HETEROLYTIC VERSUS HOMOLYTIC PEROXIDE BOND-CLEAVAGE BY SPERM WHALE MYOGLOBIN AND MYOGLOBIN MUTANTS

Despite extensive work on homolytic versus heterolytic peroxide bond cleavage by iron porphyrins, the nature of the corresponding reactions catalyzed by myoglobin (Mb) and hemoglobin (Hb) remains unclear. These hemoproteins react with peroxides to give a ferryl (Fe(IV)=O) complex and a protein radical, but the coupling of dioxygen bond cleavage to protein radical formation is obscure. This process is examined here with the help of 4-hydroperoxy-4-methyl-2,6-di-tert-butyl-cyclohexa-2,5-dien-1-one (BHTOOH), a compound that is converted heterolytically to an alcohol and homolytically to several rearranged products. The homolytic/heterolytic product ratio for recombinant sperm whale Mb (0.53) is the same as for native sperm whale Mb (0.57) and slightly higher than that for bovine Hb (0.44). Reaction of the hydroperoxide with Fe2+ and low concentrations of ascorbate results in a much lower rate of peroxide cleavage and overwhelming production of homolytic products. High concentrations of ascorbate prevent the formation of rearranged homolytic products by reducing the alkoxy radical to the alkoxide anion. Reaction of [O-18]BHTOOH with Mb or Hb yields rearranged products that retain >95% of the O-18-labeled peroxide oxygen. This suggests that oxidation of the hydroperoxide to the hydroperoxy radical (ROOH --> ROO.) is not a major reaction because the peroxy radical is expected to undergo exchange with molecular oxygen. The extent of doubly (H-2/O-18) labeled products formed in incubations of [O-18]BHTOOH and [H-2]BHTOOH with Mb suggests that 92% of the radical products arise by peroxide homolysis and 8% via the hydroperoxy radical route. Similar homolytic/heterolytic product ratios for the Tyr-151 --> Phe (0.55), Tyr-103 --> Phe/Lys-102 --> Gln (0.63), and His-64 --> Val (0.57) mutants of sperm whale Mb show that partitioning between the two pathways is insensitive to proximal hydrogen bonding or electron-donating residues. Peroxide cleavage is doubled in the His-64 --> Val mutant due to conversion of the iron to the pentacoordinated state without significantly altering the homolytic/heterolytic ratio. In sum, the homolytic/heterolytic ratio for Mb of approximately 0.5 is apparently determined by the identity of the fifth iron ligand and the general physical properties of the heme site rather than by identifiable acid-base or electron transfer interactions with specific amino acid residues.