title: DiGeorge critical region 8 (DGCR8) is a double-cysteine-ligated heme protein.
abstract: All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8. Here we further characterize the DGCR8-heme interaction using biochemical and spectroscopic methods. The DGCR8-heme complex is highly stable, with a half-life exceeding 4 days. Mutation of the conserved proline 351 to an alanine increases the rate of heme dissociation and allows the DGCR8-heme complex to be reconstituted biochemically. Surprisingly, DGCR8 binds ferric heme without CO to generate a hyperporphyrin spectrum. The electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectra of the DGCR8-heme complex suggest a ferric heme bearing two cysteine ligands. This model was further confirmed using selenomethionine-substituted DGCR8 and mercury titration. DGCR8 is the first example of a heme-binding protein with two endogenous cysteine side chains serving as axial ligands. We further show that native DGCR8 binds heme when expressed in eukaryotic cells. This study provides a chemical basis for understanding the function of the DGCR8-heme interaction in microRNA maturation.
authors: Department of Biological Chemistry, David Geffen School of Medicine, Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
authors: Ian
authors: I
authors: Barr
authors: Aaron T
authors: AT
authors: Smith
authors: Rachel
authors: R
authors: Senturia
authors: Yanqiu
authors: Y
authors: Chen
authors: Brooke D
authors: BD
authors: Scheidemantle
authors: Judith N
authors: JN
authors: Burstyn
authors: Feng
authors: F
authors: Guo
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D006736:Horses; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011166:Porphyrins / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011506:Proteins / Q000235:genetics* / Q000502:physiology*; D016601:RNA-Binding Proteins; D012645:Selenomethionine / Q000737:chemistry; D013053:Spectrophotometry / Q000379:methods; D014982:Xenopus laevis
chemical_list: C474043:DGCR8 protein, human; D035683:MicroRNAs; D011166:Porphyrins; D011506:Proteins; D016601:RNA-Binding Proteins; D006418:Heme; D012645:Selenomethionine; D011392:Proline; D003545:Cysteine