Heme-copper/dioxygen adduct formation relevant to cytochrome c oxidase: spectroscopic characterization of [(6L)FeIII-(O2 2−)-CuII]+

In the further development and understanding of heme-copper dioxygen reactivity relevant to cytochrome c oxidase O(2)-reduction chemistry, we describe a high-spin, five-coordinate dioxygen (peroxo) adduct of an iron(II)-copper(I) complex, [((6)L)Fe(II)Cu(I)](BArF(20)) (1), where (6)L is a tetraarylporphyrinate with a tethered tris(2-pyridylmethyl)amine chelate for copper. Reaction of 1 with O(2) in MeCN affords a remarkably stable [t(1/2) (rt; MeCN) approximately 60 min] adduct, [((6)L)Fe(III)-(O(2) (2-))-Cu(II)](+) (2) [EPR silent; lambda(max)=418 (Soret), 561 nm], formulated as a peroxo complex based on manometry (1:O(2)=1:1; spectrophotometric titration, -40 degrees C, MeCN), mass spectrometry {MALDI-TOF-MS: (16)O(2), m/z 1191 ([((6)L)Fe(III)-((16)O(2) (2-))-Cu(II)](+)); (18)O(2), m/z 1195}, and resonance Raman spectroscopy (nu((O-O))=788 cm(-1); Delta(16)O(2)/(18)O(2)=44 cm(-1); Delta(16)O(2)/(16/18)O(2)=22 cm(-1)). (1)H and (2)H NMR spectroscopy (-40 degrees C, MeCN) reveals that 2 is the first heme-copper peroxo complex which is high-spin, with downfield-shifted pyrrole resonances (delta(pyrrole)=75 ppm, s, br) and upfield shifted peaks at delta= -22, -35, and -40 ppm, similar to the pattern observed for the mu-oxo complex [((6)L)Fe(III)-O-Cu(II)](BAr(F)) (3) (known S=2 system, antiferromagnetically coupled high-spin Fe(III) and Cu(II)). The corresponding magnetic moment measurement (Evans method, CD(3)CN, -40 degrees C) also confirms the S=2 spin state, with mu(B)=4.9. Structural insights were obtained from X-ray absorption spectroscopy, showing Fe-O (1.83 A) and Cu-O (1.882 A) bonds, and an Fe...Cu distance of 3.35(2) A, suggestive of a mu-1,2-peroxo ligand present in 2. The reaction of 2 with cobaltocene gives 3, differing from the observed full reduction seen with other heme-Cu peroxo complexes. Finally, thermal decomposition of 2 yields 3, with concomitant release of 0.5 mol O(2) per mol 2, as confirmed quantitatively by an alkaline pyrogallol dioxygen scavenging solution.     

Resonance raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide

Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O(2) by quinols and simultaneously translocates protons across the cytoplasmic membrane. The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system. Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm(-)(1) for intermediates derived from H(2)(16)O(2)/H(2)(18)O(2). The experiments using H(2)(16)O(18)O yielded no new bands, indicating that all the bands arose from the Fe=O stretching (nu(Fe)(=)(O)) mode. Among them, the intensity of the 805/X cm(-)(1) pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome bo appeared prior to the formation of the F species at higher pH. For this intermediate, a Raman band assignable to the C-O stretching mode of a tyrosyl radical was deduced at 1489 cm(-)(1) from difference spectra. This suggests that the P intermediate of cytochrome bo contains an Fe(IV)=O heme and a tyrosyl radical like compound I of prostaglandin H synthase. The 783/753 cm(-)(1) pair, which was dominant at neutral pH and close to the nu(Fe)(=)(O) frequency of the oxoferryl intermediate of CcO, presumably arises from the F intermediate. On the contrary, the (767)/730 cm(-)(1) species has no counterpart in CcO. Its presence may support the branched reaction scheme proposed previously for O(2) reduction by cytochrome bo.     

Substrate-specific interactions with the heme-bound oxygen molecule of nitric-oxide synthase

We report the characterization by resonance Raman spectroscopy of the oxygenated complex (Fe(II)O(2)) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with N(omega)-hydroxy-l-arginine. The frequencies of the nu(Fe-O) and nu(O-O) modes were 530 and 1135 cm(-), respectively, in both the presence and absence of tetrahydrobiopterin. On the basis of a comparison of these frequencies with those of saNOS and bsNOS saturated with l-arginine (nu(Fe-O) at 517 cm(-1) and nu(O-O) at 1123 cm(-1)) and those of substrate-free saNOS (nu(Fe-O) at 517 and nu(O-O) at 1135 cm(-1)) (Chartier, F. J. M., Blais, S. P., and Couture, M. (2006) J. Biol. Chem. 281, 9953-9962), we propose two models that account for the frequency shift of nu(Fe-O) (but not nu(O-O)) upon N(omega)-hydroxy-l-arginine binding as well as the frequency shift of nu(O-O) (but not nu(Fe-O)) upon l-arginine binding. The implications of these substrate-specific interactions with respect to catalysis by NOSs are discussed.     

