Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A(278nm) / A(388nm)) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c(3) (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A, Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A(553nm) - A(570nm)](red) / A(280nm)) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization (NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c(3) from Desulfomicrobium (Des.) norvegicum (NMR spectra, and N-terminal amino acid sequence).

We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126+/-2 kDa composed of two subunits, alpha=93+/-3 kDa and beta=32+/-2 kDa, which contains 6+/-1 Fe/molecule, 0.4+/-0.1 Mo/molecule, 0.3+/-0.1 W/molecule, and 1.3+/-0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d(1) configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme.

The macrocyclic phenanthrolinophane 2,9-[2,5,8-triaza-5-(N-anthracene-9-methylamino) ethyl]-[9]-1,10-henanthrolinophane(L) bearing a pendant arm containing a coordinating amine and an anthracene group forms stable complexes with Zn(II), Cd(II) and Hg(II) in solution. Stability constants of these complexes were determined in 0.10 mol dm(-3) NMe4Cl H2O-MeCN (1:1, v/v) solution at 298.1+/-0.1 K by means of potentiometric (pH metric) titration. The fluorescence emission properties of these complexes were studied in this solvent. For the Zn(II) complex, steady-state and time-resolved fluorescence studies were performed in ethanol solution and in the solid state. In solution, intramolecular pi-stacking interaction between phenanthroline and anthracene in the ground state and exciplex emission in the excited state were observed. From the temperature dependence of the photostationary ratio (I-Exc/I-M), the activation energy for the exciplex formation (E-a) and the binding energy of the exciplex (-DeltaH) were determined. The crystal structure of the [ZnLBr](ClO4).H2O compound was resolved, showing that in the solid state both intra- and inter-molecular pi-stacking interactions are present. Such interactions were also evidenced by UV-vis absorption and emission spectra in the solid state. The absorption spectrum of a thin film of the solid complex is red-shifted compared with the solution spectra, whereas its emission spectrum reveals the unique featureless exciplex band, blue shifted compared with the solution. In conjunction with X-ray data the solid-state data was interpreted as being due to a new exciplex where no pi-stacking (full overlap of the pi-electron cloud of the two chromophores-anthracene and phenanthroline) is observed. L is a fluorescent chemosensor able to signal Zn(II) in presence of Cd(II) and Hg(II), since the last two metal ions do not give rise either to the formation of pi-stacking complexes or to exciplex emission in solution.

The synthetic flavylium salt 7-(N,N-diethylamino)-4'-hydroxyflavylium tetrafluoroborate gives rise in aqueous solution to a complex network of chemical reactions driven by pH. The system was studied by H-1 NMR, single crystal X-ray diffraction, steady state and transient UV-Vis spectrophotometry as well as stopped flow. The crystal structure shows a high degree of coplanarity between the pyrylium system and the phenyl group in position 2. Thermodynamic and kinetic constants for the pH dependent network of chemical reactions were obtained. The introduction of an amino group in position 7 allows formation of protonated species leading, in particular, to a tautomeric form of the protonated cis-chalcone, CcH(+), whose absorption spectra is rather red shifted, in comparison with the correspondent protonated trans-chalcone, CtH(+). The CcH(+) species can be rapidly converted into the flavylium cation through a first order process with lifetime of 0.2 s at pH = 2.35. This new reaction channel confers this compound a peculiar behaviour in acidic media, allowing to define an unidirectional pH driven reaction cycle.

The multistate/multifunctional properties of 4'-hydroxyflavylium in a water/ I -n-butyl-3 -methyl-imidazolium hexalluorophosphate (fbmim][PF6]) biphasic system are described. The kinetics and thermodynamics of this flavylium salt have been fully characterised in aqueous solutions and compared to those obtained in [bmim][PF6]. The trans-chalcone is thermally more stable in the ionic liquid but shows efficient photoisomerisation to the cis-chalcone, allowing to define write-read-erase cycles in this biphasic system. (C) 2004 Elsevier B.V. All rights reserved.

The chemistry and photochemistry of 3',4'-(methylenedioxy)flavylium was studied by means of UV-Vis spectrophotometry, H-1 NMR, stopped flow, and continuous irradiation, in acidic and basic aqueous solutions. Six species were identified: the flavylium cation, AH(+); the hemiketal, B; the cis- and the trans-chalcones, Cc and Ct, and their ionized forms, Cc(-) and Ct(-). These species de. ne a multiequilibria network whose kinetics and thermodynamics were completely characterized. The two pairs of chalcones de. ne two coupled photochromic systems, respectively in acidic and basic media, both allowing cycles capable of writing, reading and erasing to be defined.

The interaction of reduced rabbit cytochrome b(5) with reduced yeast iso-1 cytochrome c has been studied through the analysis of (1)H-(15)N HSQC spectra, of (15)N longitudinal ( R(1)) and transverse ( R(2)) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b(5) has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b(5).

The coordination features of the two dipyridine-containing polyamine macrocycles 2,5,8,11,14-pentaaza[ 15][ [15](2,2')[1,15]-bipyridylophane (L1) and 4,4'-(2,5,8,11,14-pentaaza[15]-[15](2,2')-bipyridylophane) (L2) toward Cu(II) and Ni(II) have been studied by means of potentiometric and spectrophotometric UV-vis titrations in aqueous solutions. While in L1 all the nitrogen donor atoms are convergent inside the macrocyclic cavity, in L2 the heteroaromatic nitrogen atoms are located outside. Ligands L1 and L2 form stable mono- and dinuclear complexes with Cu(II). In the case of Ni(II) coordination, only L1 gives dinuclear complexes, while L2 can form only mononuclear species. In the Cu(II) or Ni(II) complexes with L1 the metal(s) are lodged inside the macrocyclic cavity, coordinated to the heteroaromatic nitrogens. As shown by the crystal structure of the [CuL1](2+) and [NiL1](2+) cations, at least one of the two benzylic nitrogens is not coordinated and facile protonation of the complex takes place at neutral or slightly acidic pH values. The particular molecular architecture of L2, which displays two well-separated binding moieties, strongly affects its coordination behavior. In the mononuclear [ CuL2](2+) complex, the metal is encapsulated inside the cavity, not coordinated by the dipyridine unit. Protonation of the complex, however, occurs on the aliphatic polyamine chain and gives rise to translocation of the metal outside the cavity, bound to the heteroaromatic nitrogens. In the [NiL2](2+) complex the metal is coordinated by the dipyridine nitrogens, outside the macrocyclic cavity. Thermodynamic and/or kinetic considerations may explain the different behavior with respect to the corresponding Cu(II) complex.

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe2+ chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.