Takehiko Tosha

Publications of Takehiko Tosha

• Structural basis for nitrous oxide generation by bacterial nitric oxide reductases.

  Authors: Yoshitsugu Shiro, Hiroshi Sugimoto, Takehiko Tosha, Shingo Nagano, Tomoya Hino

  Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 05/2012; 367(1593):1195-203.

  The crystal structure of the bacterial nitric oxide reductase (cNOR) from Pseudomonas aeruginosa is reported. Its overall structure is similar to those of the main subunit of aerobic andThe crystal structure of the bacterial nitric oxide reductase (cNOR) from Pseudomonas aeruginosa is reported. Its overall structure is similar to those of the main subunit of aerobic and micro-aerobic cytochrome oxidases (COXs), in agreement with the hypothesis that all these enzymes are members of the haem-copper oxidase superfamily. However, substantial structural differences between cNOR and COX are observed in the catalytic centre and the delivery pathway of the catalytic protons, which should be reflected in functional differences between these respiratory enzymes. On the basis of the cNOR structure, we propose a possible reaction mechanism of nitric oxide reduction to nitrous oxide as a working hypothesis.

• Molecular structure and function of bacterial nitric oxide reductase.

  Authors: Tomoya Hino, Shingo Nagano, Hiroshi Sugimoto, Takehiko Tosha, Yoshitsugu Shiro

  Biochimica et biophysica acta. 04/2012; 1817(4):680-7.

  The crystal structure of the membrane-integrated nitric oxide reductase cNOR from Pseudomonas aeruginosa was determined. The smaller NorC subunit of cNOR is comprised of 1 trans-membrane helix and aThe crystal structure of the membrane-integrated nitric oxide reductase cNOR from Pseudomonas aeruginosa was determined. The smaller NorC subunit of cNOR is comprised of 1 trans-membrane helix and a hydrophilic domain, where the heme c is located, while the larger NorB subunit consists of 12 trans-membrane helices, which contain heme b and the catalytically active binuclear center (heme b(3) and non-heme Fe(B)). The roles of the 5 well-conserved glutamates in NOR are discussed, based on the recently solved structure. Glu211 and Glu280 appear to play an important role in the catalytic reduction of NO at the binuclear center by functioning as a terminal proton donor, while Glu215 probably contributes to the electro-negative environment of the catalytic center. Glu135, a ligand for Ca(2+) sandwiched between two heme propionates from heme b and b(3), and the nearby Glu138 appears to function as a structural factor in maintaining a protein conformation that is suitable for electron-coupled proton transfer from the periplasmic region to the active site. On the basis of these observations, the possible molecular mechanism for the reduction of NO by cNOR is discussed. This article is part of a Special Issue entitled: Respiratory Oxidases.

• Ferritin protein nanocage ion channels: gating by N-terminal extensions.

  Authors: Takehiko Tosha, Rabindra K Behera, Ho-Leung Ng, Onita Bhattasali, Tom Alber, Elizabeth C Theil

  The Journal of biological chemistry. 02/2012;

  Ferritin protein nanocages, self-assembled from 4-α helix bundle subunits, use Fe2+ and O to synthesize encapsulated, ferric oxide minerals. Ferritin minerals are iron concentrates stored for cellFerritin protein nanocages, self-assembled from 4-α helix bundle subunits, use Fe2+ and O to synthesize encapsulated, ferric oxide minerals. Ferritin minerals are iron concentrates stored for cell growth. Ferritins are also antioxidants, scavenging Fenton chemistry reactants. Channels for iron entry and exit consist of helical hairpin segments surrounding the three-fold symmetry axes of the ferritin nanocages. We now report structural differences caused by amino acid substitutions in the Fe2+ ion entry and exit channels and at the cytoplasmic pores, from high-resolution (1.3-1.8 angstroms) protein crystal structures of the eukaryotic model ferritin, frog M. Mutations that eliminate conserved ionic or hydrophobic interactions between R72-D122 and L110-L134 increase flexibility in the ion channels, cytoplasmic pores, and/or the N-terminal extensions of the helix bundles. Decreased ion binding in the channels and changes in ordered water are also observed. Protein structural changes coincide with increased Fe2+ exit from dissolved, ferric minerals inside ferritin protein cages; Fe2+ exit from ferritin cages depends on a complex, surface-limited process to reduce and dissolve the ferric mineral. High concentrations of bovine serum albumin or lysozyme (protein crowders) to mimic the cytoplasm, restored Fe2+ exit in the variants to wild-type. The data suggest that fluctuations in pore structure control gating. The newly identified role of the ferritin subunit N-terminal extensions in gating Fe2+ exit from the cytoplasmic pores strengthens the structural and functional analogies between ferritin ion channels in the water soluble protein assembly and membrane protein ion channels gated by cytoplasmic N-terminal peptides.

