Histidinyl radical formation in the self-peroxidation reaction of bovine copper-zinc superoxide dismutase.
In the absence of suitable oxidizable substrates, the peroxidase reaction of copper-zinc superoxide dismutase (SOD) oxidizes SOD itself, ultimately resulting in its inactivation. A SOD-centered free radical adduct of 2-methyl-2-nitrosopropane (MNP) was detected upon incubation of SOD with the spin trap and a hydroperoxide (either H(2)O(2) or peracetic acid). Proteolysis by Pronase converted the anisotropic electron paramagnetic resonance (EPR) spectrum of MNP/(center dot)SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine + HO(center dot)) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/(center dot)SOD, suggesting that the latter was centered on a histidine residue. An additional hyperfine coupling was detected when histidine specifically (13)C-labeled at C-2 of the imidazole ring was used, providing evidence for trapping at that atom. All of the experimental spectra were convincingly simulated assuming hyperfine couplings to 2 nearly equivalent nitrogen atoms and 2 different protons, also consistent with trapping at C-2 of the imidazole ring. Free histidinyl radical consumed oxygen, implying peroxyl radical formation. MNP-inhibitable oxygen consumption was also observed when cuprous SOD but not cupric SOD was added to a H(2)O(2) solution. Formation of 2-oxohistidine, the stable product of the SOD-hydroperoxide reaction, required oxygen and was inhibited by MNP. These results support formation of a transient SOD-peroxyl radical.
Gunther
Michael R
MR
Department of Biochemistry and Molecular Pharmacology, West Virginia University, Morgantown, West Virginia 26506, USA. mgunther@hsc.wvu.edu
Peters
J Andrew
JA
Sivaneri
Meena K
MK
D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006639:Histidine / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism*
D005609:Free Radicals; D017665:Hydroxyl Radical; D006639:Histidine; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D010100:Oxygen