title: Analysis of Heme Iron Coordination in DGCR8: The Heme-Binding Component of the Microprocessor Complex.
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. Binding of heme to DGCR8 is essential for pri-miRNA processing. On the basis of the split Soret ultraviolet-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 of the Fe(II) and Fe(II)-CO forms indicate discrete species exhibiting peaks with absorption coefficients substantially larger than those for ferric DGCR8 and that previously reported for a ferrous form of DGCR8. Electron-nuclear double resonance spectroscopy 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 those of previous studies of a different ferrous DGCR8 isoform.
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Girvan
authors.affiliation: School of Chemistry, University of East Anglia , Norwich Research Park, Norwich NR4 7TJ, U.K.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Bradley
authors.affiliation: School of Chemistry, University of East Anglia , Norwich Research Park, Norwich NR4 7TJ, U.K.
authors.forename: Myles R
authors.initials: MR
authors.lastname: Cheesman
authors.affiliation: Department of Chemistry, Marquette University , 535 North 14th Street, Milwaukee, Wisconsin 53233, United States.
authors.forename: James R
authors.initials: JR
authors.lastname: Kincaid
authors.affiliation: Department of Chemistry, Marquette University , 535 North 14th Street, Milwaukee, Wisconsin 53233, United States.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Marquette University , 535 North 14th Street, Milwaukee, Wisconsin 53233, United States.
authors.forename: Kazimierz
authors.initials: K
authors.lastname: Czarnecki
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Karl
authors.initials: K
authors.lastname: Fisher
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: David
authors.initials: D
authors.lastname: Leys
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.affiliation: Centre for Synthetic Biology of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
mesh_terms: D003001:Cloning, Molecular; D006418:Heme / Q000737:chemistry*; D006801:Humans; D007501:Iron / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D013057:Spectrum Analysis / Q000379:methods
chemical_list: C474043:DGCR8 protein, human; D016601:RNA-Binding Proteins; D006418:Heme; D007501:Iron