Felix Tuczek

Purification and spectroscopic studies on catechol oxidases from Lycopus europaeus and Populus nigra: evidence for a dinuclear copper center of type 3 and spectroscopic similarities to tyrosinase and hemocyanin.

Abstractu2002We purified two catechol oxidases from Lycopus europaeus and Populus nigra which only catalyze the oxidation of catechols to quinones without hydroxylating tyrosine. The molecular mass of the Lycopus enzyme was determined to 39u200a800 Da and the mass of the Populus enzyme was determined to 56u200a050 Da. Both catechol oxidases are inhibited by thiourea, N-phenylthiourea, dithiocarbamate, and cyanide, but show different pH behavior using catechol as substrate. Atomic absorption spectroscopic analysis found 1.5 copper atoms per protein molecule. Using EPR spectroscopy we determined 1.8 Cu per molecule catechol oxidase. Furthermore, EPR spectroscopy demonstrated that catechol oxidase is a copper enzyme of type 3. The lack of an EPR signal is due to strong antiferromagnetic coupling that requires a bridging ligand between the two copper ions in the met preparation. Addition of H2O2 to both enzymes leads to oxy catechol oxidase. In the UV/Vis spectrum two new absorption bands occur at 345 nm and 580 nm. In accordance with the oxy forms of hemocyanin and tyrosinase the absorption band at 345 nm is due to an O22– (πσ*)→Cu(II) (dx2–y2) charge transfer (CT) transition. The absorption band at 580 nm corresponds to the second O22– (πv*)→Cu(II) (dx2–y2) CT transition. The UV/Vis bands in combination with the resonance Raman spectra of oxy catechol oxidase indicate a μ-η2u200a:u200aη2 binding mode for dioxygen. The intense resonance Raman peak at 277 cm–1, belonging to a Cu-N (axial His) stretching mode, suggests that catechol oxidase has six terminal His ligands, as known for molluscan and arthropodan hemocyanin.

Reversible Dioxygen Binding and Phenol Oxygenation in a Tyrosinase Model System

The complex [Cu2(L-66)]2+ (L-66 = a,a-bis¿bis[2-(1-methyl-2-benzimidazolyl)ethyl]amino¿-m-xylene) undergoes fully reversible oxygenation at low temperature in acetone. The optical [lambda(max) = 362 (epsilon 15000), 455 (epsilon 2000), and 550 nm (epsilon 900M(-1)cm(-1))] and resonance Raman features (760 cm(-1), shifted to 719cm(-1)(-1) with 18O2) of the dioxygen adduct [Cu2(L-66)(O2)]2+ indicate that it is a mu-eta2:eta2-peroxodicopper(II) complex. The kinetics of dioxygen binding, studied at - 78 degrees C, gave the rate constant k1 = 1.1M(-1) 5(-1) for adduct formation, and k(-1) =7.8 x 10(-5)s(-1), for dioxygen release from the Cu2O2 complex. From these values, the O2 binding constant K= 1.4 x 10(4)M(-1) at -78 degrees C could be determined. The [Cu2(L-66)(O2)]2+ complex performs the regiospecific ortho-hydroxylation of 4-carbomethoxyphenolate to the corresponding catecholate and the oxidation of 3,5-di-tert-butylcatechol to the quinone at -60 degrees C. Therefore, [Cu2(L-66)]2+ is the first synthetic complex to form a stable dioxygen adduct and exhibit true tyrosinase-like activity on exogenous phenolic compounds.

Advances in Mössbauer Emission Spectroscopy

After effects following nuclear transformation have been extensively studied in a large variety of matrices by Mössbauer Emission Spectroscopy (MES). The branching ratios of transient atomic charge states and energetically unstable states of the environment, excitations of electronic ligand field states and populations out of equilibrium within the electronic ground state manifold have been studied. Recent developments and also new insights and understandings of different aspects of the aftereffects of radioactive decay of57Co in semiconductors and molecular crystals are reviewed. A detailed picture of the decay process within the electronic states of the nucleogenic Fe3+ species could be obtained from emission spectroscopy of sources in applied magnetic fields.

Time-differential Mössbauer emission spectroscopy: Development of a new spectrometer and first results

A spectrometer for time-differential Mössbauer spectroscopy was developed with a high count rate capability, a good time resolution, and a flexible data handling. Chemical and physical aftereffects following the57Co decay were studied within the cobalt doped systems Turnbulls blue and ZnS.

Metastable electronic states induced by nuclear decay and light

Radioactive atoms incorporated in insulating solid-state compounds create various kinds of chemical and physical “after-effects” upon nuclear disintegration. Mössbauer emission spectroscopy of57Co-labelled coordination compounds has undoubtedly become the most informative technique to detect such after-effects like aliovalent charge and spin states of the nucleogenic iron atom resulting from the57Co(EC)57Fe decay, low energy excitations of crystal field and Zeeman states, linkage isomerism, radical formation with subsequent redox reactions, and others. We have extensively studied57Co-labelled complexes with [CoIIN6] cores employing time-integral and time-resolved Mössbauer emission spectroscopy. In complexes of strong ligands fields (where the corresponding synthesized iron(II) complexes show low spin behaviour with1A1 ground slate) and in complexes of intermediate ligand field strength (where the corresponding iron(II) compounds exhibit thermal spin-crossover1A1 ⇌5T2) we have observed the population of metastable high spin states, which is strongly time- and temperature-dependent in the case of the strong-field complexes. Irradiation of iron(II) spin-crossover complexes with light also induces the formation of metastable high spin states, which are the same in nature as those resulting from decaying nuclei as an “intramolecular light source”. The mechanisms for “light-induced excited spin state trapping” (LIESST) and “nuclear decay induced excited spin state trapping” (NIESST) have been elucidated; they are strongly related to each other. In57Co-labelled LiNbO3 and other matrices, we have observed nonthermalized populations of the Zeeman sublevels of the ligand field ground state. These low energy excitations and the metastable ligand field states constitute the last stages of the slowing down process after the nuclear decay.

Mössbauer emission spectroscopy of metastable electronic states

In57Fe Mössbauer emission spectroscopy, the parent nucleus57Co is incorporated in the matrix by diffusion, implantation or co-precipitation. Mössbauer emisssion spectra often differ from the corresponding absorption spectra in several aspects. The “anomalous” features (denoted as “after-effects” of nuclear transformation) comprise mainly (a) anomalous charge states, (b) anomalous spin states, and (c) metastable electronic populations within these charge states. The first two classes of effects were extensively studied during the sixties and seventies. More recently, several investigations have shown that, under certain circumstances, the electronic systems of Fe(II) and Fe(III) have not reached the thermal equilibrium within the Mössbauer time window. These effects have been evidenced (i) within the crystal field ground-state manifolds, but also (ii) as spectral contributions from excited crystal field states. In the present paper, an overview of these experiments will be given, together with their theoretical interpretation and in comparison to results obtained with other methods (e.g. optical spectroscopy). Special emphasis will be given to the systems57Co/LiNbO3 and57Co/MgO, where nonequilibrium populations within the6A1 ground manifold of Fe(III) and Γ5g ground manifold of Fe(II) have been observed in an external magnetic field.