(A) Single-point FECH activity was determined in vitro by measuring the amount of 55Fe incorporation into the protoporphyrin IX analog, deuteroporphyrin (DP). FECH has basal activity that is significantly increased by PKA-mediated phosphorylation. This increase in activity was dependent upon both DP and ATP, highlighting the substrate specificity of the assay for DP and the dependence on phosphorylation. (B) Kinetic analyses were subsequently performed with 0.1 µM FECH, 3 µM DP, and 0.2–100 µM 55FeCl3 at 25°C. Phosphorylation of FECH leads to a statistically significant increase in maximum velocity (vmax). There was no significant difference in the Km. Please see Figure 5—source data 1 for vmax and Km values. (C and D) FECH activity was measured in isolated intact mitochondria. Samples from high-dose forskolin (FSK)-treated differentiating MEL cells have higher FECH activity (C) (*p-value<0.05, Mean ± SEM, n = 5). In contrast, AKAP10-KO mitochondria had less FECH activity (D). Very little activity was detected in samples in which DP was substituted with NMMP. (E) A schematic showing the intron 3 and exon 4 sequences of wild-type murine Fech as well as the CRISPR oligo and the single-stranded DNA (ssDNA) that were introduced as a template for DNA repair. Intronic and exonic sequences are shown in lower and upper cases, respectively. Highlighted in yellow are the three PAM (protospacer adjacent motif) sequences closest to the T115A mutation site that facilitates the potential use of multiple CRISPR oligos. The missense mutations necessary to generate the T115A substitution are in orange. Shown in blue are synonymous substitutions designed to either disrupt the PAM sequences to prevent cleavage of the newly introduced mutant allele or to facilitate genotyping using allele-specific primers near the T115A mutation site. (F) Genomic DNA was isolated from the individual clones of MEL cells and used for PCR analysis with allele-specific primers. The parental MEL cells only had the wild-type allele. The intron 3 and exon 4 sequences of these cells were sequenced to confirm these agarose gel electrophoresis results. (G) Undifferentiated and differentiated parental and mutant cells expressing only the FECHT115A allele were lysed and subjected to western analysis to examine the induction of FECH protein during erythroid maturation. FECHThr115Ala protein had very similar up regulation with differentiation. (H) Differentiated MEL cells were lysed, immunoprecipitated with the indicated antibodies, and bound proteins were subjected to western analysis. Cells expressing only mutant FECH were phosphorylation defective at Thr115. (I–K) Mitochondria isolated from differentiated MEL cells expressing only endogenous FECHT115A (Mut), generated by genome editing, has lower FECH activity than wild-type (WT) control (I). These cells expressing non-phosphorylated FECH also have reduced hemoglobinization by o-dianisidine staining (J) and increased accumulation of PPIX substrate (K) as demonstrated by HPLC analysis (*p-value<0.05, Mean ± SEM, n = 11). (L) Wild-type or mutant differentiated MEL cells treated with vehicle (MOCK) or FSK were stained with o-dianisidine. Cells expressing mutant FECH were refractory to the effects of FSK. *p-value<0.05, Mean ± SEM, n = 3, unless otherwise specified. All immunoblots were performed twice. IB-immunoblot; vi-initial velocity; Km-Michaelis-Menten constant; FSK-forskolin; NMMP:N-methyl-mesoporphyrin-IX.