important details about the dinuclear copper active site [2]. Important aspects of the enzyme reactivity remain not known, including the mode of substrate binding and dioxygen activation at the dinuclear copper center. According to biomimetic studies, three types of copper-dioxygen species are apparently suitable for the reaction: a lg2:g2-peroxidodicopper(II), a bis(l-oxido)dicopper(III), or a l-hydroperoxido-dicopper(II) species [1, 3]. Though, the activation parameters which characterize the biomimetic phenol hydroxylations are quite different from those we found for mushroom tyrosinase [4]. These data were obtained from enzymatic studies performed at variable S43 temperature in a mixed aqueous-organic solvent. Other recent developments enabled us to set up enzymatic and model studies where tyrosinase and dinuclear copper complexes perform the sulfoxidation of organic sulfides. In this case, the reaction requires the presence of a co-substrate, and therefore the enzymatic activity becomes equivalent to those of external monooxygenases like cytochrome P450 [5]. References 1. Battaini G et al. (2006) Adv Inorg Chem 58:185–233 2. Matoba Y et al. (2006) J Biol Chem 281:8981–8990 3. Lewis EA, Tolman WB (2004) Chem Rev 104:1047–1076 4. Granata A et al. (2006) Chem Eur J 12:2504–2514 5. Pievo R et al. (2008) Biochemistry 47:3493–3498 D-43 O2-activation at an asymmetric dicopper center Isaac Garcia-Bosch1, Anna Company1, Xavi Ribas, Miquel Costas 1 Department of Chemistry, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, 17071, Spain. Miquel.costas@udg.edu Understanding the intimate details of O2 binding/activation at copper sites is of interest because of the relevance of such reactions in biological and technological processes [1]. For the particular case of dicopper sites, three basic Cu2O2 core structures have been widely described [1]. CuII O O CuII CuII O O CuII CuII2(µ-η 2:η 2-O2) trans-CuII2(µ-η1:η1-O2) CuIII O O CuIII CuIII2(µ-O)2 Each specific Cu2O2 core determines particular spectroscopic and chemical properties [3]: while end-on trans-CuII2 (l-g1:g1-O2) species exhibit nucleophilic–basic behavior, side-on CuII2 (l-g2:g2-O2) and bisl-oxo dicopper(III) cores show an electrophilic–acid oxidant character. The rich and subtle chemistry exhibited by Cu2O2 cores makes asymmetric options interesting, but Cu2O2 species in systems containing distinct copper sites have been seldom observed [2], and a unique example of an asymmetric CuII2 -l-g1:g2-O2 core has been reported [3]. Herein we describe the O2 chemistry of a novel unsymmetric dicopper complex. The chemistry of the corresponding Cu2O2 species offer new insights that call into revision our current understanding of the mechanism of O2 activation at dicopper sites. References 1. Itoh S (2004) Comprehensive coordination chemistry II, vol 8. In: Que L Jr, Tolman WB (eds) Elsevier, Amsterdam, pp 369–393 2. Mirica LM, Ottenwaelder X, Stack TDP (2004) Chem Rev 114:1013 3. Lewis EA, Tolman WB (2004) Chem Rev 114:1047 4. Hatcher LQ, Karlin KD (2004) J Biol Inorg Chem 9:669 5. Battaini G, Granata A, Monzani E, Gullotti M, Casella L (2006) Adv Inorg Chem 58:185 6. Tachi Y, Aita K, Teramae S, Tani F, Naruta Y, Fukuzumi S, Itoh S (2004) Inorg Chem 43:4558 D-44 Tyrosinase reactivity in a model complexes: an alternative hydroxylation mechanism through a Cu(III)-bis-oxide intermediate Pratik Verma, Peng Kang, Liviu M. Mirica, Francois Cuenot, Michael Vance, Edward I. Solomon, T. Daniel P. Stack 123 S44 Department of Chemistry, Stanford University, California 94305, USA. stack@stanford.edu The dinuclear copper enzyme tyrosinase activates dioxygen to form a side-on