Resonance Raman studies of a dioxy-tetraaza-nickel complex containing a cofacial dimeric structure

The electronic absorption and resonance Raman (rR) spectra of a planar Ni(II) N₂O₂-core complex were measured in solution as well in the solid state. Excitation within the contour of the first CT transition produced rR enhancement mainly of internal ligand vibrations. In the solid state the electronic structure is dominated by the properties of a face-to-face dimer.     

Resonance Raman and semiempirical electronic structure studies of an odd-electron dinickel tetraiminoethylenedimacrocycle complex

Resonance Raman studies of Ni2TIED3+ (TIED = tetraiminoethylenedimacrocycle) reveal that many modes couple to the intense electronic transition centered at 725 nm, a feature that is nominally similar to the intense delocalized intervalence absorption bands observed in the same region for Fe2(TIED)L4(5+) and Ru2(TIED)L4(5+) (L is any of several axial ligands). Time-dependent spectral modeling of the Raman and absorption spectra for the nickel compound was undertaken to understand the electronic transition. We were unable to model the Raman and absorption spectra successfully with a single electronic transition, suggesting that the absorption band is made up of two overlapping transitions. Semiempirical electronic structure calculations corroborate the suggestion. Additionally, these calculations indicate that the transitions are in fact ligand-localized transitions, with little metal involvement and no charge-transfer character. Furthermore, the ground-state electronic structure is best described as an identical pair of NiII centers bridged by a radical anion rather than a three-site mixed-valence assembly. Previous EPR studies (McAuley and Xu, Inorg. Chem. 1992, 31, 5549) had indicated primarily ligand character for the radical. The assignments are consistent with the resonance Raman results where the dominant modes coupled to the transitions are assigned as totally symmetric bridge vibrations.     

Resonance Raman study of a two-chromophore system. The 2:1 complex of hexamethylbenzene with tetracyanoethylene

Raman excitation profiles are presented for the 2:1 electron donor-acceptor (EDA) complex of hexamethylbenzene (HMB) and tetracyanoethylene (TCNE) in cyclohexane. Though the absorption and Raman spectra of the 1:1 and 2:1 complexes are similar, distinct differences are found in the Raman excitation profiles (REPs) of vibrational modes common to both systems. REPs of the 2:1 complex show intensity cancellation that is taken as evidence for interference of two charge-transfer excited states. The implications of the observed spectra concerning excited state electron delocalization are considered.     

The cytidinium—cytidine complex: infrared and Raman spectroscopic studies

Using IR and Raman spectra, it is shown that the sytidinium cation hydrogen bonds to cytidine to form a stable 1:1 complex, in both aqueous solution (pH ~ 3.3) and as a solid. The spectra indicate that the proton is located asymmetrically in the NH?N bond of the complex, on the vibrational time scale, in both solution and the solid. The perdeuterated systems were also examined; their spectra support these conclusions.     

Photoreactivation of alloxanthine-inhibited xanthine oxidase

Alloxanthine-inhibited xanthine oxidase (XOD) was found to be photoreactivated by irradiation of light of wavelengths in the range of 340-430 nm. The enzyme activity can be fully controlled to be on or off by many dark-light cycles. Electron spin resonance measurement shows the appearance of the molybdenum (V) ion and the reduced form of flavin adenine dinucleotide (FADH.) radical signals after irradiation of the alloxanthine-XOD complex. Electronic-absorption spectrum also shows the bleaching of Fe/S and flavin adenine dinucleotide chromophores at 375 and 450 nm as well as broad-band absorption of FADH. in the range of 500-700 nm. The quantum yield of photoreactivation of the enzyme activity is approximately 0.06. A photoinduced intraenzyme electron-transfer model is proposed to rationalize the photoreactivation process.     

