CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity
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- 作者：Judy P. Hines ; Aaron T. Smith ; Jose P. Jacob…
- 关键词：Heme ; microRNA ; RNA processing ; Carbon monoxide ; Nitric oxide
- 刊名：Journal of Biological Inorganic Chemistry
- 出版年：2016
- 出版时间：December 2016
- 年：2016
- 卷：21
- 期：8
- 页码：1021-1035
- 全文大小：1,419 KB
- 刊物类别：Biomedical and Life Sciences
- 刊物主题：Life Sciences
Biochemistry
Microbiology - 出版者：Springer Berlin / Heidelberg
- ISSN：1432-1327
- 卷排序：21
文摘
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low-spin, and pH insensitive with the histidine ligand retained, suggesting that the protonatable lysine ligand has been replaced by CO.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.