Role of heme types in heme-copper oxidases: Effects of replacing a heme b with a heme o mimic in an engineered heme-copper center in myoglobin

To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called Cu_BMb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an ∼4 nm blue shift in the Soret band and ∼20 mV decrease in the heme reduction potential. In a control experiment, the heme b in Cu_BMb was also replaced with a mesoheme, which resulted in an ∼13 nm blue shift and ∼30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted Cu_BMb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type Cu_BMb. In reaction with O₂, Cu_BMb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by ∼19-fold in the heme o mimic-substituted Cu_BMb (from 0.028 s^-1 to 0.0015 s^-1), while the mesoheme-substituted Cu_BMb shared a similar heme degradation rate with that of Cu_BMb (0.023 s^-1). No correlation was found between the heme reduction potential and its O₂ reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.