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 approximately 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. (1994) 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 v(CO) and v(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.(ABSTRACT TRUNCATED AT 250 WORDS)
Sun
J
J
Department of Chemistry, Biochemistry, & Molecular Biology, Oregon Graduate Institute of Science & Technology, Portland 97291-1000.
Loehr
T M
TM
Wilks
A
A
Ortiz de Montellano
P R
PR
D002248:Carbon Monoxide / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D017354:Point Mutation; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D008024:Ligands; D006418:Heme; D006639:Histidine; D002248:Carbon Monoxide; D006419:Heme Oxygenase (Decyclizing)