Reaction of Human Myoglobin and H2O2

Abstract

The human myoglobin (Mb) sequence is similar to other mammalian Mb sequences, except for a unique cysteine at position 110. Reaction of wild-type recombi- nant human Mb, the C110A variant of human Mb, or horse heart Mb with H2O2 (protein/H2O2 ⴝ 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO䡠) and tyrosine phenoxyl radicals as detected by EPR spec- troscopy at 77 K. For wild-type human Mb, a second radical (g ⬃ 2.036) was detected after decay of Trp-OO䡠 that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was included in the reaction mixture at protein/DMPO ratios <1:10 mol/mol, a DMPO adduct ex- hibiting broad absorptions was detected. Hyperfine cou- plings of this radical indicated a DMPO-thiyl radical. Incubation of wild-type human Mb with thiol-blocking reagents prior to reaction with peroxide inhibited DMPO adduct formation, whereas at protein/DMPO ra- tios >1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb fol- lowing reaction with H2O2 demonstrated the formation of a homodimer (mass of 34,107 ⴞ 5 atomic mass units) sensitive to reducing conditions. The human Mb C110A variant afforded no dimer under identical conditions. Together, these data indicate that reaction of wild-type human Mb and H2O2 differs from the corresponding re- action of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolec- ular disulfide bond formation.