Protein Structure and Folding
DiGeorge Critical Region 8 (DGCR8) Is a Double-cysteine-ligated Heme Protein*

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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.

Heme
Ligand Binding Protein
Metalloproteins
MicroRNA
Oxidation Reduction
RNA Binding Protein
RNA Processing
Spectroscopy
Thiol
DiGeorge Syndrome

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*

This project is supported, in whole or in part, by National Institutes of Health Grants GM080563 (to F. G.) and HL065217 (to J. N. B.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4, Tables S1 and S2, and references.