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Mechanism of formation of the ester linkage between heme and Glu310 of CYP4B1: 18O protein labeling studies.

Cytochrome P450s in the CYP4 family covalently bind their heme prosthetic group to a conserved acidic I-helix residue via an autocatalytic oxidation. This study was designed to evaluate the source of oxygen atoms in the covalent ester link in CYP4B1 enzymes labeled with [18O]glutamate and [18O]aspartate. The fate of the heavy isotope was then traced into wild-type CYP4B1 or the E310D mutant-derived 5-hydroxyhemes. Glutamate-containing tryptic peptides of wild-type CYP4B1 were found labeled to a level of 11-13% 18O. Base hydrolysis of labeled protein released 5-hydroxyheme which contained 12.8 +/- 1.9% 18O. Aspartate-containing peptides of the E310D mutant were labeled with 6.0-6.5% 18O, but as expected, no label was transmitted to recovered 5-hydroxyheme. These data demonstrate that the oxygen atom in 5-hydroxyheme derived from wild-type CYP4B1 originates in Glu310. Stoichiometric incorporation of the heavy isotope from the wild-type enzyme supports a perferryl-initiated carbocation mechanism for covalent heme formation in CYP4B1.