IDENTIFICATION OF HISTIDINE-25 AS THE HEME LIGAND IN HUMAN LIVER HEME OXYGENASE

Electronic and resonance Raman spectroscopic studies are reported for the His25Ala mutant of human liver heme oxygenase (HO) and its complex with heme. In the oxidized (ferric) form of the enzyme.substrate complex, the heme is shown to be in a high-spin, five-coordinate state. This is distinct from the same complex in the wild-type enzyme in which the heme is six-coordinate, ligated to a proximal histidine and a water molecule in an environment reminiscent of aquometmyoglobin. The reduced (ferrous) form of the complex of the H25A heme oxygenase mutant has lost the very prominent resonance Raman band at similar to 217 cm(-1) seen in the wild-type complex that has been unambiguously assigned to the proximal Fe-N(His) vibrational frequency [Sun et al. (1993) Biochemistry 32, 14151; Takahashi et al. (1993) Biochemistry 33, 1010]. The absence of this band in the spectrum of the mutant protein definitively identifies His 25 as the proximal ligand of the heme substrate. Furthermore, this ferrous heme-H25A HO complex exists as an equilibrium mixture between a five-coordinate, high-spin species and a four-coordinate, intermediate-spin species. Although the H25A mutant protein shows no heme oxygenase activity, the heme is competent to bind carbon monoxide. Studies of the CO adduct of the H25A HO complex show nu(CO) and nu(Fe-CO) frequencies at 1960 and 529 cm(-1), respectively, that are characteristic of a hydrophobic carbon monoxide binding site on a heme with a weak proximal ligand. Furthermore, these frequencies indicate that the ligand trans to the CO in the adduct cannot be a histidine, a result that is entirely consistent with the absence of the His 25 ligand in the enzyme with the point mutation. The fifth ligand in the ferric and ferrous forms of the heme-H25A HO is ruled out as being from another histidine or a tyrosine. However, the exact identity of the weak ligand as well as the site of attachment (proximal versus distal, relative to the wild type enzyme) are not known at the present time.