8 research outputs found
Progressive Reduction of the Mn-Catalase Mn(III)-Mn(III) State to the Mn(II)-Mn(III) and Mn(II)-Mn(II) States by Nitrogen Monoxide
Photoassembly of the Tetra-Mn Site of Photosynthetic Water Oxidation: EPR Evidence for Dark Ligation of a Binuclear Mn Intermediate
Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo
X-ray Crystallography and Electron Paramagnetic Resonance Spectroscopy Reveal Active Site Rearrangement of Cold-Adapted Inorganic Pyrophosphatase
Multifrequency EPR analysis of the dimanganese cluster of the putative sulfate thiohydrolase SoxB of Paracoccus pantotrophus
Multicopper oxidase involvement in both Mn(II) and Mn(III) oxidation during bacterial formation of MnO2
CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity
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low-spin, and pH insensitive with the histidine ligand retained, suggesting that the protonatable lysine ligand has been replaced by CO.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