金屬蛋白的小分子活化與感應

Abstract

金屬蛋白的小分子活化和感應在自然界中可以有效地控制烷烴氧化或是感應一氧化氮。在這裡，我將展示了兩種模型，一種是甲烷單氧化酵素（pMMO），另一種是延胡索酸鹽和硝酸鹽還原調節蛋白（FNR）。 在第1部分中，我將研究噬甲烷菌Methylococcus capsulatus(Bath) (M. capsulatus) 甲烷單氧化酵素的B次單元（PmoB）的銅輔因子的數量跟特性。該蛋白體次單元對一價銅有相當高的親和力。為了闡述其銅親和力，我們設計全長的PmoB次單元、以及N端截斷的設計，包括PmoB33-414和PmoB55-414，以及麥芽糖結合蛋白（MBP）標籤轉殖並在大腸桿菌中大量表現。除了以N端截斷的方式觀察一價銅親和力，此外Y374F、Y374S和M300L突變蛋白也被建構。在培養過程，當大腸桿菌在1.0mM CuII溶液大量表達PmoB，甲烷單氧化酵素的B次單元表現得跟在甲烷菌中一樣。 在本實驗中，我們進一步收集這些B次單元的蛋白質，測量銅的數量、以Cu Kα邊緣X射線吸收近邊光譜（XANES）證實所有PmoB重組體都是一價銅蛋白。並根據Cu延伸的X射線吸收邊緣精細結構（EXAFS）的分析，發現PmoB蛋白顯示出「雙銅中心」的證據。當我們測量這些重組膜結合的PmoB蛋白發現並沒有甲烷和丙烯氧化的特定活性。然而其中的PmoB33-414蛋白卻可觀察到過氧化氫的顯著產生。此雙銅中心與氧氣反應產生過氧化氫的現象，會更近一步導致位於PmoB亞基的C末端亞結構域的一價銅的氧化。 在第2部分中，由於FNR蛋白是含有四鐵四硫金屬簇的轉錄因子，它的金屬簇化學結構對於氧氣與一氧化氮非常敏感。在我的研究中，試圖觀察大腸桿菌生理狀態從有氧呼吸轉換為厭氧硝酸鹽呼吸，即大腸桿菌的發酵生長，FNR蛋白在生理條件下四鐵四硫金屬簇的變化。當大腸桿菌BL21DE（PLyS）與含有fnr基因的轉化質體pET22b在厭氧條件以含有硝酸鹽的LB培養液中生長時，我們發現從SDS-Page分析中積累了大量的重組FNR。同時發現重組FNR的表現量可以通過Ni-NTA柱層析容易地純化。將這些純化的FNR進行EPR測量。我們觀察到在gav = 2.03處出現強烈的順磁信號，這表示在蛋白質內形成雙亞硝基鐵複合物。同時從含有硝酸鹽的厭氧生長中分離的單元重組FNR單體的鐵含量為2.42個鐵。再加上我們確保在大腸桿菌體內FNR蛋白亞硝基化後形成Roussin Red Ester（RRE），並且以二硫亞磺酸鈉還原觀察陰離子Roussin Red Ester EPR特徵（g = 2.005, g║ = 1.97）。 並綜合 FNR的基因調控和隨後在大腸桿菌中的蛋白質表達譜進一步證明了此亞硝化型態在硝酸鹽厭氧呼吸下於大腸桿菌中讓FNR獲得新的功能，構成了它正向的基因自動調節。

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.