Electron transfer between a tyrosyl radical and a cysteine residue in hemoproteins: spin trapping analysis.

We investigated electron transfer between a tyrosyl radical and cysteine residue in two systems, oxyhemoglobin (oxyHb)/peroxynitrite/5,5-dimethyl-1-pyrroline N-oxide (DMPO) and myoglobin (Mb)/hydrogen peroxide/DMPO, using a combination of techniques including ESR, immuno-spin trapping (IST), and ESI/MS. These techniques show that the nitrone spin trap DMPO covalently binds to one or more amino acid radicals in the protein. Treating oxyHb with peroxynitrite and Mb with H2O2 in the presence of a low DMPO concentration yielded secondary Cys-DMPO radical adduct exclusively, whereas in the presence of high DMPO, more of the primary Tyr-DMPO radical adduct was detected. In both systems studied, we found that, at high DMPO concentrations, mainly tyrosyl radicals (Hb-Tyr42/Tyr24 and Mb-Tyr103) are trapped and the secondary electron-transfer reaction does not compete, whereas in the presence of low concentrations of DMPO, the secondary reaction predominates over tyrosyl trapping, and a thiyl radical is formed and then trapped (Hb-Cys93 or Mb-Cys110). With increasing concentrations of DMPO in the reaction medium, primary radicals have an increasing probability of being trapped. MS/MS was used to identify the specific Tyr and Cys residues forming radicals in the myoglobin system. All data obtained from this combination of approaches support the conclusion that the initial site of radical formation is a Tyr, which then abstracts an electron from a cysteine residue to produce a cysteinyl radical. This complex phenomenon of electron transfer from one radical to another has been investigated in proteins by IST, ESR, and MS.