peroxodicopper(II) complex, which is capable of oxidizing phenols to catechols. Several synthetic side-on peroxodicopper(II) complexes created with simple diamine ligands will be discussed that faithfully reproduce the spectrum of oxygenated tyrosinase, yet converted to Cu(III)-bis-oxide species upon phenolate addition at extreme temperatures (153 K). These species decay with hydroxylation of the aromatic ring by a mechanism that shares the hallmarks of an electrophilic aromatic substitution mechanism, as seen with the enzyme. DFT calculations on this system support strongly that the bis-m-oxodicopper(III) species can serve as the electrophilic agent in this oxidation. Overall, the evidence for sequential O–O bond cleavage and C–O bond formation suggests an alternative mechanism to the concerted or late-stage O–O scission generally accepted for phenol hydroxylation by tyrosinase. D-45 Reduction of dioxygen to water by the multicopper oxidases Edward I. Solomon Department of Chemistry, Stanford University, Stanford, CA, USA. Edward.Solomon@stanford.edu In nature the four electron reduction of O2 to H2O is carried out by Cytochrome c Oxidase (CcO) and the multicopper oxidases (MCOs). In the former, Cytochrome c provides electrons for pumping protons to produce a gradient for ATP synthesis, while in the MCOs the function is the oxidation of substrates, either organic or metal ions. In the MCOs the reduction of O2 is carried out at a trinuclear Cu cluster (TNC). Oxygen intermediates have been trapped which exhibit unique spectroscopic features that reflect novel geometric and electronic structures. These intermediates have both intact and cleaved O– O bonds, allowing the reductive cleavage of the O–O bond to be studied in detail both experimentally and computationally. These studies show that the topology of the TNC provides a unique geometric and electronic structure particularly suited to carry out this key reaction in nature. J Biol Inorg Chem (2009) 14 (Suppl 1):S33–S44 Reference 1. Solomon EI, Augustine AJ, Yoon J (2008) Reduction of O2 to H2O by the multicopper oxidases. Dalt Trans 30:3921–3932 D-46 Formation of a bridged butterfly dicopper core and its relevance to stepwise O2-activation in biological systems Yasuhiro Funahashi, Tomoaki Toyama, Kotaro Yoshii, Tomohide Nishikawa, Tomohiko Inomata, Tomohiro Ozawa, Hideki Masuda Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Nagoya 466-8555, Japan. funahashi.yasuhiro@nitech.ac.jp In biological systems, the active dimetal center acts for dioxygen uptake and activation. In type III copper proteins, dioxygen binds to the dicopper(II) center in the l-g2:g2-type side-on mode for its transportation and substrate-oxygenation, respectively. Recently, we succeeded in synthesizing a l-g2:g2-peroxodicopper(II) complex, [CuII2 (aSp)2 (l-g2:g2-O2)(Bz2)]SbF6 (aSp = a-isosparteine, Bz2 = benzoate), and revealed its crystal structure by X-ray diffraction analysis (Fig. 1) [1]. The new butterfly-type l-peroxo dicopper(II) complex can be readily formed by oxygenation of the copper(I) state in the presence of Bz2 or conversion of the corresponding bis(l-oxo)dicopper(III) species with axial coordination of Bz2 in organic solution at -80°C. These reactions may be related to the controlled stepwise reduction of dioxygen in non-heme diiron proteins and oxidation of water to evolve dioxygen in manganese cluster of photosystem II. Fig. 1 Structure of the ‘‘Bridged-Butterfly core’’ of a O2-binding dicopper(II) complex, 1 Reference 1. Funahashi Y, Nishikawa T, Wasada-Tsutsui Y, Kajita Y, Yamaguchi S, Arii H, Ozawa T, Jitsukawa K, Tosha T, Hirota S, Kitagawa T, Masuda H (2008) J Am Chem Soc 130:16444–16445 123