Reaction of human myoglobin and H2O2 - Involvement of a thiyl radical produced at cysteine 110

The human myoglobin (Mb) sequence is similar to other mammalian Mb sequence s, except for a unique cysteine at position 110. Reaction of wild-type reco mbinant human Mb, the C110A variant of human Mb, or horse heart Mb with H2O 2 (protein/H2O2 = 1:1.2 mol/mol) resulted in formation of tryptophan peroxy l (Trp-OO.) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g similar to 2.036) was detected after decay of Trp-OO. that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DM PO) was included in the reaction mixture at protein/DMPO ratios less than o r equal to 1:10 mol/mol, a DMPO adduct exhibiting broad absorptions was det ected. Hyperfine couplings of this radical indicated a DMPO-thiyl radical. Incubation of mild-type human Mb with thiol-blocking reagents prior to reac tion with peroxide inhibited DMPO adduct formation, whereas at protein/DMPO ratios greater than or equal to 1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb following reaction with H2O2 demonstrated the formation of a homodimer (mass of 34,107 +/- 5 a tomic mass units) sensitive to reducing conditions. The human Mb C110A vari ant afforded no dimer under identical conditions. Together, these data indi cate that reaction of wild-type human Mb and H2O2 differs from the correspo nding reaction of other myoglobin species by formation of thiyl radicals th at lead to a homodimer through intermolecular disulfide bond formation.