當舎 武彦

Ferritin protein nanocages, self-assembled from four-alpha-helix bundle subunits, use Fe2+ and oxygen 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 3-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 angstrom) protein crystal structures of the eukaryotic model ferritin, frog M. Mutations that eliminate conserved ionic or hydrophobic interactions between Arg-72 and Asp-122 and between Leu-110 and Leu-134 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.

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 N2O, has been characterized at 2.5 angstrom 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.

Ferritins, ancient protein nanocages, reversibly synthesize hydrated ferric oxide concentrates; minerals with thousands of iron atoms grow in 8 nm cavities of the 12 nm cages of plant and animal ferritins. Cells use ferritin iron for iron-protein cofactor synthesis and as a trap for reactive iron from damaged iron-proteins. Recent ferritin structural studies show the iron entry path through iron ion channels, oxidoreductase sites and nucleation channels, a distance of similar to 5 nm from one end of the cage subunits (4 alpha-helix bundles) to the other. We now show that conserved L154, at the cavity entrance in a loop between helix 4 and a fifth short helix, slows mineral dissolution (50% mineral dissolution was >7 times faster in L154G ferritin). The effects on iron exit of leucine/glycine replacement an residue 154 at the end of iron entry path shows convergence of the iron entry and exit at L154 on the cage edge. The L154-dependent cage stabilization mechanism and the path that Fe(II) follows from the mineral surface to the ferritin protein are problems that remain unsolved in understanding the complex, eukaryotic ferritin protein cages that evolved for natural iron metabolism and are also used for imaging, nanocatalysis and nanomaterials.

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 are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O-2/H2O2, which accumulates in the large central cavity; for Dps, H2O2 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 H2O2 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 30x 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.

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 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 angstrom) crystal structures show (1) a line of metal ions, 15 angstrom 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 similar to 27-angstrom 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.

A new mu-eta(2):eta(2)-peroxo dicopper(II) complex, [Cu-2(II)(alpha SP)(2)(mu-eta(2):eta(2)-O-2) (Bz(.))]SbF6 (alpha Sp = alpha-isosparteine, Bz, = benzoate) was synthesized by oxygenation of [Cu-1(alpha Sp)(CH3CN)]SbF6 with Bz(.) at -80 degrees C in acetone. X-ray crystallographic analysis of the dark blue crystal revealed that the "bridged butterfly core" is formed by bridging Bz(.) bound in the axial position. The weaker sigma-electron donation of aSp causes the axial coordination of Bz(.) to stabilize the butterfly-type mu-eta(2):eta(2)-peroxo dicopper(II) species as an intermediate of stepwise O-2-activation to bis(mu-oxo) dicopper(III) species.

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 critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of eight 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) and monitored by CD spectroscopy, protein crystallography, and Fe2+ release rates. To study the 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 as follows: (i) normal protein cage assembly and mineralization, (ii) increased iron release (t(1/2) decreased 17-fold), and (iii) decreased alpha-helix (8%). Overexpression (>4-fold), in HeLa cells, showed for ferritin H L138P equal protein expression and total cell Fe-59 but increased chelatable iron, 16%, p < 0.01 (Fe-59 in the deferoxamine-containing medium), and decreased Fe-59 in ferritin, 28%, p < 0.01, compared with wild type. The coincidence of decreased Fe-59 in open pore ferritin with increased chelatable Fe-59 in cells expressing the ferritin open pore mutation suggests that ferritin pore gating influences to the amount of iron (Fe-59) in ferritin in vivo.

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 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, Delta A(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.

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 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.

