Replacement of the Proximal Histidine Iron Ligand by a Cysteine or Tyrosine Converts Heme Oxygenase to an Oxidase

Abstract

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.

Electrochemical reduction of ferrous α-verdoheme in complex with heme oxygenase-1

“…This midpoint potential value agreed closely with those reported previously, i.e. −0.076 V vs NHE for heme-rHO-1 complex [30] and −0.065 V vs NHE for heme-human HO-1 complex [31] (Table 1). The slope of the Nernst plot, ~0.084, indicated that this process is a one-electron oxidation of ferrous heme.…”

Measurement of the Heme Affinity for Yeast Dap1p, and Its Importance in Cellular Function

“…These frequencies are characteristic of a 5-coordinate high-spin ferric heme (23). In porphyrins bound to a proximal histidine, the ν 4 mode dominates the high-frequency spectra, but in Dap1p, as in other hemoproteins lacking a proximal histidine, the ν 4 and ν 3 modes have comparable intensities (24,25). Attempts to confirm the iron(III) ligation to a tyrosine with RR experiments using a 514.5-nm excitation were inconclusive (data not shown).…”

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

“…The frequencies of the phenolate modes observed for a number of heme iron-tyrosinate proteins are listed in Table 1 [20,[29][30][31][32][33][34]. In general, ferric-heme proteins with tyrosinate ligation have been found to have penta-or hexa-HS heme states.…”