Structural changes linked to proton translocation by subunit c of the ATP synthase

F1F0 ATP synthases use a transmembrane proton gradient to drive the synthesis of cellular ATP. The structure of the cytosolic F1 portion of the enzyme and the basic mechanism of ATP hydrolysis by F1 are now well established, but how proton translocation through the transmembrane F0 portion drives these catalytic changes is less clear. Here we describe the structural changes in the proton-translocating F0 subunit c that are induced by deprotonating the specific aspartic acid involved in proton transport. Conformational changes between the protonated and deprotonated forms of subunit c provide the structural basis for an explicit mechanism to explain coupling of proton translocation by F0 to the rotation of subunits within the core of F1. Rotation of these subunits within F1 causes the catalytic conformational changes in the active sites of F1 that result in ATP synthesis.     

Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase

The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.     

Highly coupled ATP synthesis by F1-ATPase single molecules

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the alpha3beta3gamma subcomplex (F1-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F1+epsilon)). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the epsilon-subunit has an essential function during ATP synthesis.     

ATP合成酶及其功能机制综述

在细胞能量转变过程中，F1F0-型ATP合成酶是一个关键酶。在ATP合成过程中，这个大的蛋白复合体利用质子梯度和相关的膜电势来合成ATP。这个酶结构的不同作用形式正在逐步阐明。一致的看法是这个酶由两个旋转发动机构成，一个在F1上，它将催化过程与内部的转子运动联系在一起，另一个在F0上，它将质子迁移与F0转子的运动联系在一起。虽然两个马达可以独立工作，但是它们必须结合在一起才能转换能量。从结构、基因和生化物理方面的研究中得出的关于这个旋转马达的功能的证据，在这里将作一个回顾，一些不确定的，关于酶机制尚留迷团的内容也将讨论如下。     

Tracking kinesin-driven movements with nanometre-scale precision

Several enzyme complexes drive cellular movements by coupling free energy-liberating chemical reactions to the production of mechanical work. A key goal in the study of these systems is to characterize at the molecular level mechanical events associated with individual reaction steps in the catalytic cycles of single enzyme molecules. Ideally, one would like to measure movements driven by single (or a few) enzyme molecules with sufficient temporal resolution and spatial precision that these events can be directly observed. Kinesin, a force-generating ATPase involved in microtubule-based intracellular organelle transport, will drive the unidirectional movement of microscopic plastic beads along microtubules in vitro. Under certain conditions, a few (less than or equal to 10) kinesin molecules may be sufficient to drive either bead movement or organelle transport. Here we describe a method for determining precise positional information from light-microscope images. The method is applied to measure kinesin-driven bead movements in vitro with a precision of 1-2 nm. Our measurements reveal basic mechanical features of kinesin-driven movements along the microtubule lattice, and place significant constraints on possible molecular mechanisms of movement.     

Anaerobic microbial metabolism can proceed close to thermodynamic limits.

Many fermentative bacteria obtain energy for growth by reactions in which the change in free energy (DeltaG') is less than that needed to synthesize ATP. These bacteria couple substrate metabolism directly to ATP synthesis, however, by classical phosphoryl transfer reactions. An explanation for the energy economy of these organisms is that biological systems conserve energy in discrete amounts, with a minimum, biochemically convertible energy value of about -20 kJ mol-1 (refs 1, 2, 3). This concept predicts that anaerobic substrate decay ceases before the minimum free energy value is reached, and several studies support this prediction. Here we show that metabolism by syntrophic associations, in which the degradation of a substrate by one species is thermodynamically possible only through removal of the end product by another species, can occur at values close to thermodynamic equilibrium (DeltaG' approximately 0 kJ mol-1). The free energy remaining when substrate metabolism halts is not constant; it depends on the terminal electron-accepting reaction and the amount of energy required for substrate activation. Syntrophic associations metabolize near thermodynamic equilibrium, indicating that bacteria operate extremely efficient catabolic systems.     

Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7?? resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.     

线粒体F型ATP合成酶的制备方法研究与探讨

科研工作者们在过去的50年前赴后继的工作中深入研究了ATP合成酶的功能,并努力尝试解析该酶的空间结构以便能够从结构基础上对ATP合成酶的催化机理进行阐明;但蛋白的纯化工作却一直是困扰研究顺利进展的最大障碍.蛋白的纯化技术是与特定阶段科技发展及科研工作者思维模式的直接反应,因而是一门不断发展的艺术.文章主要介绍了ATP合成酶的提取与纯化工作,在详细对比了现有方法的基础上,给出了纯化此酶复合体的详细方案和线粒体、亚线粒体、ATP合成酶的提取与纯化方法;并在方法讨论的基础上对线粒体F型ATP合成酶的研究提出了前瞻性的方案.     

E. coli F1-ATPase interacts with a membrane protein component of a proton channel

The ATP synthases of bacteria, mitochondria and chloroplasts, which use the energy of a transmembrane proton gradient to power the synthesis of ATP, consist of an integral membrane component F0--thought to contain a proton channel--and a catalytic component, F1. To help investigate the way F0 and F1 are coupled, we have sequenced the b-subunit of the Escherichia coli F0, which seems to be the counterpart of a thermophilic bacteria F0 subunit thought to be essential for F1 binding. We report here that its sequence is remarkable, being hydrophobic around the N-terminus and highly charged in the remainder. We propose that the N-terminal segment lies in the membrane and the rest outside. The extramembranous section contains two adjacent stretches of 31 amino acids where the sequence is very similar: in the second of these stretches there is further internal homology. These duplicated stretches of the polypeptide probably fold into two alpha-helices which have many common features able to make contact with F1 subunits. Thus protein b occupies a central position in the enzyme, where it may be involved in proton translocation. It is possibly also important in biosynthetic assembly.     

Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the alpha/beta-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the alpha/beta-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.     

杂多化合物在催化反应中的应用研究

本文分别从以下三个方面对杂多化合物的催化作用进行概括：1、主要应用在石油化工方面的烃类的烷基化和异构的酸催化功能;2、应用在配合氧化剂催化烯烃的环氧化反应和燃油脱硫氧化还原催化功能;3、在有机合成中的酯化反应方面的氧化还原催化功能.     

环氧丙烷合成技术现状与绿色合成研究进展

环氧丙烷(PO)是一种重要的有机化工中间体。本文对PO生产的主要方法(氯醇法、共氧化法、过氧乙酸法)的优缺点进行了分析;结合绿色化学的原理,对电化学催化氧化、光催化氧化、胶束催化氧化、生物酶催化氧化合成PO等生产技术进行了评述;对TS-1/H2O2催化烯烃环氧化反应机理及PO合成工艺进行了详细的介绍。结果表明,TS-1/H2O2催化环氧化工艺具有低污染、高选择性、反应条件温和等优点,代表了PO绿色合成的发展方向。     

Effects of truncated mutants of the ε subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

The ε subunit of the chloroplast ATP synthase and the truncated ε mutants which lack some amino acid residues from the N-terminus or C-terminus were overexpressed in E. coil When the ε subunit or the truncated ε proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. For the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of ε-deficient membrane reconstituted with the ε subunit or the truncated ε proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. For the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type ε protein. These results suggested that: (a) the N-terminal domain of the ε subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of ε subunit; and (b) the C-terminal domain of the ε subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase.     

团簇Co4P反应活性及催化性质的研究

利用团簇Co4P作为Co-P体系的局域结构,对其进行全参数优化计算,获得6种优化构型,对团簇Co4P各优化构型的化学反应活性及催化活性进行研究,得到以下结论：通过对能隙、柔度及前线轨道图综合分析,得出构型2(4)及3(2)均具有优良的化学反应活性,其中构型2(4)的化学反应活性优于3(2),且构型2(4)可作为电极材料的研究对象.不仅如此,通过态密度分析发现构型2(4)在催化反应中也具有良好活性,可作为优良的催化剂.     

A mechanistic principle for proton pumping by cytochrome c oxidase

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.     

磁性花生壳负载钯催化剂的制备及催化氨硼烷释氢性能

以高锰酸钾改性后的花生壳为原料,通过共沉淀法制备了磁性花生壳(PSK-Fe_3O_4)复合材料,并以其为载体制备了磁性花生壳负载钯催化剂(Pd/PSK-Fe_3O_4).以XRD、FT-IR、TEM、XPS、TG等表征手段对该催化剂进行了表征,并将其应用于催化氨硼烷水解释氢.结果表明:Pd/PSK-Fe_3O_4催化剂在催化氨硼烷水解反应中表现出较好的催化活性,其转换频率(TOF)为6.7 mol_(H2)·mol_(Pd)~(-1)·min~(-1),表观活化能(E_(app))为28.0 kJ mol~(-1).Pd/PSK-Fe_3O_4催化氨硼烷水解释氢反应对于催化剂浓度和氨硼烷浓度的反应级数分别为一级和零级.此外,循环实验表明该催化剂具有较好的循环稳定性.     

ATPG is required for the accumulation and function of chloroplast ATP synthase in Arabidopsis

The subunit Ⅱ of chloroplast ATP synthase is one of the two peripheral stalks, which associates the catalytic CF1 with mem-brane-spanning CFo . Although the structural and functional roles of chloroplast ATP synthase have been extensively examined, the physiological significance of subunit Ⅱ in vivo is still unclear. In this work, we identified one Arabidopsis T-DNA insertion mutant of atpG gene encoding the subunit Ⅱ of chloroplast ATP synthase. The atpg null mutant displayed an albino lethal pheno-type, as it could not grow photoautotrophically. Transmission electron microscopy analysis showed that chloroplasts of atpg lacked the organized thylakoid membranes. Loss of subunit Ⅱ affected the accumulation of CF1-CFo complex, however, it did not seem to have an effect on the CF1 assembly. The light induced ATP formation of atpg was significantly reduced compared with the wild type. Based on these results, we suggested that ATPG was essential for the accumulation and function of chloroplast ATP synthase.     

用于高性能超级电容器的基于蚕沙的二氧化锰/碳纳米复合材料

MnO₂/biomass carbon nanocomposite was synthesized by a facile hydrothermal reaction. Silkworm excrement acted as a carbon precursor, which was activated by ZnCl₂ and FeCl₃ combining chemical agents under Ar atmosphere. Thin and flower-like MnO₂ nanowires were in-situ anchored on the surface of the biomass carbon. The biomass carbon not only offered high conductivity and good structural stability but also relieved the large volume expansion during the charge/discharge process. The obtained MnO₂/biomass carbon nanocomposite electrode exhibited a high specific capacitance (238 F·g?1 at 0.5 A·g?1) and a superior cycling stability with only 7% degradation after 2000 cycles. The observed good electrochemical performance is accredited to the materials’ high specific surface area, multilevel hierarchical structure, and good conductivity. This study proposes a promising method that utilizes biological waste and broadens MnO₂-based electrode material application for next-generation energy storage and conversion devices.     

CO2催化加氢制乙醇研究进展

燃料乙醇是可再生的清洁燃料,具有替代汽油的应用前景.以CO2气体为碳源并通过催化加氢制燃料乙醇具有环境保护和节约能源的现实意义.主要介绍了CO2催化加氢的反应机理以及催化剂活性组分、前驱物、助剂及载体对催化活性、产物选择性的影响,同时介绍了反应条件对催化过程的影响.