Disruption of an active site hydrogen bond converts human heme oxygenase-1into a peroxidase

The crystal structure of heme oxygenase-l suggests that Asp-140 may partici pate in a hydrogen bonding network involving ligands coordinated to the hem e iron atom. To examine this possibility, Asp-140 was mutated to an alanine , phenylalanine, histidine, leucine, or asparagine, and the properties of t he purified proteins were investigated, UV-visible and resonance Raman spec troscopy indicate that the distal water ligand is lost from the iron in all the mutants except, to some extent, the D140N mutant. In the D140H mutant, the distal water ligand is replaced by the new His-140 as the sixth iron l igand, giving a bis-histidine complex, The D140A, D140H, and D140N mutants retain a trace (<3%) of biliverdin forming activity, but the D140F and D140 L mutants are inactive in this respect. However, the two latter mutants ret ain a low ability to form verdoheme, an intermediate in the reaction sequen ce. All the Asp-140 mutants exhibit a new peroxidase activity. The results indicate that disruption of the distal hydrogen bonding environment by muta tion of Asp-140 destabilizes the ferrous dioxygen complex and promotes conv ersion of the ferrous hydroperoxy intermediate obtained by reduction of the ferrous dioxygen complex to a ferryl species at the expense of its normal reaction with the porphyrin ring.