Annette Rompel

Catalytic oxidation of 3,5-Di-tert-butylcatechol by a series of mononuclear manganese complexes: synthesis, structure, and kinetic investigation.

The manganese compounds [Mn(bpia)(OAc)(OCH 3 )](PF 6 ) (1), [Mn(bipa)(OAc)(OCH 3 )](PF 6 ) (2), [Mn(bpia)(Cl) 2 ](ClO 4 ) (3), [Mn(bipa)(Cl) 2 ](ClO 4 ) (4), [Mn(Hmimppa)(Cl) 2 ].CH 3 OH (5), and [Mn(mimppa)(TCC)].2CHCl 3 (6) (bpia= bis-(picolyl)(N-methylimidazole-2-yl)amine; bipa = bis(N-methylimidazole-2-yl)(picolyl)amine; Hmimppa = ((1-meth-ylimidazole-2-yl)methyl)((2-pyridyl)methyl)(2-hydroxyphenyl)amine; TCC = tetrachlorocatechol) were synthesized and characterized by various techniques such as X-ray crystallography, mass spectrometry, IR, EPR, and UV/vis spectroscopy, cyclic voltammetry, and elemental analysis. 1 and 2 crystallize in the triclinic space group P1 (No. 2), 4 and 6 crystallize in the monoclinic space group P2 1 /n (No. 14), and 5 crystallizes in the orthorhombic space group Pna2 1 . Complexes 1-4 are structurally related to the proposed active site of the manganese-dependent extradiol-cleaving catechol dioxygenase exhibiting an N 4 O 2 donor set (1 and 2) or N 4 Cl 2 donor set (3 and 4). Cyclic voltammetric data show that the substitution of oxygen donor atoms with chloride causes a shift of redox potentials to more positive values. These compounds show high catalytic activity regarding the oxidation of 3,5-di-tert-butylcatechol to 3,5-di-tert-butylquinone exhibiting saturation kinetics at high substrate concentrations. The turnover numbers k c a t = (86 ′ 7) h - 1 (1), k c a t = (101 ′ 4) h - 1 (2), k c a t = (230 ′ 4) h - 1 (3), and k c a t = (130 ′ 4) h - 1 (4) were determined from the double reciprocal Lineweaver-Burk plot. Compounds 5 and 6 can be regarded as structural and electronic Mn analogues for substituted forms of Fe-containing intradiol-cleaving catechol dioxygenases. To our knowledge 5 is the first mononuclear Mn(II) compound featuring an N 3 OCl 2 donor set.

Latent and active abPPO4 mushroom tyrosinase cocrystallized with hexatungstotellurate(VI) in a single crystal.

Mushroom tyrosinase isoform abPPO4 (Agaricus bisporus polyphenol oxidase 4) was crystallized by means of an Anderson-type polyoxometalate. The enzyme crystallized as a crystallographic heterodimer containing the zymogen (L-TYR; 64 kDa), the 21 kDa smaller activated form (A-TYR) and the polyoxometalate (POM) within one single crystal in a 1:1:1 ratio.

Substrate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation1

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‐dihydronaphthaline (1) 6,7‐dihydroxy‐1‐(3,4‐dihydroxyphenyl)‐3‐carboxynaphthaline (5) and 2,6‐bis‐(3′,4′‐dihydroxyphenyl)‐1‐carboxy‐3‐oxacyclo‐(3,0)‐pentan‐2‐on‐1‐ene (4) were formed. A reaction mechanism for the formation of (1, 4 and 5) is discussed.

Oxidative switches in functioning of mammalian copper chaperone Cox17

Cox17, a copper chaperone for cytochrome-c oxidase, is an essential and highly conserved protein in eukaryotic organisms. Yeast and mammalian Cox17 share six conserved cysteine residues, which are involved in complex redox reactions as well as in metal binding and transfer. Mammalian Cox17 exists in three oxidative states, each characterized by distinct metal-binding properties: fully reduced mammalian Cox17(0S-S) binds co-operatively to four Cu+; Cox17(2S-S), with two disulfide bridges, binds to one of either Cu+ or Zn2+; and Cox17(3S-S), with three disulfide bridges, does not bind to any metal ions. The E(m) (midpoint redox potential) values for two redox couples of Cox17, Cox17(3S-S)<-->Cox17(2S-S) (E(m1)) and Cox17(2S-S)<-->Cox17(0S-S) (E(m2)), were determined to be -197 mV and -340 mV respectively. The data indicate that an equilibrium exists in the cytosol between Cox17(0S-S) and Cox17(2S-S), which is slightly shifted towards Cox17(0S-S). In the IMS (mitochondrial intermembrane space), the equilibrium is shifted towards Cox17(2S-S), enabling retention of Cox17(2S-S) in the IMS and leading to the formation of a biologically competent form of the Cox17 protein, Cox17(2S-S), capable of copper transfer to the copper chaperone Sco1. XAS (X-ray absorption spectroscopy) determined that Cu4Cox17 contains a Cu4S6-type copper-thiolate cluster, which may provide safe storage of an excess of copper ions.