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 to the salen ligand enables highly selective generation of three related intermediates, O = Mn-IV(salen), HO-Mn-IV(salen), and H2O-Mn-III(salen(+center dot)), each of which is thoroughly characterized using various spectroscopic techniques including UV-vis, electron paramagnetic resonance, resonance Raman, electrospray ionization mass spectrometry, H-2 NMR, and X-ray absorption spectroscopy. These intermediates are all one-electron oxidized from the starting Mn-III(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-Mn-IV(salen) (1.83 angstrom) is considerably longer than that of O = Mn-IV(salen) (1.58 angstrom); the electronic configuration of H2O-Mn-III(salen(+center dot)) is Mn-III-phenoxyl radical, while those of O = Mn-IV(salen) and HO-Mn-IV(salen) are Mn-IV-phenolate. Among O = Mn-IV(salen), HO-Mn-IV(salen), and H2O-Mn-III(salen(+center dot)), only the O = Mn-IV(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 epoxiclize olefins. The high activity of Mn(salen) is a direct consequence of the favored formation of the reactive O = Mn-IV(salen) state.

Properties of ferritin gated pores control rates of FMNH2 reduction of ferric iron in hydrated oxide minerals inside the protein nanocage, and are discussed in terms of: ( 1) the conserved pore gate residues ( ion pairs: arginine 72, aspartate 122, and a hydrophobic pair, leucine 110 - leucine 134), ( 2) pore sensitivity to heat at temperatures 30 degrees C below that of the nanocage itself, and ( 3) 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. (c) 2007 Elsevier B. V. All rights reserved.

We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17 alpha-hydroxyprogesterone. The uncoupled H2O2 formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17 alpha-hydroxyprogesterone (15% of NADPH consumed), indicating that 17 alpha-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 17 alpha-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 17 alpha-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 17 alpha-hydroxyprogesterone than in progesterone.

Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H-2, 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 ( Gln3Asn), 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.

The reactions of manganese(III) porphyrin complexes with terminal oxidants, such as m-chloroperbenzoic acid, iodosylarenes, and H2O2, 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, H-1 and F-19 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)(MnO)-O-V](+) 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)(MnO)-O-IV is generated instead of the [(Porp)(MnO)-O-V](+) species. The stability of the [(Porp)(MnO)-O-V](+) species also depends on the electronic nature of the porphyrin ligands: [(Porp)(MnO)-O-V](+) 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 PPh3 and thioanisoles, but not olefins and alkanes at room temperature. These results indicate that the oxidizing power of [(Porp)(MnO)-O-V](+) is low in the presence of base. However, when the [(Porp)(MnO)-O-V](+) 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)(MnO)-O-V16](+) and (H2O)-O-18, are also discussed.

A heme domain of coral allene oxide synthase (cAOS) catalyzes the formation of allene oxide from fatty acid hydroperoxide. Although cAOS has a similar heme active site to that of catalase, cAOS is completely lacking in catalase activity. A close look at the hydrogen-bonding possibilities around the distal His in cAOS suggested that the imidazole ring is rotated by 180 degrees relative to that of catalase because of the hydrogen bond between Thr-66 and the distal His-67. This could contribute to the functional differences between cAOS and catalase, and to examine this possibility, we mutated Thr-66 in cAOS to Val, the corresponding residue in catalase. In contrast to the complete absence of catalase activity in wild type (WT) cAOS, T66V had a modest catalase activity. On the other hand, the mutation suppressed the native enzymatic activity of the formation of allene oxide to 14% of that of WT cAOS. In the resonance Raman spectrum, whereas WT cAOS has only a 6-coordinate/high spin heme, T66V has a 5-coordinate/ high spin heme as a minor species. Because catalase adopts a 5-coordinate/ high spin structure, probably the 5-coordinate/ high spin portion of T66V showed the catalase activity. Furthermore, in accord with the fact that the CN affinity of catalase is higher than that of WTc AOS, the CN affinity of T66V was 8-fold higher than that of WT cAOS, indicating that the mutation could mimic the heme active site in catalase. We, therefore, propose that the hydrogen bond between Thr-66 and distal His-67 could modulate the orientation of distal His, thereby regulating the enzymatic activity in cAOS.