Resonance Raman characterization of the P intermediate in the reaction of bovine cytochrome c oxidase

Reduced cytochrome c oxidase binds molecular oxygen, yielding an oxygenated intermediate first (Oxy) and then converts it to water via the reaction intermediates of P, F, and O in the order of appearance. We have determined the iron-oxygen stretching frequencies for all the intermediates by using time-resolved resonance Raman spectroscopy. The bound dioxygen in Oxy does not form a bridged structure with Cu(B) and the rate of the reaction from Oxy to P (P(R)) is slower at higher pH in the pH range between 6.8 and 8.0. It was established that the P intermediate has an oxo-heme and definitely not the Fe(a(3))-O-O-Cu(B) peroxy bridged structure. The Fe(a(3))=O stretching (nu(Fe=O)) frequency of the P(R) intermediate, 804/764 cm(-1) for (16)O/(18)O, is distinctly higher than that of F intermediate, 785/750 cm(-1). The rate of reaction from P to F in D(2)O solution is evidently slower than that in H(2)O solution, implicating the coupling of the electron transfer with vector proton transfer in this process. The P intermediate (607-nm form) generated in the reaction of oxidized enzyme with H(2)O(2) gave the nu(Fe=O) band at 803/769 cm(-1) for H(2)(16)O(2)/H(2)(18)O(2) and the simultaneously measured absorption spectrum exhibited the difference peak at 607 nm. Reaction of the mixed valence CO adduct with O(2) provided the P intermediate (P(M)) giving rise to an absorption peak at 607 nm and the nu(Fe=O) bands at 804/768 cm(-1). Thus, three kinds of P intermediates are considered to have the same oxo-heme a(3) structure. The nu(4) and nu(2) modes of heme a(3) of the P intermediate were identified at 1377 and 1591 cm(-1), respectively. The Raman excitation profiles of the nu(Fe=O) bands were different between P and F. These observations may mean the formation of a pi cation radical of porphyrin macrocycle in P.     

Structures of reaction intermediates of bovine cytochrome c oxidase probed by time-resolved vibrational spectroscopy

Structures of reaction intermediates of bovine cytochrome c oxidase (CcO) in the reactions of its fully reduced form with O2 and fully oxidized form with H2O2 were investigated with time-resolved resonance Raman (RR) and infrared spectroscopy. Six oxygen-associated RR bands were observed for the reaction of CcO with O2. The isotope shifts for an asymmetrically labeled dioxygen, (16)O(18)O, has established that the primary intermediate of cytochrome a3 is an end-on type dioxygen adduct and the subsequent intermediate (P) is an oxoiron species with Fe=O stretch (nu(Fe=O)) at 804/764 cm(-1) for (16)O2/(18)O2 derivatives, although it had been long postulated to be a peroxy species. The P intermediate is converted to the F intermediate with nu(Fe=O) at 785/751 cm(-1) and then to a ferric hydroxy species with nu(Fe-OH) at 450/425 cm(-1) (443/417 cm(-1) in D2O). The rate of reaction from P to F intermediates is significantly slower in D2O than in H2O. The reaction of oxidized CcO with H2O2 yields the same oxygen isotope-sensitive bands as those of P and F, indicating the identity of intermediates. Time-resolved infrared spectroscopy revealed that deprotonation of carboxylic acid side chain takes place upon deligation of a ligand from heme a3. UV RR spectrum gave a prominent band due to cis C=C stretch of phospholipids tightly bound to purified CcO.     

The ferrous dioxygen complex of the oxygenase domain of neuronal nitric-oxide synthase

The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe(2+)O(2) nNOSoxy). We detect a line at 1135 cm(-1) in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm(-1) with (18)O(2). This line is assigned as the O-O stretching mode (nu(O-O)) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or N(omega)-hydroxy-L-arginine, in the presence or absence of (6R)-5,6, 7,8-tetrahydro-L-biopterin, reveal that the nu(O-O) line is insensitive to the presence of the substrate and the pterin. The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques.     