Crystallization and preliminary X-ray crystallographic analysis of latent isoform PPO4 mushroom (Agaricus bisporus) tyrosinase

Polyphenol oxidase 4 (PPO4) from the natural source A. bisporus was crystallized in its latent precursor form (pro-tyrosinase; Ser2–Thr565) using the 6-tungstotellurate(VI) salt Na6[TeW6O24]·22H2O as a crystallization additive.

Hen Egg-White Lysozyme Crystallisation: Protein Stacking and Structure Stability Enhanced by a Tellurium(VI)-Centred Polyoxotungstate

As synchrotron radiation becomes more intense, detectors become faster and structure‐solving software becomes more elaborate, obtaining single crystals suitable for data collection is now the bottleneck in macromolecular crystallography. Hence, there is a need for novel and advanced crystallisation agents with the ability to crystallise proteins that are otherwise challenging. Here, an Anderson–Evans‐type polyoxometalate (POM), specifically Na6[TeW6O24]⋅22 H2O (TEW), is employed as a crystallisation additive. Its effects on protein crystallisation are demonstrated with hen egg‐white lysozyme (HEWL), which co‐crystallises with TEW in the vicinity (or within) the liquid–liquid phase separation (LLPS) region. The X‐ray structure (PDB ID: 4PHI) determination revealed that TEW molecules are part of the crystal lattice, thus demonstrating specific binding to HEWL with electrostatic interactions and hydrogen bonds. The negatively charged TEW polyoxotungstate binds to sites with a positive electrostatic potential located between two (or more) symmetry‐related protein chains. Thus, TEW facilitates the formation of protein–protein interfaces of otherwise repulsive surfaces, and thereby the realisation of a stable crystal lattice. In addition to retaining the isomorphicity of the protein structure, the anomalous scattering of the POMs was used for macromolecular phasing. The results suggest that hexatungstotellurate(VI) has great potential as a crystallisation additive to promote both protein crystallisation and structure elucidation.

X-ray Absorption Near Edge Structure Spectroscopy to Resolve the in Vivo Chemistry of the Redox-Active Indazolium trans-[Tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019)

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (1, KP1019) and its analogue sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (2, KP1339) are promising redox-active anticancer drug candidates that were investigated with X-ray absorption near edge structure spectroscopy. The analysis was based on the concept of the coordination charge and ruthenium model compounds representing possible coordinations and oxidation states in vivo. 1 was investigated in citrate saline buffer (pH 3.5) and in carbonate buffer (pH 7.4) at 37 °C for different time intervals. Interaction studies on 1 with glutathione in saline buffer and apo-transferrin in carbonate buffer were undertaken, and the coordination of 1 and 2 in tumor tissues was studied too. The most likely coordinations and oxidation states of the compound under the above mentioned conditions were assigned. Microprobe X-ray fluorescence of tumor thin sections showed the strong penetration of ruthenium into the tumor tissue, with the highest concentrations near blood vessels and in the edge regions of the tissue samples.

The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of "Substrate-Guiding Residues" for Enzymatic Specificity.

Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono- and o-diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. The structure reveals that the distinction between mono- and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed.

Purification and characterization of tyrosinase from walnut leaves (Juglans regia)

Graphical abstract Tyrosinase from walnut leaves (Juglans regia) corresponding to the known jrPPO1 sequence was purified and characterized. Two major tyrosinase forms differing only in their C-termini were identified. The first form (jrPPO1(Asp101 → Pro444)) is one amino acid shorter than the second form (jrPPO1(Asp101 → Arg445)).

Cytotoxic effects of novel polyoxotungstates and a platinum compound on human cancer cell lines

The cytotoxicity of a new platinum compound Pt1 [2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedichloroplatin(II)] and six polyoxometalates (POM1–6) on two neuroblastoma cell lines (SHEP-SF and KCN) and an Ewings Sarcoma cell line (CADO–ES-1) was studied. Cisplatin [cis-diamminedichloroplatinum(II)] and carboplatin [cis-diammine(cyclobutanedicarboxylato)platinum(II)] were used as reference agents. Using MTT tests, the cytotoxicity (LD50: lethal doses 50%) of the compounds were measured at different concentrations. After 72 h exposure, the LD50 data for the platinum-containing substances ranged between 4.47×10−6 and 1.91×10−4 M. The SHEP-SF cell line displayed the highest sensitivity to cisplatin. The novel platinum agent Pt1 had a similar cytotoxic effect to the reference agent cisplatin. Both cisplatin and Pt1 were more cytotoxic than carboplatin. The POMs reduced cell viability compared to untreated cells at concentrations between 8.4×10−7 and 3.47×10−5 M. POM1 ([(CH3)4N]2Na6.5(NH4)2[SnII1.5(WO2(OH))0.5(WO2)2(SbW9O33)2]·32H2O) was the most effective polyoxoanion with a mean LD50 value of 8.83×10−6 M in the three cell lines tested. With CADO-ES-1 and KCN cells, POM1 was found to be more effective than the platinum compounds cisplatin, carboplatin and Pt1.