• Crystal structure of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus.

  Authors: Yushi Matsumoto, Takehiko Tosha, Andrei V Pisliakov, Tomoya Hino, Hiroshi Sugimoto, Shingo Nagano, Yuji Sugita, Yoshitsugu Shiro

  Nature structural & molecular biology. 02/2012; 19(2):238-45.

  The structure of quinol-dependent nitric oxide reductase (qNOR) from G. stearothermophilus, which catalyzes the reduction of NO to produce the major ozone-depleting gas N(2)O, has been characterizedThe structure of quinol-dependent nitric oxide reductase (qNOR) from G. stearothermophilus, which catalyzes the reduction of NO to produce the major ozone-depleting gas N(2)O, has been characterized at 2.5 Å resolution. The overall fold of qNOR is similar to that of cytochrome c-dependent NOR (cNOR), and some structural features that are characteristic of cNOR, such as the calcium binding site and hydrophilic cytochrome c domain, are observed in qNOR, even though it harbors no heme c. In contrast to cNOR, structure-based mutagenesis and molecular dynamics simulation studies of qNOR suggest that a water channel from the cytoplasm can serve as a proton transfer pathway for the catalytic reaction. Further structural comparison of qNOR with cNOR and aerobic and microaerobic respiratory oxidases elucidates their evolutionary relationship and possible functional conversions.

• Moving Iron through ferritin protein nanocages depends on residues throughout each four α-helix bundle subunit.

  Authors: Suranjana Haldar, Loes E Bevers, Takehiko Tosha, Elizabeth C Theil

  The Journal of biological chemistry. 05/2011; 286(29):25620-7.

  Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction andEukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe²⁺/O₂ oxidoreduction and formation of [Fe³⁺O](n) multimers within the protein cage, en route to the cavity, at sites distributed over ~50 Å. Recent NMR and Co²⁺-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe²⁺ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe³⁺O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A(650 nm)) and on mineral growth (Fe³⁺O-A(350 nm)), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe²⁺ access/selectivity to the active sites (Glu¹³⁰), (ii) distribution of Fe²⁺ to each of the three active sites near each ion channel (Asp¹²⁷), (iii) product (diferric oxo) release into the Fe³⁺O nucleation channels (Ala²⁶), and (iv) [Fe³⁺O](n) transit through subunits (Val⁴², Thr¹⁴⁹). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe²⁺ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits.

• Moving metal ions through ferritin-protein nanocages from three-fold pores to catalytic sites.

  Authors: Takehiko Tosha, Ho-Leung Ng, Onita Bhattasali, Tom Alber, Elizabeth C Theil

  Journal of the American Chemical Society. 10/2010; 132(41):14562-9.

  Ferritin nanocages synthesize ferric oxide minerals, containing hundreds to thousands of Fe(III) diferric oxo/hydroxo complexes, by reactions of Fe(II) ions with O(2) at multiple di-iron catalyticFerritin nanocages synthesize ferric oxide minerals, containing hundreds to thousands of Fe(III) diferric oxo/hydroxo complexes, by reactions of Fe(II) ions with O(2) at multiple di-iron catalytic centers. Ferric-oxy multimers, tetramers, and/or larger mineral nuclei form during postcatalytic transit through the protein cage, and mineral accretion occurs in the central cavity. We determined how Fe(II) substrates can access catalytic sites using frog M ferritins, active and inactivated by ligand substitution, crystallized with 2.0 M Mg(II) ± 0.1 M Co(II) for Co(II)-selective sites. Co(II) inhibited Fe(II) oxidation. High-resolution (<1.5 Å) crystal structures show (1) a line of metal ions, 15 Å long, which penetrates the cage and defines ion channels and internal pores to the nanocavity that link external pores to the cage interior, (2) metal ions near negatively charged residues at the channel exits and along the inner cavity surface that model Fe(II) transit to active sites, and (3) alternate side-chain conformations, absent in ferritins with catalysis eliminated by amino acid substitution, which support current models of protein dynamics and explain changes in Fe-Fe distances observed during catalysis. The new structural data identify a ∼27-Å path Fe(II) ions can follow through ferritin entry channels between external pores and the central cavity and along the cavity surface to the active sites where mineral synthesis begins. This "bucket brigade" for Fe(II) ion access to the ferritin catalytic sites not only increases understanding of biological nanomineral synthesis but also reveals unexpected design principles for protein cage-based catalysts and nanomaterials.

• CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers.

  Authors: Jennifer K Schwartz, Xiaofeng S Liu, Takehiko Tosha, Adrienne Diebold, Elizabeth C Theil, Edward I Solomon

  Biochemistry. 10/2010; 49(49):10516-25.

  DNA protection during starvation (Dps) proteins are miniferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites areDNA protection during starvation (Dps) proteins are miniferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O(2)/H(2)O(2), which accumulates in the large central cavity; for Dps, H(2)O(2) is the more common Fe(II) oxidant contrasting with eukaryotic maxiferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi- versus miniferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two Bacillus anthracis miniferritins: one in which two Fe(II) react with O(2) exclusively (Dps1) and a second in which both O(2) or H(2)O(2) can react with two Fe(II) (Dps2). Both result in the formation of iron oxybiomineral. The data show a single 5- or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30× more stable than Dps1; and the lower limit of K(D) for binding a second Fe(II), in the absence of oxidant, is 2-3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O(2). The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.

• Oxidation Reactivity of Bis(mu-oxo) Dinickel(III) Complexes: Arene Hydroxylation of the Supporting Ligand.

  Authors: Kaoru Honda, Jaeheung Cho, Takahiro Matsumoto, Jungyun Roh, Hideki Furutachi, Takehiko Tosha, Minoru Kubo, Shuhei Fujinami, Takashi Ogura, Teizo Kitagawa, Masatatsu Suzuki

  Angewandte Chemie (International ed. in English). 05/2009;

• Formation of a bridged butterfly-type mu-eta2:eta2-peroxo dicopper core structure with a carboxylate group.

  Authors: Yasuhiro Funahashi, Tomohide Nishikawa, Yuko Wasada-Tsutsui, Yuji Kajita, Syuhei Yamaguchi, Hidekazu Arii, Tomohiro Ozawa, Koichiro Jitsukawa, Takehiko Tosha, Shun Hirota, Teizo Kitagawa, Hideki Masuda

  Journal of the American Chemical Society. 01/2009; 130(49):16444-5.

• Formation of a Bridged Butterfly-Type mu-eta(2):eta(2)-Peroxo Dicopper Core Structure with a Carboxylate Group.

  Authors: Yasuhiro Funahashi, Tomohide Nishikawa, Yuko Wasada-Tsutsui, Yuji Kajita, Syuhei Yamaguchi, Hidekazu Arii, Tomohiro Ozawa, Koichiro Jitsukawa, Takehiko Tosha, Shun Hirota, Teizo Kitagawa, Hideki Masuda

  Journal of the American Chemical Society. 12/2008;

• The ferritin Fe2 site at the diiron catalytic center controls the reaction with O2 in the rapid mineralization pathway.

  Authors: Takehiko Tosha, Mohammad R Hasan, Elizabeth C Theil

  Proceedings of the National Academy of Sciences of the United States of America. 11/2008;

  Oxidoreduction in ferritin protein nanocages occurs at sites that bind two Fe(II) substrate ions and O(2), releasing Fe(III)(2)-O products, the biomineral precursors. Diferric peroxo intermediatesOxidoreduction in ferritin protein nanocages occurs at sites that bind two Fe(II) substrate ions and O(2), releasing Fe(III)(2)-O products, the biomineral precursors. Diferric peroxo intermediates form in ferritins and in the related diiron cofactor oxygenases. Cofactor iron is retained at diiron sites throughout catalysis, contrasting with ferritin. Four of the 6 active site residues are the same in ferritins and diiron oxygenases; ferritin-specific Gln(137) and variable Asp/Ser/Ala(140) substitute for Glu and His, respectively, in diiron cofactor active sites. To understand the selective functions of diiron substrate and diiron cofactor active site residues, we compared oxidoreductase activity in ferritin with diiron cofactor residues, Gln(137) --> Glu and Asp(140) --> His, to ferritin with natural diiron substrate site variations, Asp(140), Ser(140), or Ala(140). In Gln(137) --> Glu ferritin, diferric peroxo intermediates were undetectable; an altered Fe(III)-O product formed, DeltaA(350) = 50% of wild type. In Asp(140) --> His ferritin, diferric peroxo intermediates were also undetectable, and Fe(II) oxidation rates decreased 40-fold. Ferritin with Asp(140), Ser(140), or Ala(140) formed diferric peroxo intermediates with variable kinetic stabilities and rates: t(1/2) varied 1- to 10-fold; k(cat) varied approximately 2- to 3-fold. Thus, relatively small differences in diiron protein catalytic sites determine whether, and for how long, diferric peroxo intermediates form, and whether the Fe-active site bonds persist throughout the reaction cycle (diiron cofactors) or break to release Fe(III)(2)-O products (diiron substrates). The results and the coding similarities for cofactor and substrate site residues-e.g., Glu/Gln and His/Asp pairs share 2 of 3 nucleotides-illustrate the potential simplicity of evolving active sites for diiron cofactors or diiron substrates.

