Identification of histidine 45 as the axial heme iron ligand of heme oxygenase-2

Kazunobu Ishikawa, Kathryn Mansfield Matera, Hong Zhou, Hiroshi Fujii, Michihiko Sato, Tetsuhiko Yoshimura, Masao Ikeda-Saito, Tadashi Yoshida

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Abstract

A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (ΔHHO2) was expressed in Escherichia coli. To identify the axial heine ligand of HO-2, His-45 to Ala (ΔH45A) and His-152 to Ala (ΔH152A) mutants have been prepared using this expression system. ΔH45A could form a 1:1 complex with hemin but was completely devoid of the heme degradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-ΔH45A complex. The ΔH152A mutant was expressed as an inclusion body and was recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 column chromatography since it eluted out of the column at the void volume. The gel filtration-purified ΔH152A exhibited spectroscopic and enzymatic properties identical to those of wild- type. We conclude, in contrast to the previous reports (McCoubrey and Maines (1993) Arch. Bioshem. Biophys. 302, 402-408; McCoubrey, W. K. Jr., Huang, T. J., and Maines, M. (1997) J. Biol. Chem. 272, 12568-12574), that His-45, but not His-152, in heine oxygenase isoform-2 is the proximal ligand of the heme and is essential for the heme degradation activity of the enzyme. His-152 appears to play a structural role in stabilization of the heme oxygenase protein.

Original languageEnglish
Pages (from-to)4317-4322
Number of pages6
JournalJournal of Biological Chemistry
Volume273
Issue number8
DOIs
Publication statusPublished - 1998 Feb 20

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Ishikawa, K., Matera, K. M., Zhou, H., Fujii, H., Sato, M., Yoshimura, T., Ikeda-Saito, M., & Yoshida, T. (1998). Identification of histidine 45 as the axial heme iron ligand of heme oxygenase-2. Journal of Biological Chemistry, 273(8), 4317-4322. https://doi.org/10.1074/jbc.273.8.4317