A weak Fe-O bond in the oxygenated complex of the nitric-oxide synthase of Staphylococcus aureus

Little is known about the intermediates formed during catalysis by nitric-oxide synthase (NOS). We report here the characterization by resonance Raman spectroscopy of the oxygenated complex of the NOS from Staphylococcus aureus (saNOS) as well as the kinetics of formation and decay of the complex. An oxygenated complex transiently formed after mixing reduced saNOS with oxygen and decayed to the ferric enzyme with kinetics that were dependent on the substrate L-arginine and the cofactor H(4)B. The oxygenated complex displayed a Soret absorption band centered at 430 nm. Resonance Raman spectroscopy revealed that it can be described as a ferric superoxide form (Fe(III)O(2)(-)) with a single nu(O-O) mode at 1135 cm(-1). In the presence of L-arginine, an additional nu(O-O) mode at 1123 cm(-1) was observed, indicating an increased pi back-bonding electron donation to the bound oxygen induced by the substrate. With saNOS, this is the first time that the nu(Fe-O) mode of a NOS has been observed. The low frequency of this mode, at 517 cm(-1), points to an oxygenated complex that differs from that of P450(cam). The electronic structure of the oxygenated complex and the effect of L-arginine are discussed in relation to the kinetic properties of saNOS and other NOS.     

Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.     

Ferryl-oxo heme intermediate in the antimalarial mode of action of artemisinin

Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopies have been employed to investigate the reductive cleavage of the O-O bond of the endoperoxide moiety of the antimalarial drug artemisinin and its analog trioxane alcohol by hemin dimer. We have recorded FTIR spectra in the nu(O-O) and nu(as)(Fe-O-Fe) regions of artemisinin and of the hemin dimer that show the cleavage of the endoperoxide and that of the hemin dimer, respectively. We observed similar results in the trioxane alcohol/hemin dimer reaction. The RR spectrum of the artemisinin/hemin dimer reaction displays a vibrational mode at 850 cm(-1) that shifts to 818 cm(-1) when the experiment is repeated with (18)O-O(18) endoperoxide enriched trioxane alcohol. The frequency of this vibration and the magnitude of the (18)O-O(18) isotopic shift led us to assign the 850 cm(-1) mode to the Fe(IV) = O stretching vibration of a ferryl-xoxo heme intermediate that occurs in the artemisinin/hemin dimer and trioxane alcohol/hemin reactions. These results provide the first direct characterization of the antimalarial mode of action of artemisinin and its trioxane analog, and suggest that artemisinin appears to react with heme molecules that have been incorporated into hemozoin and subsequently the heme performs cytochrome P450-type chemistry.     

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

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.     

A novel diiron complex as a functional model for hemerythrin

Diiron(II) complexes with a novel dinucleating polypyridine ligand, N,N,N',N'-tetrakis(6-pivalamido-2-pyridylmethyl)-1,3-diaminopropan-2-ol (HTPPDO), were synthesized as functional models of hemerythrin. Structural characterization of the complexes, [Fe2II(Htppdo)(PhCOO)](ClO4)3 (1), [Fe2II(Htppdo)((p-Cl)PhCOO)](ClO4)3 (2), [Fe2II(Htppdo)((p-Cl)PhCOO)](BF4)3 (2') and [Fe2II(tppdo)((p-Cl)PhCOO)](ClO4)2 (3), were accomplished by electronic absorption, and IR spectroscopic, electrochemical, and X-ray diffraction methods. The crystal structures of 1 and 2' revealed that the two iron atoms are asymmetrically coordinated with HTPPDO and bridging benzoate. One of the iron centers (Fe(1)) has a seven-coordinate capped octahedral geometry comprised of an N3O4 donor set which includes the propanol oxygen of HTPPDO. The other iron center (Fe(2)) forms an octahedron with an N3O3 donor set and one vacant site. The two iron atoms are bridged by benzoate (1) or p-chlorobenzoate (2). On the other hand, both Fe atoms of complex 3 are both symmetrically coordinated with N3O4 donors and two bridging ligands; benzoate and the propanolate of TPPDO. Reactions of these complexes with dioxygen were followed by electronic absorption, resonance Raman and ESR spectroscopies. Reversible dioxygen-binding was demonstrated by observation of an intense LMCT band for O2(2-) to Fe(III) at 610 (1) and 606 nm (2) upon exposure of dioxygen to acetone solutions of 1 and 2 prepared under an anaerobic conditions at -50 degrees C. The resonance Raman spectra of the dioxygen adduct of 1 exhibited two peaks assignable to the nu(O-O) stretching mode at 873 and 887 cm(-1), which shifted to 825 and 839 cm(-1) upon binding of (18)O2. ESR spectra of all dioxygen adducts were silent. These findings suggest that dioxygen coordinates to the diiron atoms as a peroxo anion in a mu-1,2 mode. Complex 3 exhibited irreversible dioxygen binding. These results indicate that the reversible binding of dioxygen is governed by the hydrophobicity of the dioxygen-binding environment rather than the iron redox potentials.     

