title: Histidine-132 does not stabilize a distal water ligand and is not an important residue for the enzyme activity in heme oxygenase-1.
abstract: Heme oxygenase is a key enzyme in the oxygen-dependent heme catabolism pathway. In order to clarify the role of highly conserved His132 in heme oxygenase isoform-1, we have prepared 30 kDa truncated rat heme oxygenase mutants in which His132 has been replaced by Ala, Gly, and Ser. The expressed recombinant mutant proteins were isolated in inclusion bodies and were 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 gel filtration column chromatography, as it eluted out of the column at the void volume. The gel filtration-purified heme oxygenase mutants have spectroscopic and enzymatic properties identical to those of wild type. The hemin complex of the H132A mutant exhibits a transition between a high-spin acid form and a low-spin alkaline form with a pKa value of 7.6 identical to that in the wild-type complex. These results demonstrate that His132 in heme oxygenase does not link to the coordinated water molecule and is not an essential residue for the enzyme activity. These results are in accordance with our previous preliminary results [Ito-Maki, M., Ishikawa, K., Mansfield Matera, K., Sato, M., Ikeda-Saito, M., & Yoshida, T. (1995) Arch. Biochem. Biophys. 317, 253-258] but contradict a recent report that His132 is the distal base linked to the coordinated water molecule and an important residue for enzyme catalysis [Wilks, A., Ortiz de Montellano, P. R., Sun, J., & Loehr, T. M. (1996) Biochemistry 35, 930-936]. Prolonged storage of the solubilized fraction from the inclusion bodies of the mutants, H132S in particular, results in an increase in the void volume fraction with a concomitant decrease of the 30 kDa fraction. We infer that His132 plays a structural role in stabilization of the heme oxygenase protein.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Matera
authors.forename: H
authors.initials: H
authors.lastname: Zhou
authors.forename: C T
authors.initials: CT
authors.lastname: Migita
authors.forename: S E
authors.initials: SE
authors.lastname: Hobert
authors.forename: K
authors.initials: K
authors.lastname: Ishikawa
authors.forename: K
authors.initials: K
authors.lastname: Katakura
authors.forename: H
authors.initials: H
authors.lastname: Maeshima
authors.forename: T
authors.initials: T
authors.lastname: Yoshida
authors.forename: M
authors.initials: M
authors.lastname: Ikeda-Saito
mesh_terms: D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D009154:Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D014867:Water / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D014867:Water; D006639:Histidine; D006419:Heme Oxygenase (Decyclizing)