• Ferritin contains less iron (59Fe) in cells when the protein pores are unfolded by mutation.

  Authors: Mohammad R Hasan, Takehiko Tosha, Elizabeth C Theil

  The Journal of biological chemistry. 09/2008;

  Ferric minerals in ferritins are protected from cytoplasmic reductants and Fe2+ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two criticalFerric minerals in ferritins are protected from cytoplasmic reductants and Fe2+ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of 8 ferritin protein pores controls reactions between external reductant and the ferritin mineral; pore gating is altered by mutation, low heat and physiological urea (1 mM), monitored by CD-spectroscopy, protein crystallography and Fe release rates. To study effects of a ferritin pore gating mutation in living cells, we cloned/expressed human ferritin H and H L138P, homologous to the frog open pore model that was unexpressable in human cells. Human ferritin H L138P behaved like the open pore ferritin model in vitro: i. normal protein cage assembly and mineralization, ii. increased iron release (T1/2 decreased 17-fold), and iii. decreased -helix (8%). Overexpression (> 4-fold), in HeLa cells, showed for ferritin H L138P, equal protein expression and total cell 59Fe, but increased chelatable iron, 16%, P<0.01, (59Fe in the deferoxamine containing medium), and decreased 59Fe in ferritin, 28%, P<0.01, compared to wild type. The coincidence of decreased 59Fe in open pore ferritin with increased chelatable 59Fe in cells expressing the ferritin open pore mutation suggests that ferritin pore gating influences the amount of iron (59Fe) in ferritin in vivo.

• Spectroscopic definition of the ferroxidase site in M ferritin: comparison of binuclear substrate vs cofactor active sites.

  Authors: Jennifer K Schwartz, Xiaofeng S Liu, Takehiko Tosha, Elizabeth C Theil, Edward I Solomon

  Journal of the American Chemical Society. 08/2008; 130(29):9441-50.

  Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing freeMaxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a nonheme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly antiferromagnetically coupled, consistent with a mu-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure--including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin--coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites.

• Transient intermediates from Mn(salen) with sterically hindered mesityl groups: interconversion between MnIV-phenolate and MnIII-phenoxyl radicals as an origin for unique reactivity.

  Authors: Takuya Kurahashi, Akihiro Kikuchi, Takehiko Tosha, Yoshitsugu Shiro, Teizo Kitagawa, Hiroshi Fujii

  Inorganic chemistry. 04/2008; 47(5):1674-86.

  In order to reveal structure-reactivity relationships for the high catalytic activity of the epoxidation catalyst Mn(salen), transient intermediates are investigated. Steric hindrance incorporated toIn order to reveal structure-reactivity relationships for the high catalytic activity of the epoxidation catalyst Mn(salen), transient intermediates are investigated. Steric hindrance incorporated to the salen ligand enables highly selective generation of three related intermediates, OMnIV(salen), HO-Mn IV(salen), and H2O-MnIII(salen (+*)), each of which is thoroughly characterized using various spectroscopic techniques including UV-vis, electron paramagnetic resonance, resonance Raman, electrospray ionization mass spectrometry, 2H NMR, and X-ray absorption spectroscopy. These intermediates are all one-electron oxidized from the starting MnIII(salen) precursor but differ only in the degree of protonation. However, structural and electronic features are strikingly different: The Mn-O bond length of HO-MnIV(salen) (1.83 A) is considerably longer than that of OMnIV(salen) (1.58 A); the electronic configuration of H2O-MnIII(salen (+*)) is MnIII-phenoxyl radical, while those of OMnIV(salen) and HO-MnIV(salen) are MnIV-phenolate. Among OMnIV(salen), HO-MnIV(salen), and H2O-MnIII(salen (+*)), only the OMnIV(salen) can transfer oxygen to phosphine and sulfide substrates, as well as abstract hydrogen from weak C-H bonds, although the oxidizing power is not enough to epoxidize olefins. The high activity of Mn(salen) is a direct consequence of the favored formation of the reactive OMnIV(salen) state.

