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

Efficient and Selective Isotopic Labeling of Hemes to Facilitate the Study of Multiheme Proteins

“…This modified strain was then transformed with the vector pEC86 containing the ccmABCDEFGH (cytochrome c maturation) genes, that endows E. coli with the ability to correctly incorporate hemes in multiheme cytochromes c , when growing aerobically (23). This increases the versatility of this novel hemA E. coli strain with respect to the type of cytochromes it can express efficiently, versus previous methods (30,32,33). …”

“…Although Woodward et al (30) and Bryson et al (33) reported strategies to isotopically label hemes, using mutants of E. coli incapable of synthesizing either the heme or dALA, respectively, these methods are limited with respect to the type of cytochromes that can be expressed. In particular, c -type cytochromes could not be obtained.…”

“…Toward this end, several approaches have been developed involving the supplementation of the growth media with specifically labeled heme cofactors (30,31) or with isotopically labeled heme precursor -aminolevulinic acid (dALA) (3133). Also, to guarantee that the uptake of these substituents is efficient, bacterial strains that are incapable of synthetizing hemes cofactors were created by deleting genes that are responsible for biosynthesis of dALA in the cell, such as the hemA gene (33,34). …”