UV Raman monitoring of histidine protonation and H-(2)H exchange in plastocyanin

UV resonance Raman bands of Cu-bound and protonated histidine residues have been detected in (2)H(2)O solutions of poplar plastocyanin. For the Cu(II) protein, slow NH-(2)H exchange of the His37 ligand was monitored via the growth of bands at 1389 and 1344 cm(-1) when Pcy was exchanged into (2)H(2)O, or via their diminution when the protein was exchanged back into H(2)O; the rate constant is 7 x 10(-4)/s at pH (p(2)H) 7.4 at room temperature. The slow exchange is attributed to imidazole H-bonding to a backbone carbonyl. Nearby bands at 1397 and 1354 cm(-1), appear and disappear within the mixing time, and are assigned to the solvent-exposed His87 ligand. The approximately 10 cm(-1) differences between His37 and His87 are attributed to the effect of H-bonding on the imidazole ring modes. The UVRR spectra of the Cu(I) protein in (2)H(2)O reveal a 1408 cm(-1) band, characteristic of NH-(2)H-exchanged histidinium, which grows in as the p(2)H is lowered. Its intensity follows a titration curve with pK(a)=4.6. This protonation is assigned to the His87 residue, whose bond to the Cu(I) is known from crystallography to be broken at low pH. As the 1408 cm(-1) band grows, a band at 1345 cm(-1) diminishes, while another, at 1337 cm(-1) stays constant. These are assigned to modes of bound His87 and His37, respectively, shifted down 7-9 cm(-1) from their Cu(II) positions.     

RNA binding efficacy of theophylline,theobromine and caffeine

The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.     

关帝山黄刺玫灌丛群落结构与生物量的研究

本文研究了关帝山东坡海拔1700m处的黄刺玫灌丛的群落结构及其生物量,并探讨了生物量与群落结构、动态演替和群落性质的关系。灌丛的生物量是用回归模型来预测的,其为:黄刺玫(?)_(枝)=2.5289(D~2H)~(1.8988),(?)_(叶)=0.1521(D~2H)~(1.9236),(?)_(根)=1.9884(D~2H)~(1.9304);沙棘(?)_(枝)=0.2993(D~2H)~(2.1344),(?)_(叶)=0.1988(D~2H)~(2.1332),(?)_(根)=2.0269(D~2H)~(1.6113);其余灌木和草本植物用实测数据估算。最后得出,6月份黄刺玫灌丛的总生物量为6.231t/ha,其中灌木层4.867t/ha,草本层1.364 t/ha。     

3-D-QSAR analysis of N-(3-acyloxy-2-benzylpropyl)-N'-dihydroxytetrahydrobenzazepine and tetrahydroisoquinoline and N-(3-acyloxy-2-benzylpropyl)-N'-(4-hydroxy-3-methoxybenzyl) thioureas analogues as potent vanilloid receptor ligands

3-D-Quantitative structure--activity relationships of N-(3-acyloxy-2-benzylpropyl)-N'-dihydroxytetrahydro-benzazepine and tetrahydroisoquinoline and N-(3-acyloxy-2-benzylpropyl)-N'-(4-hydroxy-3-methoxybenzyl) thiourea analogues as potent vanilloid receptor ligands were investigated using the CoMFA and the COMSIA methods. The best CoMFA model obtained in this study from 29 substituted thiourea analogues is a two-component model with the following statistics. R(2)((cv))=0.407 and RMSE((cv))=0.532 for the cross-validation, and R(2)=0.705 and RMSE=0.375 for the fitted. The best COMSIA model obtained from the same 29 compounds is a two-component model with the following statistics: R(2)((cv))=0.336 and RMSE((cv))=0.563 for the cross-validation, and R(2)=0.693 and RMSE=0.382 for the fitted.     

