氧氣和糖是動物、植物、真菌和許多細菌的生命之本。"呼吸"這一新陳代謝過程使得細胞能夠?qū)⑹澄镛D(zhuǎn)化為能量。生物化學家Carola Hunte教授和她來自弗萊堡大學卓越中心CIBSS的團隊借助冷凍電鏡，以極高的精度首次可視化了那些為人類提供能量的蛋白質(zhì)系統(tǒng)的結(jié)構(gòu)和功能。該小組研究了由兩個呼吸鏈復合物結(jié)合成的一組超復合物放線菌。

Oxygen and sugar are the basis of life for animals, plants, fungi and many bacteria. The metabolic process called respiration makes it possible to convert food into energy for the cells. Biochemist Prof. Dr. Carola Hunte and her team from the Cluster of Excellence CIBSS at the University of Freiburg have now visualized for the first time with unparalleled precision how an assembly of protein machines, which also supplies energy to humans, is structured and functions. The team studied two respiratory chain complexes fused into a supercomplex in a group of bacteria called Actinobacteria.

除了提供呼吸過程的基本闡釋外，冷凍電鏡的解析可以助力結(jié)核病或白喉治療的新藥研發(fā)。亨特解釋說："這些結(jié)構(gòu)圖像讓我們體驗了一次分子內(nèi)部之旅，觀察到他們的工作方式及一些奇特的現(xiàn)象。闡明結(jié)構(gòu)的同時，也解釋了超復合物是如何工作的。" 這項研究的結(jié)果發(fā)表在Nature Communications上，該研究是與法國國家科學研究中心的綜合生物學中心（CBI）、遺傳學和分子與細胞生物學研究所（IGBMC）的研究主任Bruno Klaholz博士、法國國家健康與醫(yī)學研究院(Inserm)和法國斯特拉斯堡大學合作完成的。

In addition to providing a basic elucidation of respiratory processes, the cryogenic electron microscope analysis could aid in the development of new drugs to treat tuberculosis or diphtheria. "These images are like a journey into our molecular inner workings and its peculiar rules," Hunte explains, "Elucidating the structure simultaneously illuminates how the supercomplex works." The results of the study appeared in the journal?Nature Communications?and were produced in collaboration with Dr. Bruno Klaholz, research director at the Centre for Integrative Biology (CBI) / Institute of Genetics and of Molecular and Cellular Biology (IGBMC) of the CNRS, Inserm and the University of Strasbourg/France.

本文轉(zhuǎn)載自Science Daily:

"Researchers visualize protein machinery of respiration for the first time"

細胞的能量貨幣

The energy currency of the cell

?

三磷酸腺苷(ATP)是細胞的能量貨幣——ATP由呼吸產(chǎn)生，并將能量從食物轉(zhuǎn)移到細胞內(nèi)以進行所有的細胞工作。受益于呼吸鏈的轉(zhuǎn)化，二磷酸腺苷(ADP)被轉(zhuǎn)化為富含能量的ATP。為了完成這一轉(zhuǎn)化，呼吸鏈的蛋白質(zhì)復合物在一個復雜的生化過程中用電子和質(zhì)子形成跨越膜的電化學驅(qū)動力，該生化過程的動力由糖的燃燒提供。

Adenosine triphosphate (ATP) is the energy currency of the cell - the molecule is obtained during respiration and transfers energy from food to all processes in the cell. Thanks to the processes on the respiratory chain, adenosine diphosphate is turned into the energy-rich ATP. To do this, protein complexes of the respiratory chain build up an?electrochemical driving force across a membrane with electrons and protons in a complicated chemical-physical process that is powered by the combustion of sugar.

該研究的第一作者Hunte團隊的Wei-Chun Kao博士解釋說："我們分析了呼吸系統(tǒng)細胞色素bcc-aa3超復合物，二十六個蛋白質(zhì)組成了這個蛋白質(zhì)系統(tǒng)。在這之前，我們并不清楚他們的分子力和動力的具體互動。" 研究人員發(fā)現(xiàn)，該復合物的質(zhì)子泵與人類非常相似，但電子被電子載體醌接管的部分在細菌中顯示出明顯的差異。"這個差異使我們可以借機開發(fā)特定的藥劑，通過干擾呼吸鏈來殺死致病放線菌，如結(jié)核分枝桿菌或白喉桿菌，"Hunte補充說。

"We analyzed the respiratory cytochrome bcc-aa3 supercomplex. Twenty-six proteins make up the protein machine. The exact interaction of molecular forces and dynamics is not well understood yet, and this is where such a detailed description helps us," explains the study's first author Dr. Wei-Chun Kao of Hunte's team. The proton pump of the complex is very similar to humans, the researchers find, but the part where electrons are taken over by the electron carrier quinone shows clear differences in the bacterium. "This is where we could tie in and develop specific agents that kill pathogenic actinobacteria such as?Mycobacterium tuberculosis?or?Corynebacterium diphtheriae?by interfering with the respiratory chain," Hunte adds.

