title: Interactions of superoxide anion with enzyme radicals: kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer.
abstract: The kinetics of O2*- reaction with semi-oxidized tryptophan radicals in lysozyme, Trp*(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp*(Lyz) radicals were formed by Br2*- oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 x 10(8) M(-1) s(-1); but drops to 8 x 10(7) M(-1) s(-1) or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp*(Lyz) + Trp*(Lyz) recombination reaction is about (4-7 x 10(6) M(-1) s(-1)) at/or below pH 6.2 then drops to 1.3-1.6 x 10(6) M(-1) s(-1) at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semi-oxidized Trp* residues upon Br2*- reaction. The kinetics of Trp*(Lyz) with ascorbate, a reducing species rather larger than O2*- have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals--leading to the repair of the semi-oxidized Trp*(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp*(Lyz) + O2*- reaction, as is most of the Trp*(Lyz) + Trp*(Lyz) reaction. However, the kinetic behavior of Trp*(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O2*-. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O2*- reactivity. To our knowledge, this is the first report on the kinetics of a protein-O2*- reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.
authors.affiliation: Laboratoire de Photobiologie, Muséum National d'Histoire Naturelle, Paris, France. santus@mnhn.fr
authors.forename: R
authors.initials: R
authors.lastname: Santus
authors.forename: L K
authors.initials: LK
authors.lastname: Patterson
authors.forename: G L
authors.initials: GL
authors.lastname: Hug
authors.forename: M
authors.initials: M
authors.lastname: Bazin
authors.forename: J C
authors.initials: JC
authors.lastname: Mazière
authors.forename: P
authors.initials: P
authors.lastname: Morlière
mesh_terms: D000838:Anions; D001205:Ascorbic Acid / Q000494:pharmacology; D004579:Electron Transport; D005609:Free Radicals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008823:Micelles; D009113:Muramidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000494:pharmacology; D011675:Pulse Radiolysis; D013481:Superoxides / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D000838:Anions; D005609:Free Radicals; D008823:Micelles; D013481:Superoxides; D014443:Tyrosine; D014364:Tryptophan; D009113:Muramidase; D001205:Ascorbic Acid; D010100:Oxygen