Title
Analysis of Heme Iron Coordination in DGCR8: The Heme-Binding Component of the Microprocessor Complex
Author
Hazel M Girvan, Justin M. Bradley, Myles R. Cheesman, James R. Kincaid, Yilin Liu, Kazimierz Czarnecki, Karl Fisher, David Leys, Stephen E. J. Rigby, Andrew William Munro
Year
2016
Journal
Biochemistry
Abstract
DGCR8 is the RNA binding partner of the nuclease Drosha. Their complex (the “Microprocessor”) is essential for processing of long, primary microRNAs (pri-miRNAs) in the nucleus. Heme binding to DGCR8 is essential for pri-miRNA processing. Based on the split Soret UV-visible (UV-vis) spectrum of ferric DGCR8, bis-thiolate sulfur (cysteinate, Cys-) heme iron coordination of DGCR8 heme iron was proposed. We have characterized DGCR8 heme ligation using the Δ276 DGCR8 variant and combined electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), electron nuclear double resonance, resonance Raman and electronic absorption spectroscopy. These studies indicate DGCR8 bis-Cys heme iron ligation, with conversion from bis-thiolate (Cys-/Cys-) axial coordination in ferric DGCR8 to bis-thiol (CysH/CysH) coordination in ferrous DGCR8. Pri-miRNA binding does not perturb ferric DGCR8’s optical spectrum, consistent with the axial ligand environment being separated from the substrate binding site. UV-vis absorption spectra for the FeII and FeII-CO forms indicate discrete species exhibiting peaks with absorption coefficients substantially larger than those for ferric DGCR8, and for that previously reported for a ferrous form of DGCR8. ENDOR data exclude histidine or water as axial ligands for ferric DGCR8, and favor bis-thiolate coordination in this form. UV-vis MCD and near-infrared MCD provide data consistent with this conclusion. UV-vis MCD data for ferrous DGCR8 reveal features consistent with bis-thiol heme iron coordination, and resonance Raman data for the ferrous-CO form are consistent with a thiol ligand trans to the CO. These studies support retention of DGCR8 cysteine coordination upon reduction, a conclusion distinct from previous studies on a different ferrous DGCR8 isoform.
Instrument
J-810
Keywords
Magnetic circular dichroism, Coordination chemistry, Ligand binding, Biochemistry