Image@ Wei-Chun Kao/CIBSS/University of Freiburg

具有原子分辨率的冷凍電鏡

Cryogenic microscope with atomic resolution

冷凍電鏡（Cryo-EM）是一種在零下183攝氏度的低溫下用高分辨率顯微鏡檢查樣品的技術(shù)，可以將結(jié)構(gòu)解析到單原子水平。在這個過程中，機器學習算法被用來進一步完善收集到的數(shù)據(jù)。

Cryogenic electron microscopy (Cryo-EM) is a technique that examines samples at low temperatures of - 183 Celsius in a high-resolution microscope and can resolve structures to the level of single atoms. In the process, machine learning algorithms are used to further refine the collected data.

?"有了這些(冷凍電鏡)數(shù)據(jù)，我們可以更好地了解新陳代謝和信號傳導的相互作用，這是CIBSS卓越中心目前的重點課題。"—Carola Hunte教授，弗萊堡大學CIBSS卓越中心

“With this data, we can also better understand the interplay of metabolism and signaling, which is a particular focus in the Cluster of Excellence CIBSS."—Prof. Dr. Carola Hunte, Cluster of Excellence CIBSS, University of Freiburg? ?

支持性內(nèi)容

Supportive?content

相關(guān)Nature?Communications文章:?

"Structural basis for safe and efficient energy conversion in a respiratory super-complex",?DOI:?10.1038/s41467-022-28179-x

學界認為，質(zhì)子轉(zhuǎn)運呼吸復合物組成超復合物的舉動是為了提高能量轉(zhuǎn)換的效率，限制有氧細胞呼吸過程中有害活性氧的產(chǎn)生。細胞色素bc復合體和細胞色素aa3氧化酶是質(zhì)子動力的主要驅(qū)動力，通過呼吸作用為ATP的產(chǎn)生提供動力，但在超復合物的狀態(tài)下，用何種方法轉(zhuǎn)移消耗掉的電子和質(zhì)子以提高安全性和效率還不清楚。

Proton-translocating respiratory complexes assemble into supercomplexes that are proposed to increase the efficiency of energy conversion and limit the production of harmful reactive oxygen species during aerobic cellular respiration. Cytochrome?bc?complexes and cytochrome?aa3?oxidases are major drivers of the proton motive force that fuels ATP generation via respiration, but how wasteful electron- and proton transfer is controlled to enhance safety and efficiency in the context of supercomplexes is not known.?

A.?呼吸體蛋白超復合物中能量轉(zhuǎn)換的示意圖。細胞基數(shù)交換復合物在Qo處與甲基萘酚氧化一起運行Q循環(huán)，與質(zhì)子釋放到正電膜側(cè)的位點耦合（H+P），并在 Q 時減少甲萘醌我從電負性膜側(cè)吸收質(zhì)子的位點（H+N），通過分叉電子轉(zhuǎn)移鏈接。氧化酶作為氧化還原驅(qū)動的質(zhì)子泵運行。

A.?Schematic presentation of energy conversion in the obligate respiratory supercomplex. The cyt?bcc?complex operates a Q cycle with menaquinol oxidation at the Qo?site coupled to proton release to the electropositive membrane side (H+P), and menaquinone reduction at the Qi?site with?proton uptake from the?electronegative membrane side (H+N), linked through bifurcated electron transfer. The oxidase operates as a redox-driven proton pump.

B. 細胞性密閉鎖細胞的冷凍電鏡結(jié)構(gòu)3超級復雜。同源二聚體的原子模型平行于透明表面顯示的膜且疊加。亞基采用顏色編碼，并帶有匹配的下劃線標簽。?

B.?Cryo-EM structure of cyt?bcc-aa3?supercomplex. The atomic model of the homodimer is viewed parallel to the membrane shown in transparent surface and superimposed in cartoon representation. Subunits are colour-coded with matching underlined labels.

C. 具有去垢劑的超復合物的3D重建。P和N分別表示膜的正極和負極。實驗圖的等值線水平設(shè)置為3.5均方根偏差（rmsd）。?

C.?3D reconstruction of supercomplex with dimensions and detergent micelle. P and N denote the electro-positive and -negative sides of the membrane, respectively. The contour level of the experimental map was set to 3.5 root mean square deviation (rmsd).

我們通過解析谷氨酸放線菌的細胞色素bcc-aa3（III2-IV2）超復合物的2.8 ?分辨率冷凍電鏡結(jié)構(gòu)，解決了甲萘醌、底物模擬物、番茄紅素、意外的Qc位點、分子氧、質(zhì)子轉(zhuǎn)移路線以及關(guān)鍵質(zhì)子化殘基的構(gòu)象狀態(tài)等問題。我們的研究結(jié)果闡釋了在呼吸體蛋白超復合物中，如何通過控制電子和質(zhì)子轉(zhuǎn)移實現(xiàn)安全和高效的能量轉(zhuǎn)換。該冷凍電鏡結(jié)構(gòu)可以指導引起白喉和肺結(jié)核的放線菌的針對性藥物的合理設(shè)計。

Here, we address this question with the 2.8?? resolution cryo-EM structure of the cytochrome?bcc-aa3?(III2-IV2)?supercomplex from?the actinobacterium?Coryne-bacterium glutamicum. Menaquinone, substrate mimics, lycopene, an unexpected Qc?site, dioxygen, proton transfer routes, and conformational states of key protonable residues are resolved. Our results show how safe and efficient energy conversion is achieved in a respiratory supercomplex through controlled electron and proton transfer. The structure may guide the rational design of drugs against actinobacteria that cause diphtheria and tuberculosis.

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