Resonance Raman spectroscopic studies of hydroperoxo-myoglobin at cryogenic temperatures

In agreement with previous reports (Gasyna, Z. FEBS Lett. 1979, 106, 213-218 and Leibl, W.; Nitschke, W.; Huettermann, J. Biochim. Biophys. Acta 1986, 870, 20-30) radiolytically reduced samples of oxygenated myoglobin at cryogenic temperatures have been shown by optical absorption and EPR studies to produce directly the peroxo-bound myoglobin at 77 K. Annealing to temperatures near 185 K induces proton transfer, resulting in the formation of the hydroperoxo heme derivative. Resonance Raman studies of the annealed samples has permitted, for the first time, the direct observation of the key nu(Fe-O) stretching mode of the physiologically important Fe-OOH fragment of this ubiquitous intermediate. The assignment of this mode to a feature appearing at 617 cm(-1) is strongly supported by documentation of a 25 cm(-1) shift to lower energy upon substitution with (18)O(2) and by a 5 cm(-1) shift to lower energy for samples prepared in solutions of deuterated solvent.     

Resonance Raman studies of beta-substituted porphyrin systems with unusual electronic absorption properties.

Zn(II) and Cu(II) porphyrins with beta-conjugated barbiturate functional groups have low-energy electronic transitions which are unusual in that there are two strong bands in the Soret region. Resonance excitation of the two bands shows that each has features characteristic of both the porphyrin and barbiturate groups, with some perturbation to these features caused by the interaction of the two chromophores. The resonance Raman (RR) spectrum (lambda(exc)=413.1 nm) of the 412 nm band shows two bands at 1722 and 1743 cm(-1) attributable to C==O stretches in the substituent. Changes in frequency of porphyrin core modes due to the differing metal centres are reproduced by density functional theory calculations. The Q band RR spectra show modes with anomalous polarization which may be attributed to A(2g) modes, however no overtone or combination bands are observed.     

Tautomerism of xanthine and alloxanthine: A model for substrate recognition by xanthine oxidase

Summary Tautomerism of neutral xanthine and alloxanthine has been examined both in the gas phase and in aqueous solution. The tautomeric preference in the gas phase has been studied by means of semiempirical and ab initio quantum-mechanical computations with inclusion of correlation effects at the Møller-Plesset level, and from density-functional calculations. The influence of solvent on the relative stability between tautomers has been estimated from self-consistent reaction field calculations performed with different models. The results provide a detailed picture of tautomerism for these biologically relevant purine bases. The functional implications in the recognition by xanthine oxidase are analyzed from inspection of the interaction patterns of the most stable tautomeric forms. A model for the recognition of these purine derivatives in the enzyme binding site is discussed.     

Comparison of the kinetic behaviour of xanthine oxidase and xanthine dehydrogenase

In the oxidation of hypoxanthine and xanthine by chicken liver xanthine dehydrogenase and bovine liver and milk xanthine oxidase, activation appears at high substrate concentrations. Hypoxanthine concentrations greater than 0.04 mM inhibit the bovine enzyme; concentrations greater than 0.09 mM inhibit the milk enzyme. On the other hand, uric acid is a mixed non-competitive inhibitor of bovine liver xanthine dehydrogenase. The phosphate (Na⁺ or K⁺) and chloride (Na⁺ or K⁺) ions do not affect bovine live xanthine oxidase activation, but modify the K and V values.     

Studies on the mechanism of aldehyde oxidase and xanthine oxidase

DFT calculations support a concerted mechanism for xanthine oxidase and aldehyde oxidase hydride displacement from the sp(2) carbon of 6-substituted 4-quinazolinones. The variations in transition state structure show that C-O bond formation is nearly complete in the transition state and the transition state changes are anti-Hammond with the C-H and C-O bond lengths being more product-like for the faster reactions. The C-O bond length in the transition state is around 90% formed. However, the C-H bond is only about 80% broken. This leads to a very tetrahedral transition state with an O-C-N angle of 109 degrees. Thus, while the mechanism is concerted, the antibonding orbital of the C-H bond that is broken is not directly attacked by the nucleophile and instead hydride displacement occurs after almost complete tetrahedral transition state formation. In support of this the C=N bond is lengthened in the transition state indicating that attack on the electrophilic carbon occurs by addition to the C=N bond with negative charge increasing on the nitrogen. Differences in experimental reaction rates are accurately reproduced by these calculations and tend to support this mechanism.     

