Polyphenol oxidases exhibit promiscuous proteolytic activity.

Tyrosinases catalyse both the cresolase and catecholase reactions for the formation of reactive compounds which are very important for industrial applications. In this study, we describe a proteolytic activity of tyrosinases. Two different tyrosinases originating from mushroom and apple are able to cleave the carboxylesterase EstA. The cleavage reaction correlates with the integrity of the active site of tyrosinase and is independent of other possible influencing factors, which could be present in the reaction. Therefore, the cleavage of EstA represents a novel functionality of tyrosinases. EstA was previously reported to degrade synthetic polyesters, albeit slowly. However, the EstA truncated by tyrosinase shows higher degradation activity on the non-biodegradable polyester polyethylene terephthalate (PET), which is a well-established environmental threat.

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution.

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.

S K- and Mo L-edge X-ray absorption spectroscopy to determine metal-ligand charge distribution in molybdenum-sulfur compounds.

Mo L-edge and S K-edge X-ray absorption spectroscopy were applied to investigate the charge distribution between Mo and S in a series of Mo thiolate compounds, which serve as amide-sulfur H-bonding models and exhibit different redox potentials arising from polar group effects and ligand hydrogen bonds near the redox center. For all oxidized complexes, the S K-edge spectra exhibit a thiolate-based pre-edge feature centered at 2470.2 eV and the inflection point oCCurs at 2472.0 eV. No intense pre-edge feature is observed in the spectra for the reduced Mo model compounds and the energy shift of the S K-edge position depends on the S-ligand. Correlations between ligand charge density and the redox potential of the Mo-S cores are observed.

Counting the number of disulfides and thiol groups in proteins and a novel approach for determining the local pKa for cysteine groups in proteins in vivo.

X-ray Absorption Spectroscopy (XAS) is a powerful tool to investigate sulfur in biological molecules. The spectral features are sensitive to the local electronic and geometric environment of the atom; thus, they constitute a fingerprint of the different chemical forms in which the sulfur is present. This allows straightforward detection of the ratio between free thiols and disulfides. Intra- or inter-molecular disulfide bond formation between residues plays an important role in structural and conformational changes in proteins, and such changes can be investigated using sulfur XAS. Also, a thiolate-disulfide equilibrium is involved in the regulation of the redox potential in the cells by means of modulating the concentrations of the reduced (thiolate) and oxidized (disulfide) form of the tripeptide glutathione. Thus, we can monitor the redox state of a cell by means of sulfur XAS. Thiols also exhibit an acid-base equilibrium, and sulfur XAS can be used to determine the local pKa of the -SH group. Here we report examples of how sulfur XAS has been used for these applications.

Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed.

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.

We 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 39,800 Da and the mass of the Populus enzyme was determined to 56,050 Da. Both catechol oxidases are inhibited by thiourea, N-phenylthiourea, dithiocarbamate, and cyanide, but show different pH behavior using catechol as substrate. Atomic absorption spectrosopic 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 to 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 O2(2-) (pi sigma *)-->Cu(II) (dx2 - y2) charge transfer (CT) transition. The absorption band at 580 nm corresponds to the second O2(2)- (pi v*)-->Cu(II) (dx2 - y2) CT transition. The UV/Vis bands in combination with the resonance Raman spectra of oxy catechol oxidase indicate a mu-eta 2:eta 2 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.

Substrate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation.

The substrate specificity of catechol oxidase from Lycopus europaeus towards phenols is examined. The enzyme catalyzes the oxidation of o-diphenols to o-quinones without hydroxylating monophenols, the additional activity of tyrosinase. Substrates containing a -COOH group are inhibitors for catechol oxidase. The products of enzymic oxidation of caffeic acid were analyzed and isolated by HPLC with diode array detection. The neolignans of the 2,3-dihydro-1,4-benzodioxin type (3, 6-8), 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-2,3-dicarboxy-1,2-dihydro naphthaline (1) 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-3-carboxynaphthaline (5) and 2,6-bis-(3',4'-dihydroxyphenyl)-1-carboxy-3-oxacyclo-(3,0)-pent an-2-on-1-ene (4) were formed. A reaction mechanism for the formation of (1, 4 and 5) is discussed.

Sulfur K-edge x-ray absorption spectroscopy: a spectroscopic tool to examine the redox state of S-containing metabolites in vivo.

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.

Oxidation states of the manganese cluster during the flash-induced S-state cycle of the photosynthetic oxygen-evolving complex.

The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flash-induced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR) multiline signal oscillation pattern to the Kok model. The edge spectra of samples given 0, 1, 2, or 3 flashes were combined with EPR information to calculate the pure S-state edge spectra. The edge positions (defined as the zero-crossing of the second derivatives) are 6550.1, 6551.7, 6553.5, and 6553.8 eV for S0, S1, S2, and S3, respectively. In addition to the shift in edge position, the S0--> S1 and S1--> S2 transitions are accompanied by characteristic changes in the shape of the edge, both indicative of Mn oxidation. The edge position shifts very little (0.3 eV) for the S2--> S3 transition, and the edge shape shows only subtle changes. We conclude that probably no direct Mn oxidation is involved in this transition. The proposed Mn oxidation state assignments are as follows: S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).