Interaction between the Bacterial Iron Response Regulator and Ferrochelatase Mediates Genetic Control of Heme Biosynthesis  Zhenhao Qi, Mark R O'Brian  Molecular Cell  Volume 9, Issue 1, Pages 155-162 (January 2002) DOI: 10.1016/S1097-2765(01)00431-2

Figure 1 Interaction of Irr with Ferrochelatase (A) Ferrochelatase coimmunoprecipitates with Irr in B. japonicum cell extract. Cells of strain I110(pBJH) grown under low iron were lysed and immunoprecipitated using anti-Irr (α-Irr), anti-ferrochelatase (α-FC) antibodies, or preimmune serum (Pre). Immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting using either anti-Irr or anti-ferrochelatase antibodies. (B) Pull-down of Irr or Fur by a GST-Ferrochelatase fusion protein. The GST-FC fusion protein was used to precipitate Irr in B. japonicum extracts (top), pure Irr in solution (middle), or pure His-tagged Fur in solution (bottom). GST was used as a negative control. Ten micrograms each of GST, GST-FC, purified Irr, or Fur protein was used for the corresponding individual experiment. The proteins were visualized with antibodies directed against Irr or the His tag of Fur. Molecular Cell 2002 9, 155-162DOI: (10.1016/S1097-2765(01)00431-2)

Figure 2 Effect of Expression of an Inactive Ferrochelatase on Irr Degradation in Wild-Type B. japonicum Cells (A) A GST-ferrochelatase (GST-FC) or GST-ferrochelatase-H215L (GST-H215L) fusion protein was used to precipitate Irr in B. japonicum cell extracts. GST was used as a negative control. The experiment shows that the H215L mutant retains the ability to interact with Irr. (B) The hemH(H215L) gene encoding an inactive ferrochelatase or the wild-type hemH gene was ligated into the vector pVK102 (pH215L and pHemH, respectively) and introduced into B. japonicum strain I110. The pVK102 vector without an insert was used as a control. The transconjugants contained a wild-type ferrochelatase gene (hemH) in the chromosome. Cells were grown under low iron media, then treated with 6 μM FeCl3 (+Fe) or an equivalent volume of buffer (−Fe) at time zero. Aliquots were taken at various times, and Irr was detected by immunobloting using anti-Irr antibodies. Fifty micrograms of protein was loaded per lane. Molecular Cell 2002 9, 155-162DOI: (10.1016/S1097-2765(01)00431-2)

Figure 3 Effects of hemA or hemH Deletion or hemA hemH Double Deletion on Irr Activity (A) Abbreviated heme pathway showing ALA synthase (ALAS) encoded by the hemA gene and ferrochelatase (FC) encoded by hemH. The substrates of ALA synthase are glycine and succinyl coenzyme A. “Proto” designates protoporphyrin IX. (B) Cells of strains WT (I110), hemA (MLG1), hemH (I110ek4), and hemA hemH (ΔhemAH) were grown in modified GSY medium containing either low (L) or high (H) iron. Irr and ALAD were analyzed in cells by immunoblots using anti-Irr or anti-ALAD antibodies, respectively. Fifty micrograms of protein was loaded per lane. Molecular Cell 2002 9, 155-162DOI: (10.1016/S1097-2765(01)00431-2)

Figure 4 Effects of ALA on Irr Activity in Cells of the Wild-Type and hemA Mutant Cells were grown in modified GSY media under low iron conditions supplemented with either 0 (−) or 50 μg/ml ALA (+). Irr and ALAD in cells were analyzed by immunoblots using anti-Irr or anti-ALAD antibodies, respectively. Fifty micrograms of protein was loaded per lane. Molecular Cell 2002 9, 155-162DOI: (10.1016/S1097-2765(01)00431-2)