Specific substrate-induced structural changes in the heme pocket are proposed for human cytochrome P450 aromatase (P450arom) which undergoes three consecutive oxygen activation steps. We have experimentally investigated this heme environment by resonance Raman spectra of both substrate-free and substrate-bound forms of the purified enzyme. The Fe-CO stretching mode (v(Fe)-(CO)) of the CO complex and Fe3+-S stretching mode (v(Fe-S)) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The v(Fe-CO) mode was upshifted from 477 to 485 and to 490 cm(-1) by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas v(Fe-CO) was not observed for P450arom with 19-hydroxyandrostenedione, a substrate for the second step, indicating that the heme distal site is very flexible and changes its structure depending on the substrate. The 19-aldehyde-androstenedione binding could reduce the electron donation from the axial thiolate, which was evident from the low-frequency shift of v(Fe-s) by 5 cm(-1) compared to that of androstenedione-bound P450arom. Changes in the environment in the heme distal site and the reduced electron donation from the axial thiolate upon 19-aldehyde-androstenedione binding might stabilize the ferric peroxo species, an active intermediate for the third step, with the suppression of the formation of compound I (Fe4+=O porphyrin(+center dot)) that is the active species for the first and second steps. We, therefore, propose that the substrates can regulate the formation of alternative reaction intermediates by modulating the structure on both the heme distal and proximal sites in P450arom.

Mononuclear nonheme oxoiron(IV) complexes bearing 15-membered macrocyclic ligands were generated from the reactions of their corresponding iron(II) complexes and iodosylbenzene (PhIO) in CH3CN. The oxoiron(IV) species were characterized with various spectroscopic techniques such as UV-vis spectrophotometer, electron paramagnetic resonance, electrospray ionization mass spectrometer, and resonance Raman spectroscopy. The oxoiron(IV) complexes were inactive in olefin epoxidation. In contrast, when iron(II) or oxoiron(IV) complexes were combined with PhIO in the presence of olefins, high yields of epoxide products were obtained. These results indicate that in addition to the oxoiron(IV) species, there must be at least one more active oxidant (e.g., Fe-IV-OIPh adduct or oxoiron(V) species) that effects the olefin epoxidation. We have also demonstrated that the ligand environment of iron catalysts is an important factor in controlling the catalytic activity as well as the product selectivity in the epoxidation of olefins by PhIO. (c) 2006 Elsevier Inc. All rights reserved.

O-17 NMR spectroscopy of oxo ligand of oxo metalloporphyrin can be considered as an excellent means to derive information about structure, electronic state, and reactivity of the metal bound oxo ligand. To show the Utility Of O-17 NMR spectroscopy of oxo ligand of oxo metalloporphyrin. O-17 NMR spectra of oxo ligands of dioxo ruthenium(VI), oxo chromium(IV), and oxo titanium(IV) porphyrins are measured. For all oxo metalloporphyrins, well-resolved O-17 NMR signals are detected in far high frequency region. The O-17 NMR signal of the metal bound oxo ligand shifts high frequency in order of Ru(VI) < Ti(IV) < Cr(IV), thus the O-17 NMR chemical shift does not directly correlate with the oxo-transfer reactivity, Ti(IV) < Cr(IV) < Ru(VI). On the other hand, the O-17 NMR shift of oxo ligand correlates with the bond strength of metal-oxo bond. This suggests that the O-17 NMR signal of metal bound oxo ligand is a sensitive probe to study the nature of metal-oxo bond in oxo metalloporphyrin. The effect of the electron-withdrawing meso-substituent on the O-17 NMR shift of the oxo ligand is also investigated. With increase in the electron-withdrawing effect of the meso-substituent, the O-17 NMR signal of the oxo ligand of oxo chromium(IV) porphyrin shifts high frequency while that or dioxo ruthenium(VI) porphyrin hardly change resonance position. The changes in metal-oxo bonds induced by the electron-withdrawing meso-substituent are discussed on the basis of the O-17 NMR shifts, the strengths of the metal-oxo bonds, and the oxo-transfer reaction rates. (c) 2006 Elsevier Inc. All rights reserved.

