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, memb rane-anchoring segment, and its His25 --> Ala and His132 --> Ala mutan ts have been prepared by site-directed mutagenesis and expression in E scherichia coli. We found that wild-type enzyme can degrade heme to bi liverdin, but its specific activity was about one-fifth that of the na tive, full-length enzyme, suggesting that the C-terminal segment is im portant 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 hem e-heme oxygenase H25A complex. Hence, we conclude that His25 is the pr oximal axial ligand of the heme iron and is essential for the heme deg radation activity of the enzyme. (C) 1995 Academic Press, Inc.