Formation of hemoprotein-alkylperoxide complexes demonstrated by ESR and optical absorption spectroscopy

Reactions between alkylhydroperoxides and hemoglobin or myoglobin in the ferric form (met-Hb and -Mb) were investigated by means of simultaneous ESR and optical absorption measurements at 77 K. The optical absorption spectrum observed for the frozen solution, prepared by mixing whale met-Mb and t- or n-butylhydroperoxide (t- and n-BHPO) in the presence of tetramethylammonium hydroxide (TMAOH), shows characteristic absorption maxima at 415, 542 and 574 nm. On the other hand, the ESR spectrum recorded for the same frozen solution reveals the formation of a ferric low-spin complex ( B : g 1 = 1.936, g 2 = 2.196, g 3 = 2.350). The results obtained from ESR titration suggest that complex B has t- or n-BHPO at the sixth coordinate of the heme chromophore. The observed absorption maxima of complex B are similar to the values of relevant t- and n-butylperoxide complexes of (5,10,15,20- tetraphenylporphyrinato)iron(III) (Fe(III)TPP). In addition, the observed g parameters of the complex agree well with those of the Fe(III)TPP-butylperoxide complex, which have the t- or n-butylperoxide anion at the axial position of Fe(III)TPP. Based on the crystal field analysis carried out for complex B and related Fe(III)TPP-peroxide complexes, the axial ligand set of complex B is assigned to be the deprotonated peroxide anion derived from t- or n-BHPO, and the nitrogenous ligand derived from the proximal histidine. The probable coordination structure of complex B is proposed to be the six-coordinate Fe(III)-hemoprotein- − OO-butyl complex. The present complex B is a possible model for transient hemoprotein-peroxide complexes, and could be proposed as the reaction intermediate in the reaction processes of heme containing oxidases, such as P-450 and peroxidases.