大腸桿菌中鐵調控因子與其經亞硝基轉換後之生物無機化學研究

林奇歐

doi:10.6844/NCKU.2013.00255

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大腸桿菌中鐵調控因子與其經亞硝基轉換後之生物無機化學研究

Identification and Characterization of Ferric Iron Uptake Regulatory Protein (Fur) and its Protein Bound Dinitrosyl Iron Complex (DNIC) in Escherichia coli

林奇歐 , Masters　　Advisor：俞聖法　　Co-advisor ：黃守仁

繁體中文 DOI： 10.6844/NCKU.2013.00255

雙亞硝基鐵化合物；鐵調控因子；轉錄因子；大腸桿菌；一氧化氮； Escherichia coli ； ferric ion regulatory protein (Fur) ； nitrosylation ； DiNitrosyl-Iron-Complexes (DNIC)

Abstract │ Reference (51) │ Altmetrics

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A fur gene (447 bp) from the chromosomal DNA in Escherichia coli K12 were constructed into a pBAD--TOPOR vector (Invitrogen). After its transformation into Escherichia coli TOPO, we overexpressed and purified the recombinant Fur protein (MW=21.2kDa with His-tag) by nickel-charged histidine-binding resin column. Eluted fractions of Fur were further characterized by 14% SDS-PAGE, Native-page, LC-MS/MS, and elemental analysis via ICP-OES.

Active Fur performs as a homo-dimeric protein.  Each of Fur monomer contains a ferrous iron, and a zinc ion. Its iron core could be further modulated to form a dinitrosyl iron complex (DNIC) after the treatment of gaseous NO or the NO donor of DEANO(diethylamine dinitric oxide). This protein bound DNIC is appeared with gav=2.03 observed in EPR spectroscopy.

Dimeric Fur can recognize a DNA element within Fur promoters with a 19-bp AT-rich palindromic sequence called a Fur-box. Here, we have determined that Fur protein can bind to the Fur-box with high affinity(Kd=142.5 nM) by EMSA(Electrophoretic Mobility Shift Assays) and fluorescence spectroscopy. After the treatment of NO, nitroylated Fur cannot bind with Fur-box.

Circular Dichroism spectra revealed that the secondary structure of nitrosylated Fur is different from that of Fur. More-turn structural component has been converted (5.5%) from part of -sheet structure. The iron core in Fur is presented as ferrous state whereas the nitrosylated Fur possess {Fe(NO)2}9. The symmetry of iron center in nitrosylated Fur protein is changed from trigonal bipyramidal or octahedral to tetrahedral geometry after treating with NO in X-ray Absorption Near-Edge Structures (XANES). Extended X-ray Absorption Fine Structure (EXAFS) results show that, the coordination numbers of iron center at the active site will be changed from five or six-coordinated environment with N/O ligands from His, Asp/Glu in distances of 2.16Å and 2.43Å to four-coordinated with N/O ligands from His, Asp/Glu upon the addition of DEANO. Finally, we attempt to incorporated MTSL(S-(2,2,5,5-tetramethyl-2,5-dihydro-1H- pyrrol-3-yl)methyl methanesulfonothioate) at appropriate site(s) with free cysteine or mutated by Cys in Fur, after nitrosylation, the spin-spin interaction detected by CW-EPR (Continuous Wave-Electron Paramagnetic Resonance) between the organic free radical and dinitrosyl iron complex within a distance in 5-15 Å should allow us to identify the location of the ferrous iron in the three dimensional structure of Fur.

Reference ( 51 ) 〈TOP〉

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