The ferrous iron transport (Feo) system is the predominant mode of bacterial Fe²⁺import. Advancements in the structure and function of FeoB provide glimpses into the mechanism of Fe²⁺uptake.

The RNA-binding protein DiGeorge Critical Region 8 (DGCR8) and its partner nuclease Drosha are essential for processing of microRNA (miRNA) primary transcripts (pri-miRNAs) in animals. Previous work showed that DGCR8 forms a highly stable and active complex with ferric [Fe(III)] heme using two endogenous cysteines as axial ligands. Here we report that reduction of the heme iron to the ferrous [Fe(II)] state in DGCR8 abolishes the pri-miRNA processing activity. The reduction causes a dramatic increase in the rate of heme dissociation from DGCR8, rendering the complex labile. Electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies indicate that reduction of the heme iron is accompanied by loss of the cysteines as axial ligands. ApoDGCR8 dimers, generated through reduction and removal of the heme, show low levels of activity in pri-miRNA processing in vitro. Importantly, ferric, but not ferrous, heme restores the activity of apoDGCR8 to the level of the native ferric complex. This study demonstrates binding specificity of DGCR8 for ferric heme, provides direct biochemical evidence for ferric heme serving as an activator for miRNA maturation, and suggests that an intracellular environment increasing the availability of ferric heme may enhance the efficiency of pri-miRNA processing.

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.