Abstract
A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (DeltaHHO2) was expressed in Escherichia coli. To identify the axial heme ligand of HO-2, His-45 to Ala (DeltaH45A) and His-152 to Ala (DeltaH152A) mutants have been prepared using this expression system. DeltaH45A 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-DeltaH45A complex. The DeltaH152A 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 DeltaH152A exhibited spectroscopic and enzymatic properties identical to those of wild-type. We conclude, in contrast to the previous reports (McCoubrey and Maines (1993) Arch. Biochem. 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 heme 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.
Highlights
Heme oxygenase (HO),1 a microsomal protein, catalyzes the regiospecific oxidative degradation of iron protoporphyrin IX to biliverdin, CO, and Fe in the presence of NADPH-cytochrome P-450 reductase, which functions as an
Amino acid sequence similarity between the two isoforms is only about 40% with an extra N-terminal 20-amino acid sequence present in HO-2; there are two stretches of highly conserved sequences with matched predicted secondary structure [11]. Both HO-1 and HO-2 have hydrophobic sequences at their C-terminal ends which have been proposed to be involved in binding to microsomal membranes [12,13,14]
Our spectroscopic and enzymatic examinations of the 28-kDa tryptic HO-2 fragment-heme complex has shown that both isoforms display the same enzymatic activity and that the two isoforms have homologous active site structures: the proximal ligand of the heme HO-2 complex is a neutral imidazole of the histidine residue [18]
Summary
Heme oxygenase (HO),1 a microsomal protein, catalyzes the regiospecific oxidative degradation of iron protoporphyrin IX (heme hereafter) to biliverdin, CO, and Fe in the presence of NADPH-cytochrome P-450 reductase, which functions as an. A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (⌬HHO2) was expressed in Escherichia coli.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