Structural characterization of the [Pco/o(2)] compound of cytochrome c oxidase

The structural properties of a key transient oxygen intermediate of cytochrome c oxidase, P(R), remain an enigma, although inferences have been drawn from its equilibrium analogues, [Pco/o(2)] , P(H) and P(M). With resonance Raman spectroscopy, an oxygen isotope-sensitive band at 806 cm(-1) was observed in [Pco/o(2)] produced by adding CO and O(2) to the resting enzyme. The vibrational band shifted to 771 cm(-1) upon isotopic substitution of (16)O(2) with (18)O(2). The same modes at 806 and 771 cm(-1) were present simultaneously when the mixed isotope, (18)O(16)O, was employed, indicating that in [Pco/o(2)] the O-O bond is cleaved, resulting in a Fe(4+)O(2-) structure. This result unifies the nature of the three equilibrium analogues of the P(R) intermediate.     

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.     

A functional model for pMMO (particulate methane monooxygenase): Hydroxylation of alkanes with H2O2 catalyzed by beta-diketiminatocopper(II) complexes

The reaction of copper(II) complexes supported by a series of beta-diketiminate ligands ((R1,R2)L, [(Dipp)N-C(R(2))-C(R(1))-C(R(2))-N(Dipp)](-), Dipp=2,6-diisopropylphenyl; see ) and H(2)O(2) has been examined spectroscopically at a low temperature. The beta-diketiminatocopper(II) complexes with R(2)=H (no substituent on the beta-carbon) provided a copper-oxygen intermediate that exhibited the same spectroscopic features as those of the bis(mu-oxo)dicopper(III) complex generated by the reaction of corresponding beta-diketiminatocopper(I) complex and O(2). On the other hand, the beta-diketiminatocopper(II) complexes with methyl substituent on the beta-carbon (R(2)=Me) did not produce such an intermediate in the same reaction. The beta-diketiminatocopper(II) complexes carrying an electron-withdrawing substituent on the alpha-carbon (R(1)=NO(2) or CN) but no beta-substituent (R(2)=H) exhibited a high catalytic activity in the oxygenation reaction of alkanes with H(2)O(2). Mechanism of the catalytic oxygenation reaction as well as the substituent effects of the ligands on the copper(II)-H(2)O(2) reactivity is discussed.     

Spectroscopic behavior of copper complexes of nonsteroidal anti-inflammatory drugs

The proposed curative properties of Cu-based nonsteroidal anti-inflammatory drugs (NSAIDs) have led to the development of numerous Cu(II) complexes of NSAIDs with enhanced anti-inflammatory activity. Crystalline complexes, Cu(II)-NSAID (ibuprofen, naproxen, tolmetin, and diclofenac), with a carboxylic function have been studied by means of infrared and Raman spectroscopy. All NSAIDs bind the metal through the carboxylate group. On the basis of the comparison between the wavenumber of the COO(-) group vibrations and Delta nu (nu(asimm)COO(-) - nu(simm)COO(-)) between Na salts and Cu(II) complexes, conclusions on the probable structure of the complexes have been drawn. The spectroscopic data support the formation of dimeric [Cu(2)L(4)(H(2)O)(2)] complexes in which the COO(-) group behaves as a bridging bidentate ligand. The low wavenumber region of the Raman spectrum provided information on Cu-O and Cu-Cu bonds in the complexes. Thermogravimetric results gave further support to the vibrational data.     

Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water-perchlorate helical network

A new octanuclear copper(II) complex has been synthesized and structurally characterized by X-ray crystallography: [Cu(8)(HL)(4)(OH)(4)(H(2)O)(2)(ClO(4))(2)].(ClO(4))(2).2H(2)O (1) (H(3)L=2,6-bis(hydroxyethyliminoethyl)-4-methyl phenol). The complex is formed by the linkage of two terminal bimetallic cationic units and a tetranuclear mu(3)-hydroxo bridged dicubane core by a very short intramolecular hydrogen bond (O-H...O, 1.48(3)A and the angle 175 degrees). The coordination sphere of the terminal copper atoms is square pyramidal, the apical positions being occupied by water and a perchlorate ion. Complex 1 self-assembles to form a new type of water-perchlorate helical network [(H(2)O)(2)(ClO(4))](infinity) involving oxygen atoms of coordinated perchlorate ion and the two lattice water molecules through hydrogen-bonding interaction. The variable temperature-dependent susceptibility measurement (2-300K) of 1 reveals a strong antiferromagnetic coupling, J(1)=-220cm(-1) and J(2)=-98cm(-1) (J(1) and J(2) representing the exchange constant within [Cu(2+)](4) and [Cu(2+)](2) units, respectively). The complex binds to double-stranded supercoiled plasmid DNA giving a K(app) value of 1.2x10(7)M(-1) and displays efficient oxidative cleavage of supercoiled DNA in the presence of H(2)O(2) following a hydroxyl radical pathway.