Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the p roximal iron ligand is replaced by a cysteine or tyrosine, have been expres sed and characterized. Resonance Raman studies indicate that the ferric hem e complexes of these proteins, like the complex of the H25A mutant but unli ke that of the wild type, are 5-coordinate high-spin. Labeling of the iron with Fe-54 confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate, Resonance-enhanced tyrosinate modes in the resonance R aman spectrum of the H25Y heme complex provide direct evidence for tyrosina te ligation in this protein. The H25C and H25Y heme complexes are reduced t o the ferrous state by cytochrome P450 reductase but do not catalyze alpha- meso-hydroxylation of the hemt or its conversion to biliverdin. Exposure of the ferrous heme complexes to O-2 does not give detectable ferrous-dioxy c omplexes and leads to the uncoupled reduction of O-2 to H2O2. Resonance Ram an studies show that the ferrous H25C and H25Y heme complexes are present i n both 5-coordinate high-spin and 4-coordinate intermediate-spin configurat ions. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon redu ction to the ferrous state. This is confirmed by the spectroscopic properti es of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observ ed, is thermodynamically allowed. The two proximal ligand mutations thus de stabilize the ferrous-dioxy complex and uncouple the reduction of O-2 from oxidation of the heme group. The proximal histidine ligand, for geometric o r electronic reasons, is specifically required for normal heme oxygenase ca talysis.