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with the crystal structure, the ferric low spin state of P450SP␣
was retained irrespective of the substrate binding. These results
also indicate that the shift in redox potential of the heme that is
induced by substrate binding, which is generally indispensable
for the reductive activation of molecular oxygen, is not essential
for the H2O2-dependent P450s. Crystallographic studies on
substrate binding revealed that the C␣ carbon of the bound
palmitic acid in Conformation B is situated close to the heme
iron (4.5 Å). This conformation explains the highly selective
␣-hydroxylation of fatty acid. Surprisingly, mutations at
the active site and at the F–G loop of P450SP␣ did not impair the
high regioselectivity. The crystal structures of the L78F and
F288G mutants revealed that the location of the bound palmitic
acid was not affected by these mutations. These results imply
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JOURNAL OF BIOLOGICAL CHEMISTRY 29949