• GATED PORES IN THE FERRITIN PROTEIN NANOCAGE.

  Authors: Elizabeth C Theil, Xiaofeng S Liu, Takehiko Tosha

  Inorganica chimica acta. 04/2008; 361(4):868-874.

  Synopsis and pictogram: Gated pores in the ferritin family of protein nanocages, illustrated in the pictogram, control transfer of ferrous iron into and out of the cages by regulating contact betweenSynopsis and pictogram: Gated pores in the ferritin family of protein nanocages, illustrated in the pictogram, control transfer of ferrous iron into and out of the cages by regulating contact between hydrated ferric oxide mineral inside the protein cage, and reductants such as FMNH(2) on the outside. The structural and functional homology between the gated ion channel proteins in inaccessible membranes and gated ferritin pores in the stable, water soluble nanoprotein, make studies of ferritin pores models for gated pores in many ion channel proteins.Properties of ferritin gated pores, which control rates of FMNH(2) reduction of ferric iron in hydrated oxide minerals inside the protein nanocage, are discussed in terms of the conserved pore gate residues (arginine 72-apspartate 122 and leucine 110-leucine 134), of pore sensitivity to heat at temperatures 30 degrees C below that of the nanocage itself, and of pore sensitivity to physiological changes in urea (1-10 mM). Conditions which alter ferritin pore structure/function in solution, coupled with the high evolutionary conservation of the pore gates, suggest the presence of molecular regulators in vivo that recognize the pore gates and hold them either closed or open, depending on biological iron need. The apparent homology between ferrous ion transport through gated pores in the ferritin nanocage and ion transport through gated pores in ion channel proteins embedded in cell membranes, make studies of water soluble ferritin and the pore gating folding/unfolding a useful model for other gated pores.

• Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome P450 steroid 21-hydroxylase proved by Raman spectroscopy.

  Authors: Takehiko Tosha, Norio Kagawa, Miharu Arase, Michael R Waterman, Teizo Kitagawa

  The Journal of biological chemistry. 03/2008; 283(7):3708-17.

  We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone.We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone. The uncoupled H(2)O(2) formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17alpha-hydroxyprogesterone (15% of NADPH consumed), indicating that 17alpha-hydroxyprogesterone can better facilitate the O-O bond scission. In relation to this, it is noted that the O-O stretching mode (nu(O-O)) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17alpha-hydroxyprogesterone-bound enzyme gave single (at 1137 cm(-1)) or split nu(O-O) bands (at 1124 and 1138 cm(-1)), respectively, demonstrating the presence of two forms for the latter. In contrast to nu(O-O), no corresponding difference was observed for the Fe-O(2) stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm(-1)) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17alpha-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower nu(O-O) frequency with higher reactivity for O-O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17alpha-hydroxyprogesterone than in progesterone.

• Unique peroxidase reaction mechanism in prostaglandin endoperoxide H synthase-2: compound I in prostaglandin endoperoxide H synthase-2 can be formed without assistance by distal glutamine residue.

  Authors: Shizuo Ichimura, Takeshi Uchida, Shuhei Taniguchi, Shusuke Hira, Takehiko Tosha, Isao Morishima, Teizo Kitagawa, Koichiro Ishimori

  The Journal of biological chemistry. 06/2007; 282(22):16681-90.

  Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H2, but one of the highly conserved amino acid residues inProstaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H2, but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal glutamine (Gln-189) mutants, Arg (Gln-->Arg), Asn (Gln-->Asn), and Val (Gln-->Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574) suggested that the Gln residue might serve as a functionally equivalent residue to Arg, our current results clearly showed that the peroxidase activity of the Val and Asn mutants was comparable with that of the wild-type enzyme. In addition, the Fe-C and C-O stretching modes in the CO adduct were almost unperturbed by the mutation, implying that Gln-189 might not directly interact with the heme-ligated peroxide. Rather, the peroxidase activity of the Arg mutant was depressed, concomitant with the heme environmental change from a six-coordinate to a five-coordinate structure. Introduction of the bulky amino acid residue, Arg, would interfere with the ligation of a water molecule to the heme iron, suggesting that the side chain volume, and not the amide group, at position 189 is essential for the peroxidase activity of PGHS-2. Thus, we can conclude that the O-O bond cleavage in PGHS-2 is promoted without interactions with charged side chains at the peroxide binding site, which is significantly different from that in typical plant peroxidases.

