Demonstration that histidine-25, but not histidine-132, is the axial heme ligand in rat heme oxygenase-1

A truncated, soluble rat heme oxygenase-1 lacking its C-terminal, membrane-anchoring segment, and its His25 → Ala and His132 → Ala mutants have been prepared by site-directed mutagenesis and expression in Escherichia coli. We found that wild-type enzyme can degrade heme to biliverdin, but its specific activity was about one-fifth that of the native, full-length enzyme, suggesting that the C-terminal segment is important for accepting electrons from NADPH cytochrome P450 reductase. His132 → Ala mutant had an enzyme activity comparable to that of the wild-type enzyme; hence, the highly conserved His132 is not essential for the display of the heme oxygenase activity. In contrast, His25 → Ala mutation completely abolished the enzyme′s catalytic activity. A five-coordinate type ferrous NO EPR spectrum was observed for the heme-heme oxygenase H25A complex. Hence, we conclude that His25 is the proximal axial ligand of the heme iron and is essential for the heme degradation activity of the enzyme.