An EPR study of the peroxyl radicals induced by hydrogen peroxide in the haem proteins

The reaction of hydrogen peroxide H2O2 With horse heart metmyoglobin (HH me tMb), sperm whale metmyoglobin (SW metMb) and human metHb (metHbA) was stud ied at pH 6-8 by low temperature (10 K) EPR spectroscopy with the emphasis on the peroxyl radicals formed during the reaction. The same type of peroxy l radical was found in both myoglobin systems, as was concluded from close similarities in the spectroscopic properties of the radicals and in their k inetic dependences. This is consistent with previous reports of the peroxyl radical being localised on the Trp14 of SW and I-III myoglobins. There are two types of peroxyl radical found in the metHbA/H2O2 system, one (ROO-I) having spectral parameters, kinetic and pH dependences similar to those of the peroxyl radical found in both myoglobin systems. The other peroxyl radi cal (ROO-II) found in metHbA treated with H2O2 has slightly different, thou gh distinguishable, spectral parameters and a significantly different kinet ic dependence as compared to those of the peroxyl radical common for all th ree proteins studied (ROO-I). The concentration of ROO-I radical formed in the three proteins on addition of H2O2 correlates with the effectiveness of incorporating molecular oxygen into styrene oxide reported before for thes e three proteins. It is shown that a different distance from Trp14 to haem iron in the three proteins might be the structural basis for the different yield of the peroxyl radical and the different efficiency of incorporation of molecular oxygen into styrene. The site of the peroxyl radical found onl y in metHbA (ROO-II) is speculated to be the Trp37 residue of the beta -sub unit of HbA. (C) 2001 Elsevier Science B.V. All rights reserved.