Investigation of sulfur containing amino acids at the lipoxygenase active site using a platinum complex.

International audienceInactivation of native soybean lipoxygenase-1 was observed upon preincubation with (NEt4)[PtCl3(P(Bun)3)]. Removal of the platinum complex(es) from the inactivated enzyme by treatment with sodium diethyldithiocarbamate (Naddtc) which reverses methionine but not cysteine binding, restores most of the activity. Linoleic acid, an enzyme substrate, protects it from inactivation. The quenching of the fluorescence of the putative active site tryptophans which accompanies inactivation disappears after Naddtc reactivation. The (NEt4)[PtCl3(P(Bun)3)]-inactivated enzyme iron(II) cannot be oxidized at variance with that of the native or Naddtc reactivated enzyme, as checked by EPR spectroscopy. These results show that at least one methionine is close to the iron binding site in soybean lipoxygenase-1

Cu Polyimidazole Thioether Complexes : Comparison of RDF's Reconstructed from XAFS and XRD Data

Radial distribution functions (RDF) reconstructed from XAFS data (Cu K-edge at low and room temperatures) are compared with RDFs, constructed from XRD crystallographic data of Cu polyimidazole thioether complexes. New EDA software package possibilities are used to reconstruct RDFs: (i) model independent RDF reconstruction, and (ii) multi shell - cumulant fitting. RDFs obtained from XRD data are broadened with Debye Waller factors calculated from XAFS. This way the structural disorder (set of distances) and thermal (vibrational) disorder are quantitatively taken into account in each RDF reconstruction approach. Within experimental error the three sets of RDFs agree well with each other for the Cu-N bonds (N = imidazole) and the Cu-S bonds (S = thioether), which are in the range of 1.8 - 2.3 Å. However, they differ significantly for weakly bound ligands whose bond lengths are in the range 2.3-2.7 Å

Soybean lipoxygenases-1, -2a, -2b and -2c do not contain PQQ.

International audienceSoybean isoenzymes lipoxygenases-1, -2a, -2b and -2c were examined spectroscopically for the presence of covalently bound pyrrolo quinoline quinone (PQQ) after derivatization by phenylhydrazine (PH), 2,4-dinitrophenylhydrazine (DNPH) and 3-methyl-2-benzothiazolinone hydrazone (MBTH). DNPH derivatization of PQQ after a pronase digestion step of lipoxygenase-1 in the presence of an anion exchange gel fixing the cofactor was also investigated. None of these experiments provided evidence for the presence of PQQ contrary to previous report by Van der Meer et al (1). We have checked, by EPR spectroscopy, that the three reactants used were able to reduce the active site ferric iron. Our results were confirmed by the absence of enzyme inhibition by cis- and trans-1,2-diaminocyclohexane or benzylamine in the presence of NaBH3CN which have been reported to react with PQQ and to inactivate quinoproteins (2,3)