A mononuclear copper(II)-hydroperoxo species has been generated by the reaction of Cu(I)-H(2)BPPA complex with dioxygen, which illustrates the enzymatic reaction process of the Cu-B site in the D beta M and PHM.

Oxidizing intermediates are generated from nonheme iron(III) complexes to investigate the electronic structure and the reactivity, in comparison with the oxoiron(IV) porphyrin pi-cation radical (compound 1) as a heme enzyme model. Sterically hindered iron salen complexes, bearing a fifth ligand Cl (1), OH2 (2), OEt (3), and OH (4), are oxidized both electrochemically and chemically. Stepwise one-electron oxidation of 1 and 2 generates iron(Ill)mono- and diphenoxyl radicals, as revealed by detailed spectroscopic investigations, including UV-vis, EPR, Mossbauer, resonance Raman, and ESIMS spectroscopies. In contrast to the oxoiron(IV) formation from the hydroxoiron(III) porphyrin upon one-electron oxidation, the hydroxo complex 4 does not generate oxoiron(IV) species. Reaction of 2 with mCPBA also results in the formation of the iron(Ill)-phenoxyl radical. One-electron oxidation of 3 leads to oxidative degradation of the fifth DO ligand to liberate acetaldehyde even at 203 K. The iron(Ill)phenoxyl radical shows high reactivity for alcoxide on iron(III) but exhibits virtually no reactivity for alcohols including even benzyl alcohol without a base to remove an alcohol proton. This study explains unique properties of mononuclear nonheme enzymes with Tyr residues and also the poor epoxidation activity of Fe salen compared to Mn and Cr salen compounds.

A new tetradentate tripodal ligand (L3) containing sterically bulky imidazolyl groups was synthesized, where L3 is tris(1-methyl-2-phenyl-4-imidazolylmethyl)amine. Reaction of a bis(mu-hydroxo)dicopper(II) complex, [Cu-2(L3)(2)(OH)(2)](2+) (1), with H2O2 in acetonitrile at -40 degrees C generated a (mu-1,1-hydroperoxo)dicopper(II) complex [Cu-2(L3)(2)(OOH)(OH)](2+) (2), which was characterized by various physicochemical measurements including X-ray crystallography. The crystal structure of 2 revealed that the complex cation has a Cu-2(mu-1, 1-OOH)(mu-OH) core and each copper has a square pyramidal structure having an N3O2 donor set with a weak ligation of a tertiary amine nitrogen in the apex. Consequently, one pendant arm of L3 in 2 is free from coordination, which produces a hydrophobic cavity around the Cu-2(mu-1,1-OOH)(mu-OH) core. The hydrophobic cavity is preserved by hydrogen bondings between the hydroperoxide and the imidazole nitrogen of an uncoordinated pendant arm in one side and the hydroxide and the imidazole nitrogen of an uncoordinated pendant arm in the other side. The hydrophobic cavity significantly suppresses the H/D and O-16/O-18 exchange reactions in 2 compared to that in 1 and stabilizes the Cu-2(mu-1, 1-OOH)(mu-OH) core against decomposition. Decomposition of 2 in acetonitrile at 0 degrees C proceeded mainly via disproportionation of the hydroperoxo ligand and reduction of 2 to [Cu(L3)](+) by hydroperoxo ligand. In contrast, decomposition of a solid sample of 2 at 60 degrees C gave a complex having a hydroxylated ligand [Cu-2(L3)(L3-OH)(OH)(2)](2+) (2-(L3-OH)) as a main product, where L3-OH is an oxidized ligand in which one of the methylene groups of the pendant arms is hydroxylated. ESI-TOF/MS measurement showed that complex 2-(L3-OH) is stable in acetonitrile at -40 degrees C, whereas warming 2-(L3-OH) at room temperature resulted in the N-dealkylation from L3-OH to give an N-dealkylated ligand, bis(1-methyl-2-phenyl-4-imidazolylmethyl)amine (L2) in similar to 80% yield based on 2, and 1-methyl-2-phenyl-4-formylimidazole (PhimCHO) Isotope labeling experiments confirmed that the oxygen atom in both L3-OH and Phim-CHO come from OOH. This aliphatic hydroxylation performed by 2 is in marked contrast to the arene hydroxylation reported for some (mu-1,1-hydroperoxo)dicopper(II) complexes with a xylyl linker.

