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.
Baer
Brian R
BR
Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.
Kunze
Kent L
KL
Rettie
Allan E
AE
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics; D001224:Aspartic Acid; D001665:Binding Sites; D004952:Esters; D018698:Glutamic Acid; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D010446:Peptide Fragments; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D014357:Trypsin
D004952:Esters; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006418:Heme; D001189:Aryl Hydrocarbon Hydroxylases; C095029:cytochrome P-450 CYP4B1; D014357:Trypsin