Isotopic labeling of the heme cofactor in cytochrome p450 and other heme proteins

摘要

A recombinant bacterial expression system that generates ~(13)C-labeled heme or ~(15)N-labeled heme in functional cytochrome P450 enzymes and other heme-containing systems is reported here using a mutant strain of Escherichia coli (HU227) in which theHemA gene is inactive. By synthesizing several isotopomers of aminolevulinic acid with ~(13)C or ~(15)N at different locations, isotopes have been incorporated with high abundance into the heme cofactor of five different cytochrome P450 isoforms, alongwith one peroxidase. Confirmed both ~(13)C- and ~(15)N-incorporation; spectral and catalytic assays show the labeled enzymes produced in this system are functional.