To gain insights into the molecular basis of the design for the selective azole anti-fungals, we compared the binding properties of azole-based inhibitors for cytochrome P450 sterol 14alpha-demethylase (CYP51) from human (HuCYP51) and Mycobacterium tuberculosis (MtCYP51). Spectroscopic titration of azoles to the CYP51s revealed that HuCYP51 has higher affinity for ketoconazole (KET), an azole derivative that has long lipophilic groups, than MtCYP51, but the affinity for fluconazole (FLU), which is a member of the anti-fungal armamentarium, was lower in HuCYP51. The affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 was quite low compared with that to HuCYP51. In the resonance Raman spectra for HuCYP51, the FLU binding induced only minor spectral changes, whereas the prominent high frequency shift of the bending mode of the heme vinyl group was detected in the KET- or 4-PhIm-bound forms. On the other hand, the bending mode of the heme propionate group for the FLU-bound form of MtCYP51 was shifted to high frequency as found for the KET- bound form, but that for 4-PhIm was shifted to low frequency. The EPR spectra for 4-PhIm-bound MtCYP51 and FLU-bound HuCYP51 gave multiple g values, showing heterogeneous binding of the azoles, whereas the single g(x) and g(z) values were observed for other azole-bound forms. Together with the alignment of the amino acid sequence, these spectroscopic differences suggest that the region between the B' and C helices, particularly the hydrophobicity of the C helix, in CYP51s plays primary roles in determining strength of interactions with azoles; this differentiates the binding specificity of azoles to CYP51s.

The structure and dioxygen-reactivity of copper(I) complexes 2(R) supported by N,N-bis(6-methylpyridin-2-ylmethyl)amine tridentate ligands L2(R) [R (N-alkyl substituent) = - CH2Ph (Bn), - CH2CH2Ph (Phe) and - CH2CHPh2 (PhePh)] have been examined and compared with those of copper(I) complex 1(Phe) of N,N-bis[2-(pyridin-2-yl) ethyl] amine tridentate ligand L1(Phe) and copper( I) complex 3(Phe) of N,N-bis( pyridin-2-ylmethyl) amine tridentate ligand L3(Phe). Copper( I) complexes 2(Phe) and 2(PhePh) exhibited a distorted trigonal pyramidal structure involving a d-pi interaction with an eta(1)-binding mode between the metal ion and one of the ortho-carbon atoms of the phenyl group of the N-alkyl substituent [ - CH2CH2Ph (Phe) and - CH2CHPh2 (PhePh)]. The strength of the d-pi interaction in 2(Phe) and 2(PhePh) was weaker than that of the d-pi interaction with an eta(2)-binding mode in 1(Phe) but stronger than that of the eta(1) d - pi interaction in 3(Phe). Existence of a weak d - pi interaction in 2(Bn) in solution was also explored, but its binding mode was not clear. Redox potentials of the copper(I) complexes (E-1/2) were also affected by the supporting ligand; the order of E-1/2 was 1(Phe) > 2(R) > 3(Phe). Thus, the order of electron-donor ability of the ligand is L1(Phe) < L2(R) < L3(Phe). This was reflected in the copper( I) - dioxygen reactivity, where the reaction rate of copper(I) complex toward O-2 dramatically increased in the order of 1(R) < 2(R) < 3(R). The structure of the resulting Cu-2/O-2 intermediate was also altered by the supporting ligand. Namely, oxygenation of copper( I) complex 2(R) at a low temperature gave a (mu-eta(2):eta(2)-peroxo) dicopper(II) complex as in the case of 1(Phe), but its O - O bond was relatively weakened as compared to the peroxo complex derived from 1(Phe), and a small amount of a bis(mu-oxo) dicopper(III) complex co-existed. These results can be attributed to the higher electron-donor ability of L2(R) as compared to that of L1(Phe). On the other hand, the fact that 3(Phe) mainly afforded a bis(mu-oxo) dicopper(III) complex suggests that the electron-donor ability of L2(R) is not high enough to support the higher oxidation state of copper(III) of the bis(mu-oxo) complex.

