title: CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity.
abstract: The RNA-binding heme protein DiGeorge critical region 8 (DGCR8) and its ribonuclease partner Drosha cleave primary transcripts of microRNA (pri-miRNA) as part of the canonical microRNA (miRNA) processing pathway. Previous studies show that bis-cysteine thiolate-coordinated Fe(III) DGCR8 supports pri-miRNA processing activity, while Fe(II) DGCR8 does not. In this study, we further characterized Fe(II) DGCR8 and tested whether CO or NO might bind and restore pri-miRNA processing activity to the reduced protein. Fe(II) DGCR8 RNA-binding heme domain (Rhed) undergoes a pH-dependent transition from 6-coordinate to 5-coordinate, due to protonation and loss of a lysine ligand; the ligand bound throughout the pH change is a histidine. Fe(II) Rhed binds CO and NO from 6- and 5-coordinate states, forming common CO and NO adducts at all pHs. Fe(II)-CO Rhed is 6-coordinate, low-spin, and pH insensitive with the histidine ligand retained, suggesting that the protonatable lysine ligand has been replaced by CO. Fe(II)-NO Rhed is 5-coordinate and pH insensitive. Fe(II)-NO also forms slowly upon reaction of Fe(III) Rhed with excess NO via a stepwise process. Heme reduction by NO is rate-limiting, and the rate would be negligible at physiological NO concentrations. Importantly, in vitro pri-miRNA processing assays show that both CO- and NO-bound DGCR8 species are inactive. Fe(II), Fe(II)-CO, and Fe(II)-NO Rhed do not bear either of the cysteine ligands found in the Fe(III) state. These data support a model in which the bis-cysteine thiolate ligand environment of Fe(III) DGCR8 is necessary for establishing proper pri-miRNA binding and enabling processing activity.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, WI, 53706-1322, USA.
authors.forename: Judy P
authors.initials: JP
authors.lastname: Hines
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD, 21250-0001, USA.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Smith
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, 90095-3075, USA.
authors.forename: Jose P
authors.initials: JP
authors.lastname: Jacob
authors.affiliation: Department of Chemistry and Molecular Biology, North Dakota State University, Fargo, ND, 58108-6050, USA.
authors.forename: Gudrun S
authors.initials: GS
authors.lastname: Lukat-Rodgers
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, 90095-3075, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Barr
authors.affiliation: Department of Chemistry and Molecular Biology, North Dakota State University, Fargo, ND, 58108-6050, USA.
authors.forename: Kenton R
authors.initials: KR
authors.lastname: Rodgers
authors.affiliation: Department of Biological Chemistry, David Geffen School of Medicine, Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, 90095-3075, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Guo
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, WI, 53706-1322, USA. burstyn@chem.wisc.edu.
authors.forename: Judith N
authors.initials: JN
authors.lastname: Burstyn
mesh_terms: D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism*; D002942:Circular Dichroism / Q000379:methods; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D013059:Spectrum Analysis, Raman
chemical_list: C474043:DGCR8 protein, human; D005290:Ferric Compounds; D005296:Ferrous Compounds; D008024:Ligands; D035683:MicroRNAs; D016601:RNA-Binding Proteins; C545158:cysteine thiolate; D009569:Nitric Oxide; D006418:Heme; D006639:Histidine; D002248:Carbon Monoxide; D008239:Lysine; D003545:Cysteine
keywords: Carbon monoxide; Heme; Nitric oxide; RNA processing; microRNA