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 O·-2 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 Br·-2 oxidation of the 3–4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 × 108M-1s-1; but drops to 8 × 107M-1s-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 × 106M-1s-1) at/or below pH 6.2 then drops to 1.3-1.6 × 106M-1s-1 at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semioxidized Trp· residues upon Br·-2 reaction. The kinetics of Trp·(Lyz) with ascorbate, a reducing species rather larger than O·-2 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)+O·-2 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 O·-2. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O·-2 reactivity. To our knowledge, this is the first report on the kinetics of a protein-O·-2 reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.