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

Yi Liu, Pierre Moenne-Loccoz, Dean P. Hildebrand, Angela Wilks, Thomas M. Loehr, A. Grant Mauk, Paul R. Ortiz De Montellano

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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 α-meso-hydroxylation of the heine 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.

Original languageEnglish (US)
Pages (from-to)3733-3743
Number of pages11
JournalBiochemistry
Volume38
Issue number12
DOIs
StatePublished - Mar 23 1999

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Heme Oxygenase (Decyclizing)
Heme
Histidine
Cysteine
Tyrosine
Oxidoreductases
Iron
Ligands
NADPH-Ferrihemoprotein Reductase
Biliverdine
Hydroxylation
Proteins
Heme Oxygenase-1
Carbon Monoxide
Catalysis
Labeling
Ligation
Raman scattering
Oxidation
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Liu, Y., Moenne-Loccoz, P., Hildebrand, D. P., Wilks, A., Loehr, T. M., Mauk, A. G., & Ortiz De Montellano, P. R. (1999). Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase. Biochemistry, 38(12), 3733-3743. https://doi.org/10.1021/bi982707s

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase. / Liu, Yi; Moenne-Loccoz, Pierre; Hildebrand, Dean P.; Wilks, Angela; Loehr, Thomas M.; Mauk, A. Grant; Ortiz De Montellano, Paul R.

In: Biochemistry, Vol. 38, No. 12, 23.03.1999, p. 3733-3743.

Research output: Contribution to journalArticle

Liu, Y, Moenne-Loccoz, P, Hildebrand, DP, Wilks, A, Loehr, TM, Mauk, AG & Ortiz De Montellano, PR 1999, 'Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase', Biochemistry, vol. 38, no. 12, pp. 3733-3743. https://doi.org/10.1021/bi982707s
Liu, Yi ; Moenne-Loccoz, Pierre ; Hildebrand, Dean P. ; Wilks, Angela ; Loehr, Thomas M. ; Mauk, A. Grant ; Ortiz De Montellano, Paul R. / Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase. In: Biochemistry. 1999 ; Vol. 38, No. 12. pp. 3733-3743.
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AU - Loehr, Thomas M.

AU - Mauk, A. Grant

AU - Ortiz De Montellano, Paul R.

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N2 - 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 α-meso-hydroxylation of the heine 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.

AB - 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 α-meso-hydroxylation of the heine 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.

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