Figure 5 A His-215 to Leu Substitution of Ferrochelatase Retains Its Inhibitory Effect on Irr Activity (A) Ferrochelatase (FC) activity, protoporphyrin levels (Proto), and fluorescent colony phenotype due to protoporphyrin accumulation (Colony Fluor.) were measured in cells of strain I110 (Wt), I110ek4 (ΔhemH), and HemH(H215L) (H215L). Ferrochelatase activity is expressed as nmol mesoheme formed hr−1 mg protein−1. Protoporphyrin extracted from cells is expressed as nmol protoporphyrin mg protein−1. Colony fluorescence was assessed by visual inspection of cells on plates under UV light. (B) Cells of strain I110 (Wt), I110ek4 (ΔhemH), and HemH(H215L) (H215L) were grown in modified media containing low iron (L; no added iron) or high iron (H; 6 μM FeCl3). Ferrochelatase (FC), Irr, or ALA dehydratase (ALAD) in cells was analyzed by immunoblots using anti-ferrochelatase, anti-Irr, or anti-ALAD antibodies, respectively. Fifty micrograms of protein was loaded in each lane. Molecular Cell 2002 9, 155-162DOI: (10.1016/S1097-2765(01)00431-2)

Figure 6 The Effects of Porphyrin Substrates on Ferrochelatase-Irr Interaction The GST-FC fusion protein (10 μg protein) was used to precipitate Irr from B. japonicum extract in the presence of mesobilirubin (MB), deutoroporphyrin IX (Deu), mesoporphyrin IX (Meso), heme, or none of these(−). The top panel shows Irr pulled down by GST-FC under various conditions. The bottom panel shows Irr in extracts and demonstrates that porphyrins do not interfere with the antigenicity of Irr. The precipitates (top panel) or the crude extracts (bottom panel) were resolved by 12% SDS-PAGE and analyzed by immunoblotting using anti-Irr antibodies. GST was used as a negative control. Molecular Cell 2002 9, 155-162DOI: (10.1016/S1097-2765(01)00431-2)

Figure 7 Binding of Heme to Purified Recombinant Irr and Mutant Protein IrrC29A Binding to Irr was quantified using a fluorimetric assay by measuring the quenching of intrinsic fluorescence of Irr upon binding to heme. Recombinant Irr (closed circles) or IrrC29A (open circles; 0.5 μM) was incubated with various concentrations of heme for 3 min before measuring fluorescence emission at 338 nm using an excitation wavelength of 263 nm. Panels (A) and (B) show the data from 0 to 0.4 μM and 0 to 0.1 μM heme, respectively. The binding curve was fitted by nonlinear regression analysis using the Prism2.01 program. The best fit for wild-type Irr was for two-site binding with Kd values of 1 nM and 80 nM. For IrrC29A, the data best fit one binding site with a Kd value of 124 nM. Molecular Cell 2002 9, 155-162DOI: (10.1016/S1097-2765(01)00431-2)

Figure 8 Model for Regulation of Irr Expression and Activity at the Site of Ferrochelatase FC denotes ferrochelatase. The light porphyrin is protoporphyrin. The dark porphyrin with the inserted iron molecule is heme (protoheme). (A) Ferrochelatase activity proceeds when iron is sufficient, resulting in heme formation. Irr is inactivated, then subsequently degraded. (B) Under iron limitation, ferrochelatase is bound by only one of its substrates, protoporphyrin. This causes Irr and ferrochelatase to dissociate, thereby relieving inhibition of Irr activity for repression of the hemB gene. (C) The hemA strain cannot synthesize heme, therefore Irr does not degrade. However, the mutant does not synthesize protoporphyrin, thus the inhibition of Irr by ferrochelatase cannot be relieved. (D). Irr is constitutively present and active in a hemH mutant because there is no heme to degrade it and no ferrochelatase to inhibit activity. The status of protoporphyrin is not relevant in a ferrochelatase (hemH) mutant; therefore, a hemA strain and a hemA hemH double mutant have the same phenotype with respect to Irr function. Molecular Cell 2002 9, 155-162DOI: (10.1016/S1097-2765(01)00431-2)