Small Molecule Activation and Sensing by Metalloproteins

• Main Authors: Mu-Cheng Hung, 洪木成
• Other Authors: Steve Sheng-Fa Yu
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/9x3m8y

博士 === 國立臺灣大學 === 生化科學研究所 === 107 === Small molecule activation and sensing by metalloproteins play important roles in controlled alkane oxidation or nitric oxide sensing in nature. Here, I show two protien systems, one is particulate methane monooxygenase (pMMO), the other is the fumerate and nitrate reduction regulator (FNR). In part 1, we describe efforts to clarify the role of the copper cofactors associated with subunit B (PmoB) of the pMMO from Methylococcus capsulatus (Bath) (M. capsulatus). This subunit exhibits strong affinity toward CuI ions. To elucidate the high copper affinity of the subunit, the full-length PmoB, and the N-terminal truncated mutants PmoB33–414 and PmoB55–414, each fused to the maltose-binding protein (MBP), are cloned and over-expressed into Escherichia coli(E. coli) K12 TB1 cells. The Y374F, Y374S and M300L mutants of these protein constructs are also studied. When this E. coli grown with the pmoB gene in 1.0 mM CuII, it behaves like M. capsulatus (Bath) cultured under high copper stress with abundant membrane accumulation and high Cu I content. The recombinant PmoB proteins are verified by Western blotting of antibodies directed against the MBP sub-domain in each of the copper-enriched PmoB proteins. Cu K-edge X-ray absorption near edge spectroscopy (XANES) of the copper ions confirms that all the PmoB recombinants are CuI proteins. All the PmoB proteins show evidence of a “dicopper site” according to analysis of the Cu extended X-ray absorption edge fine structure (EXAFS) of the membranes. No specific activities toward methane and propene oxidation are observed with the recombinant membrane-bound PmoB proteins. However, significant production of hydrogen peroxide is observed in the case of the PmoB33–414mutant. Reaction of the dicopper site with dioxygen produces hydrogen peroxide and leads to oxidation of the CuI ions residing in the C-terminal sub-domain of the PmoB subunit In part 2, FNR protein is a transcriptional factor containing 4Fe-4S cluster, which is sensitive to the presence of dioxygen molecules and can switch the physiological status from aerobic respiration to anaerobic nitrate respiration, i.e., the fermentated growth of E. coli. When E. coli BL21DE (PLyS) grown with transformed plasmid pET22b containing fnr gene insert in anaerobic conditions by the presence of the nitrate salts in LB buffer, significant amounts of recombinant FNR are accumulated from the SDS-Page analysis. The recombinant FNR with poly-histidine can be easily purified through Ni-NTA column chromatography. The FNR is subjected for EPR measurement. A strong paramagnetic signal appeared at gav = 2.03 indicates the formation of iron dinitrosylated complexes within the proteins. The iron contents of unit recombinant FNR monomer isolated from the anaerobic growth with the nitrate salts was 2.42. We ensure that there is formation of Roussin’s Red ester (RRE) after the nitrosylation of FNR protein in vivo with the further reduction mediated by dithionites for the observation of EPR characteristic (g = 2.005, g║ = 1.97) of anionic Roussin’s Red ester. The gene regulation of FNR and subsequent protein expression profiling in E. coli have further indicated that nitrosylated FNR in E. coli under anaerobic respiratory are auto-regulated.