Replacement of the distal glycine 139 transforms human heme oxygenase-1 into a peroxidase

The human heme oxygenase-l crystal structure suggests that Gly-139 and Gly- 143 interact directly with iron-bound ligands, We have mutated Gly-139 to a n alanine, leucine, phenylalanine, tryptophan, histidine, or aspartate, and Gly-143 to a leucine, lysine, histidine, or aspartate, All of these mutant s bind heme, but absorption and resonance Raman spectroscopy indicate that the water coordinated to the iron atom is lost in several of the Gly-139 mu tants, giving rise to mixtures of hexacoordinate and pentacoordinate ligati on states. The active site perturbation is greatest when large amino acid s ide chains are introduced. Of the Gly-139 mutants investigated, only G139A catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme t o biliverdin, but most of them exhibit a new H2O2-dependent guaiacol peroxi dation activity. The Gly-143 mutants, all of which have lost the water liga nd, have no heme oxygenase or peroxidase activity, The results establish th e importance of Gly-139 and Gly-143 in maintaining the appropriate environm ent for the heme oxygenase reaction and show that Gly-139 mutations disrupt this environment, probably by displacing the distal helix, converting heme oxygenase into a peroxidase. The principal role of the heme oxygenase acti ve site may be to suppress the ferryl species formation responsible for per oxidase activity.