Related Experiment Video
Updated: Mar 16, 2026
Measurement of Heme Synthesis Levels in Mammalian Cells
Published on: July 9, 2015
Analysis of Heme Iron Coordination in DGCR8: The Heme-Binding Component of the Microprocessor Complex
Hazel M Girvan1, Justin M Bradley2, Myles R Cheesman2
1Centre 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.
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.
Insights
DGCR8 protein uses bis-cysteine heme iron coordination, changing from thiolate to thiol forms upon reduction. This heme ligation is crucial for microRNA processing by the Microprocessor complex.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- DGCR8 is the RNA-binding partner of Drosha, forming the Microprocessor complex essential for microRNA biogenesis.
- Heme binding to DGCR8 is critical for pri-miRNA processing.
- Previous studies proposed bis-thiolate heme iron coordination in DGCR8 based on UV-vis spectra.
Purpose of the Study:
- To characterize the heme ligation environment of DGCR8 using multiple biophysical techniques.
- To investigate the changes in heme coordination upon reduction from ferric to ferrous states.
- To determine the role of DGCR8 cysteine residues in heme iron coordination.
Main Methods:
- Electron paramagnetic resonance (EPR) spectroscopy
- Magnetic circular dichroism (MCD) spectroscopy
- Electron nuclear double resonance (ENDOR) spectroscopy
- Resonance Raman spectroscopy
- Electronic absorption spectroscopy
- Utilized a Δ276 DGCR8 variant
Main Results:
- Confirmed bis-cysteine heme iron ligation in DGCR8.
- Observed a conversion from bis-thiolate (Cys(-)/Cys(-)) in ferric DGCR8 to bis-thiol (CysH/CysH) in ferrous DGCR8.
- Pri-miRNA binding did not affect the ferric DGCR8 optical spectrum.
- Spectroscopic data excluded histidine or water as axial ligands for ferric DGCR8.
- Ferrous DGCR8 and its CO-bound form exhibited distinct spectral properties consistent with bis-thiol coordination.
Conclusions:
- DGCR8 heme iron coordination involves bis-cysteine ligands.
- Heme ligation undergoes a thiol-to-thiolate conversion upon reduction.
- The axial heme environment is distinct from the pri-miRNA binding site.
- These findings clarify the heme coordination in DGCR8, impacting understanding of Microprocessor function.
More Related Videos
Related Concept Videos
02:51Metal-Ligand Bonds
In these complexes, transition metals form coordinate covalent bonds, a kind of Lewis acid-base interaction in which both of the electrons in the bond are contributed by a donor (Lewis base) to an electron acceptor (Lewis acid). The Lewis acid in...
03:02Colors and Magnetism
When atoms or molecules absorb light at the proper frequency, their electrons are excited to higher-energy orbitals. For many main group atoms and molecules, the absorbed photons are in the ultraviolet range of the electromagnetic spectrum, which cannot be detected by the human eye. For coordination compounds, the energy difference between the d orbitals often allows photons in the visible range to be absorbed and emitted, which is seen as colors by the human...
02:42Valence Bond Theory
02:57Coordination Number and Geometry
02:58Crystal Field Theory - Octahedral Complexes
To explain the observed behavior of transition metal complexes (such as colors), a model involving electrostatic interactions between the electrons from the ligands and the electrons in the unhybridized d orbitals of the central metal atom has been developed. This electrostatic model is crystal field theory (CFT). It helps to understand, interpret, and predict the colors, magnetic behavior, and some structures of coordination compounds of transition metals.
CFT focuses on...
02:34Structural Isomerism
Isomers are different chemical species that have the same chemical formula. Structural isomerism of coordination compounds can be divided into two subcategories, the linkage isomers and coordination-sphere isomers.
Linkage isomers occur when the coordination compound contains a ligand that can bind to the transition metal center through two different atoms. For example, the CN− ligand can bind through the carbon atom or through the nitrogen atom. Similarly, SCN− can...