Demonstration that histidine 25, but not 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.
Ito-Maki
M
M
Department of Biochemistry, Yamagata University School of Medicine, Japan.
Ishikawa
K
K
Matera
K M
KM
Sato
M
M
Ikeda-Saito
M
M
Yoshida
T
T
D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D051381:Rats; D013057:Spectrum Analysis
D006639:Histidine; D006419:Heme Oxygenase (Decyclizing)