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His132 in heme oxygenase isoform-1, we have prepared 30 kDa truncated rat heme oxygenase mutants in which His132 has been replaced by Ala, Gly, and Ser.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.

Use of Heme Compounds as Iron Sources by Pathogenic Neisseriae Requires the Product of the hemO Gene

“…This phenomenon was observed with other, eukaryotic heme oxygenases and C. diphtheriae HmuO when they were overexpressed in E. coli (4,55). (1997) did not confirm the role of His-132 in heme degradation (23). The corresponding part of neisserial proteins is not well conserved and contains a tryptophan residue instead of a histidine at position 132.…”

Identification of an Escherichia coli O157:H7 heme oxygenase with tandem functional repeats

“…Bacterial and mammalian HOs use the CPR-NADPH system, ferredoxin, flavodoxin, or ascorbic acid as reducing partners in vitro (11,34,(38)(39)(40)(41). To effectively monitor and quantify the HO activity of ChuS, 50 M ascorbic acid was used.…”

The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases

“…Although the overall similarity is only 20-30% between AtHO1 and 2 and the related HOs, a number of conserved amino acid clusters can be seen throughout their coding regions. One cluster contains a positionally conserved His (His-198 in AtHO1) shown to be important for structural stability in human HOs (43). A second cluster near the N terminus of human HOs (residues 76-96 in AtHO1) is required for the oxidative cleavage and employs a His to bind the heme substrate (44).…”