Resonance Raman spectroscopy of nitric oxide reductase and cbb(3) heme-copper oxidase

Elucidating the structure and properties of the active sites in cbb3 heme-copper oxidase and in nitric oxide reductase (Nor) is crucial in understanding the reaction mechanisms of oxygen and nitric oxide reduction by both enzymes. In the work here, we have applied resonance Raman (RR) spectroscopy to investigate the structure and properties of the binuclear heme b3-CuB center of cbb3 heme-copper oxidase from Pseudomonas stutzeri and the dinuclear heme b3-FeB center of Nor from Paracoccus denitrificans in the ligand-free and CO-bound forms and in the reactions with O2 and NO. The RR data demonstrate that in the Nor/NO reaction, the formation of the N-N bond occurs with the His-Fe heme b3 bond intact, and reformation of the heme b3-O-FeB dinuclear center causes the rupture of the proximal His-Fe heme b3 bond. In the reactions of Nor and cbb3 with O2, distinct oxidized heme b3 species, which differ from the as-isolated oxidized forms, have been characterized. The activation and reduction of O2 and NO by cbb3 oxidase and nitric oxide reductase are compared and discussed.     

Oxidation of quinazoline and quinoxaline by xanthine oxidase and aldehyde oxidase

Quinazoline is oxidized by xanthine oxidase initially (and rapidly) to 4-hydroxyquinazoline which subsequently is oxidized more slowly to 2,4-dihydroxyquinazoline. Both oxidative reactions are inhibited strongly by allopurinol. Quinazoline is oxidized by aldehyde oxidase to 4-hydroxyquinazoline but within a short time (3–5 minutes) the reaction ceases; the proposal that cessation of reaction is due to product inhibition is rendered untenable by our observation that 4-hydroxyquinazoline is rapidly oxidized by aldehyde oxidase to 2,4-dihydroxyquinazoline. Preincubation of aldehyde oxidase with quinazoline results in complete inhibition of the ability of the enzyme to oxidize 4-hydroxyquinazoline and the standard substrate N-methylnicotinamide. It appears therefore that quinazoline is able to react with aldehyde oxidase and inactivate it. Quinoxaline and 2-hydroxyquinoxaline are not oxidized by xanthine oxidase but are converted by aldehyde oxidase to 2,3-dihydroxyquinoxaline; all oxidations mediated by aldehyde oxidase are inhibited completely by menadione.     

Resonance Raman detection of a ferrous five-coordinate nitrosylheme b(3) complex in cytochrome cbb(3) oxidase from Pseudomonas stutzeri

Understanding the chemical nature of the nitric oxide (NO) moiety of nitrosylheme copper oxidases is crucial for elucidation of the NO activation process. In the present work, direct resonance Raman spectroscopic observation of both the Fe(2+)-NO and the N-O stretching modes unambiguously establishes the vibrational characteristics of the NO-bound heme moiety in cytochrome cbb(3) from Pseudomonas stutzeri. Addition of NO to fully reduced enzyme causes the rupture of the proximal His-heme b(3) bond resulting in the formation of a five-coordinate heme b(3)(2+)-NO species with nu(Fe-NO) and nu(NO) at 524 and 1679 cm(-1), respectively. The frequencies of the nitrosyl species we detect are very similar to those obtained in other model- and protein heme-NO complexes. To account for this observation, we propose a model describing the oxidation and ligand-binding states in fully reduced cytochrome cbb(3) upon addition of NO.     

Resonance Raman imaging of the NADPH oxidase subunit cytochrome b558 in single neutrophilic granulocytes

We have employed confocal resonance Raman (RR) imaging to visualize the subcellular distribution of the NADPH oxidase subunit cytochrome b558 in both resting and phagocytosing neutrophils. Our Raman microscopic technique is a label-free, chemical (vibrational) imaging method that can be applied to individual, intact cells, thus probing cytochrome b558 in its native environment. The Raman signal from cytochrome b558 is resonantly and selectively enhanced in neutrophils by using 413 nm excitation. Experiments on resting neutrophils show a cytoplasmic distribution of cytochrome b558, with several areas of high content. Upon phagocytosis of polystyrene particles, we found that part of the cytochrome b558 is translocated toward the ingested beads. This is in accordance with immunocytochemistry studies combined with electron and fluorescence microscopy. As compared to these methods, RR microscopy requires minimal sample preparation and disturbance. Moreover, it allows the determination of the redox state of cytochrome b558 inside the cell, which reflects its NADPH oxidase activity.