Peroxidase-like activity of L29H myoglobin with two cooperative distal histidines on electrode using O2 as an oxidant

We recently showed that by introduction of a second distal histidine (Leu29 to His29 mutation) in the heme pocket of sperm whale myoglobin (Mb), L29H Mb mutant exhibits an enhanced peroxidase activity compared to wild type (WT) Mb using H2O2 as an oxidant. In this study, we further showed that when L29H Mb was immobilized in didecyldimethylammonium bromide (DDAB) on glassy carbon electrode (GCE), it can efficiently reduce O2 in aerobic buffer solution to H2O2, followed by electrode-driven oxidation of guaiacol, a common peroxidase substrate, which is more effective (∼3.6-fold at pH = 7.0) than that of WT Mb with a single distal His64. Moreover, the peroxidase-like activity of immobilized L29H Mb was found to be affected by pH values, i.e., the slope of the plot of reduction peak vs. guaiacol concentration was increased from (0.83 ± 0.04) × 10−3 μA/mM at pH = 8.0 to (3.08 ± 0.07) × 10−3 μA/mM at pH = 6.0, which suggests that the distal histidines play a key role in reductive activation of O2 by providing protons. Since there is no native peroxidase with two distal histidines and native peroxidase usually uses H2O2 as an oxidant, this study is thus instructive for creating functional enzymes beyond those in nature.