A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide

Abstract

The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N 2- and O 2-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H 2O 2 were calculated using hyperfine coupling constants of a H2,6 = 1.3 G and a H3,5 = 7.0 G for the ring and a β H1 = 16.7 G and a β H2 = 14.2 G for the methylene protons, and are consistent with a highly constrained, conformationally unstable tyrosyl radical. Spectra obtained at later time points contained a mixture of the 3 s signal and another signal that was insufficiently resolved for simulation. Efficient spin trapping with 3,5-dibromo-4-nitrosobenzenesulfonic acid was observed only when the spin trap was present at the time of H 2O 2 addition. Spin trapping experiments with either 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or perdeuterated 2-methyl-2-nitrosopropane (MNP- d 9 ), which have been shown to trap tyrosyl radicals, were nearly equally effective when the spin trap was added before or 10 min after the addition of H 2O 2. The superhyperfine structure of the ESR spectra obtained from Pronase-treated MNP- d 9 /•metMb confirmed the assignment to a tyrosyl radical. Delayed spin trapping experiments with site-directed mutant myoglobins in which either Tyr-103 or Tyr-146 was replaced by phenylalanine indicated that radical adduct formation with either DMPO or MNP- d 9 requires the presence of Tyr-103 at all time points, implicating that residue as the radical site.