• Synthesis, characterization, and reactivities of manganese(V)-oxo porphyrin complexes.

  Authors: Woon Ju Song, Mi Sook Seo, Serena DeBeer George, Takehiro Ohta, Rita Song, Min-Jung Kang, Takehiko Tosha, Teizo Kitagawa, Edward I Solomon, Wonwoo Nam

  Journal of the American Chemical Society. 03/2007; 129(5):1268-77.

  The reactions of manganese(III) porphyrin complexes with terminal oxidants, such as m-chloroperbenzoic acid, iodosylarenes, and H(2)O(2), produced high-valent manganese(V)-oxo porphyrins in theThe reactions of manganese(III) porphyrin complexes with terminal oxidants, such as m-chloroperbenzoic acid, iodosylarenes, and H(2)O(2), produced high-valent manganese(V)-oxo porphyrins in the presence of base in organic solvents at room temperature. The manganese(V)-oxo porphyrins have been characterized with various spectroscopic techniques, including UV-vis, EPR, 1H and 19F NMR, resonance Raman, and X-ray absorption spectroscopy. The combined spectroscopic results indicate that the manganese(V)-oxo porphyrins are diamagnetic low-spin (S = 0) species with a longer, weaker Mn-O bond than in previously reported Mn(V)-oxo complexes of non-porphyrin ligands. This is indicative of double-bond character between the manganese(V) ion and the oxygen atom and may be attributed to the presence of a trans axial ligand. The [(Porp)Mn(V)=O](+) species are stable in the presence of base at room temperature. The stability of the intermediates is dependent on base concentration. In the absence of base, (Porp)Mn(IV)=O is generated instead of the [(Porp)Mn(V)=O](+) species. The stability of the [(Porp)Mn(V)=O](+) species also depends on the electronic nature of the porphyrin ligands: [(Porp)Mn(V)=O](+) complexes bearing electron-deficient porphyrin ligands are more stable than those bearing electron-rich porphyrins. Reactivity studies of manganese(V)-oxo porphyrins revealed that the intermediates are capable of oxygenating PPh(3) and thioanisoles, but not olefins and alkanes at room temperature. These results indicate that the oxidizing power of [(Porp)Mn(V)=O](+) is low in the presence of base. However, when the [(Porp)Mn(V)=O](+) complexes were associated with iodosylbenzene in the presence of olefins and alkanes, high yields of oxygenated products were obtained in the catalytic olefin epoxidation and alkane hydroxylation reactions. Mechanistic aspects, such as oxygen exchange between [(Porp)Mn(V)=16O](+) and H(2)(18)O, are also discussed.

• Regioselective arene hydroxylation mediated by a (mu-peroxo)diiron(III) complex: a functional model for toluene monooxygenase.

  Authors: Mai Yamashita, Hideki Furutachi, Takehiko Tosha, Shuhei Fujinami, Wataru Saito, Yonezo Maeda, Kenji Takahashi, Koji Tanaka, Teizo Kitagawa, Masatatsu Suzuki

  Journal of the American Chemical Society. 02/2007; 129(1):2-3.

• Mononuclear copper(II)-hydroperoxo complex derived from reaction of copper(I) complex with dioxygen as a model of DbetaM and PHM.

  Authors: Tatsuya Fujii, Syuhei Yamaguchi, Yasuhiro Funahashi, Tomohiro Ozawa, Takehiko Tosha, Teizo Kitagawa, Hideki Masuda

  Chemical communications (Cambridge, England). 12/2006;

  A mononuclear copper(II)-hydroperoxo species has been generated by the reaction of Cu(I)-H2BPPA complex with dioxygen, which illustrates the enzymatic reaction process of the CuB site in the DbetaMA mononuclear copper(II)-hydroperoxo species has been generated by the reaction of Cu(I)-H2BPPA complex with dioxygen, which illustrates the enzymatic reaction process of the CuB site in the DbetaM and PHM.