IDENTIFICATION OF HISTIDINE-45 AS THE AXIAL HEME IRON LIGAND OF HEME OXYGENASE-2

A truncated, soluble, and enzymatically active form of human heme oxyg enase-2 (Delta HHO2) was expressed in Escherichia coli, To identify th e axial heme ligand of HO-2, His-45 to Ala (Delta H45A) and His-152 to Ala (Delta H152A) mutants have been prepared using this expression sy stem. Delta H45A could form a 1:1 complex with hemin but was completel y devoid of the heme degradation activity, A 5-coordinate-type ferrous NO EPR spectrum was ob served for the heme-Delta H45A complex, The De lta H152A 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 wa s removed by Sephadex G-75 column chromatography since it eluted out o f the column at the void volume, The gel filtration-purified Delta H15 2A exhibited spectroscopic and enzymatic properties identical to those of wild-type. We conclude, in contrast to the previous reports (McCou brey 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- a is the proximal ligand of the heme and is essential for the heme deg radation activity of the enzyme, His-152 appears to play a structural role in stabilization of the heme oxygenase protein.