The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata- Ariga, Y., Egawa, T., Suematsu, M., Park, S.- Y., Adachi, S., Shiro, Y., and Ishimura, Y. ( 2003) Biochemistry 42, 14507 - 14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying H-1 NMR paper ( Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. ( 2004) J. Biol. Chem. 279, 42836 - 42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous ( Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with nonphysiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.

To investigate the functional and structural characterization of a crucial cytochrome P450cam ( P450cam)putidaredoxin (Pdx) complex, we utilized a mutant whose spectroscopic property corresponds to the properties of the wild type P450cam in the presence of Pdx. The H-1 NMR spectrum of the carbonmonoxy adduct of the mutant, the Leu- 358 --> Pro mutant (L358P), in the absence of Pdx showed that the ring current-shifted signals arising from D-camphor were upfield-shifted and observed as resolved signals, which are typical for the wild type enzyme in the presence of Pdx. Signals from the beta-proton of the axial cysteine and the gamma-methyl group of Thr-252 were also shifted upfield and downfield, respectively, in the L358P mutant as observed for Pdx-bound wild type P450cam. The close similarity in the NMR spectra suggests that the heme environment of the L358P mutant mimics that of the Pdx-bound enzyme. The functional analysis of the L358P mutant has revealed that the oxygen adduct of the L358P mutant can promote the oxygenation reaction for D-camphor with nonphysiological electron donors such as dithionite and ascorbic acid, showing that oxygenated L358P is "activated" to receive electron from the donor. Based on the structural and functional characterization of the L358P mutant, we conclude that the Pdx-induced structural changes in P450cam would facilitate the electron transfer from the electron donor, and the Pdx binding to P450cam would be a trigger for the electron transfer to oxygenated P450cam.

To elucidate molecular mechanisms for the enhanced oxygenation activity in the three mutants of cytochrome P450cam screened by 'laboratory evolution' [Nature 399 (1999) 670], we purified the mutants and characterized their functional and structural properties. The electronic absorption and resonance Raman spectra revealed that the structures of heme binding site of all purified mutants were quite similar to that of the wild-type enzyme, although the fraction of the inactivated form, called "P420," was increased. In the reaction with H2O2, only trace amounts of the naphthalene hydroxylation product were detected by gas chromatography. We, therefore, conclude that the three mutants do not exhibit significant changes in the structural and functional properties from those of wild-type P450cam except for the stability of the axial ligand in the reduced form. The enhanced fluorescence in the whole-cell assay would reflect enhancement in the oxygenation activity below the detectable limit of the gas chromatography and/or contributions of other reactions catalyzed by the heme iron. (C) 2004 Elsevier Inc. All rights reserved.

We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the beta-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 Angstrom. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo- and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15-0.7 Angstrom. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the gamma-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by similar to0.25 Angstrom. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction.

Structural and functional roles of the hydrogen bonding network that surrounds the heme-thiolate coordination of P450(cam) from Pseudomonas putida were investigated. A hydrogen bond between the side chain amide of Gln360 and the carbonyl oxygen of the axial Cys357 was removed in Q360L. The side chain hydrogen bond and the electrostatic interaction between the polypeptide amide proton of Gln360 and the sulfur atom of Cys357 were simultaneously removed in Q360P. The increased electron donation of the axial thiolate in Q360L and Q360P was evidenced by negative shifts of their reduction potentials by 45 and 70 mV, respectively. Together with the results on L358P in which the amide proton at position 358 was removed (Yoshioka, S., Takahashi, S., Ishimori, K., Morishima, I.J. Inorg. Biochem. 2000, 81, 141-151), we propose that the side chain hydrogen bond and the electrostatic interaction of the amide proton with the thiolate ligand cause similar to45 and similar to35 mV of positive shifts, respectively, of the redox potential of the heme in P450(cam). The resonance Raman spectra of the ferrous-CO form of the Q360 mutants showed a downshifted Fe-CO stretching mode at 482similar to483 cm(-1) compared with that of wild-type P450(cam) at 484 cm(-1). The Q360 mutants also showed the upshift by 4similar to5 cm-1 of the Fe-NO stretching mode in the ferrous-NO form. These Raman results indicate the increase in the sigma-electron donation of the thiolate ligand in the reduced state of the 0360 mutants and were in contrast to the increased pi-back-do nation of the thiolate in L358P having an upshifted Fe-CO stretching mode at 489 cm(-1). The catalytic activities of the Q360 mutants for the unnatural substrates were similar to those of the wild-type enzyme, indicating that the increased sigma-electron donation does not promote the O-O bond heterolysis in the Q360 mutants, although the increased pi-electron donation in L358P promoted the heterolysis of the O-O bond. We conclude that the functions of the proximal hydrogen bonding network in P450(cam) are to stabilize the heme-thiolate coordination, and to regulate the redox potential of the heme iron. Furthermore, we propose that the pi-electron donation, not the sigma-electron donation, of the thiolate ligand promotes the heterolysis of the O-O bond of dioxygen.

We characterized electron transfer (ET) from putidaredoxin (Pdx) to the mutants of cytochrome P450(cam) (P450(cam)), in which one of the residues located on the putative binding site to Pdx, Gln360, was replaced with Glu, Lys, and Len. The kinetic analysis of the ET reactions from reduced Pdx to ferric P450(cam) (the first ET) and to ferrous oxygenated P450(cam) (the second ET) showed the dissociation constants (K) that were moderately perturbed for the Lys and Len mutants and the distinctly increased for the Glu mutant. Although the alterations in K-m indicate that Gln360 is located at the Pdx binding site, the effects of the Gln360 mutations (0.66-20-fold of that of wild type) are smaller than those of the Arg112 mutants (25-2500-fold of that of wild type) [Unno, M., et al. (1996) J. Biol. Chem. 271, 17869-17874], allowing us to conclude that Gln360 much less contributes to the complexation with Pdx than Arg112. The first ET rate (35 s(-1) for wild-type P450(cam)) was substantially reduced in the Ght mutant (5.4 s(-1)), while less perturbation was observed for the Lys (53 s(-1)) and Leu (23 s(-1)) mutants. In the second ET reaction, the retarded ET rate was detected only in the Ght mutant but not in the Lys and Len mutants. These results showed the smaller mutational effects of Gln360 on the ET reactions than those of the Arg112 mutants. In contrast to the moderate perturbations in the kinetic parameters, the mutations at Gln360 significantly affected both the standard enthalpy and entropy of the redox reaction of P450(cam), which cause the negative shift of the redox potentials for the Fe3+/Fe2+ couple by 20-70 mV. Since the arnide group of Gln360 is located near the carbonyl oxygen of the amide group of the axial cysteine, it is plausible that the mutation at Gln360 perturbs the electronic interaction of the axial ligand with heme iron, resulting in the reduction of the redox potentials. We, therefore, conclude that Gln360 primarily regulates the ET reaction of P450cam by modulating the redox potential of the heme iron and not by the specific interaction with Pdx or the formation of the ET pathway that are proposed as the regulation mechanism of Arg 112.