The chloroplast ATP synthase: Structural changes during catalysis

This article summarizes some of the evidence for the existence of light-driven structural changes in the and subunits of the chlorplast ATP synthase. Formation of a transmembrane proton gradient results in: (1) a change in the position of the subunit such that it becomes exposed to polyclonal antibodies and to reagents which selectively modifyLys109; (2) enhanced solvent accessibility of several sulfhydryl residues on the subunit; and (3) release/ exchange of tightly bound ADP from the enzyme. These and related experimental observations can, at least partially, be explained in terms of two different bound conformational states of the subunit. Evidence for structural changes in the enzyme which are driven by light or nucleotide binding is discussed with special reference to the popular rotational model for catalysis.     

Subunit movement during catalysis by F1-F0-ATP synthases

The catalytic portion (F₁) of ATP synthases have the subunit composition ₃, ₃, , , . This composition imparts structural asymmetry to the entire complex that results in differences in nucleotide binding affinity among the six binding sites. Evidence that two or more sites participate in catalysis, alternating their properties, led to the notion that the interactions of individual pairs with the small subunits must change as binding site properties alternate. A rotation of the subunit within the ₃₃ hexamer has been proposed as a means of alternating the properties of catalytic sites. Evidence argues that the rotation of the complete subunit during ATP hydrolysis is not mandatory for activity. The subunit of chloroplast F₁ may be cleaved into three large fragments that remain bound to F₁. This cleavage enhances ATPase activity without loss of evidence of site-site interactions. Complexes of ₃₃ have been shown to have significant ATPase activity in the absence of . Mg²⁺ATP affects the interaction of with the different subunits, and induces other changes in F₁, but whether these changes are induced by catalysis, or are fast enough to be involved in the catalytic turnover of the enzyme has not been established. Likewise, changes in structure and in binding site properties induced in thylakoid membrane bound CF₁ by formation of an electrochemical proton gradient may activate the enzyme rather than be apart of catalysis. Mechanisms other than rotary catalysis should be considered.     

ATP合成酶的结合变化机制和旋转催化——1997年诺贝尔化学奖的部分工作介绍

保罗.博耶(P.D.Boyer)教授为阐明ATP酶作用机制所提出的结合变化机制有两个基本要点：一是ATP合成所需要的能量原则上是用于促进酶上紧密结合的ATP的释放和无机磷、ADP的结合;二是在净ATP形成过程中,酶上的各催化部位是高度协同地顺序起作用的.γ亚基在F₁-ATP酶中的旋转运动使三个催化部位构象不对称是实现结合变化的基础.高分辨率牛心线粒体F₁-ATP酶的晶体结构发表以后,出现了一些支持旋转催化机制的直接实验证据.     

ATP合成酶的结合变化机制和旋转催化──1997年诺贝尔化学奖的部分工作介绍

保罗博耶（Ｐ．Ｄ．Ｂｏｙｅｒ）教授为阐明ＡＴＰ酶作用机制所提出的结合变化机制有两个基本要点：一是ＡＴＰ合成所需要的能量原则上是用于促进酶上紧密结合的ＡＴＰ的释放和无机磷、ＡＤＰ的结合;二是在净ＡＴＰ形成过程中,酶上的各催化部位是高度协同地顺序起作用的．γ亚基在Ｆ１ＡＴＰ酶中的旋转运动使三个催化部位构象不对称是实现结合变化的基础．高分辨率牛心线粒体Ｆ１ＡＴＰ酶的晶体结构发表以后,出现了一些支持旋转催化机制的直接实验证据     

Energy, Life, and ATP

The mechanism by which ATP is synthesized during oxidative and photophosphorylation has been elucidated by oxygen exchange and other studies: a novel form of catalysis--termed rotary catalysis--is involved.     

The mechanism of action of mitochondrial ATPase (ATP synthase)

The F₁ part of the ATP synthase contains 6 nucleotide binding sites, four of which can be occupied and covalently labeled with 8-azido-adenine nucleotides. The other two sites contain tightly bound nucleotides that cannot be replaced by 8-azido-adenine nucleotides. Of the four exchangeable sites two are directly ivolved in catalysis and these are located on -subunits, while the other two are located at - interfaces and have probably a regulatory role by influencing the affinity of the catalytic sites for substrate and product. When only one catalytic site contains substrate the affinity is very high, the rate of hydrolysis is slow, and the dissociation of products is even slower (single-site catalysis). When the second site also becomes occupied, the affinity decreases enormously, and the rate of hydrolysis and dissociation of products increases several orders of magnitude. When, however, the second site is occupied by substrate in such a way that turnover is not possible at this site (e.g., covalent linkage of nitreno-ATP), the first site is no longer active, apart from the very slow single-site catalysis. The two nonexchangeable, tightly bound nucleotides that cannot be replaced by 8-azido-nucleotides, can be replaced by 2-azido-nucleotides, due to their anticonfiguration. This anticonfiguration of the substrate is also required for binding with high affinity to a catalytic site. A picture emerges in which one of the three - pairs of F₁ contains tightly bound, nonexchangeable nucleotides, while the other two contain both one catalytic site (on ) and one regulatory site (at the - interface). Cooperativity exists both between the two catalytic sites and between the catalytic and the regulatory sites.     

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [¹⁴C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [¹⁴C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m^–2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.Abbreviations AdN adenine nucleotide - Chl chlorophyll - DTT dithiothreitol - FCCP carbonylcianide p-trifluoromethoxyphenilhydrazone - P_i inorganic orthophosphate - PMS phenazine methosulfate - TLC thin-layer chromatography - Tricine N-[tris(hydroxymethyl)methyl] glycine     

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F₁F_o-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F₁F_o-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F₁F_o-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F₁F_o-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F₁ catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F₁ to the lipid monolayer and the F_o membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F_o by electron microscopy and AFM.     

Post-translational modifications of ATP synthase in the heart: biology and function

The ATP synthase complex is a critical enzyme in the energetic pathways of cells because it is the enzyme complex that produces the majority of cellular ATP. It has been shown to be involved in several cardiac phenotypes including heart failure and preconditioning, a cellular protective mechanism. Understanding the regulation of this enzyme is important in understanding the mechanisms behind these important phenomena. Recently there have been several post-translational modifications (PTM) reported for various subunits of this enzyme complex, opening up the possibility of differential regulation by these PTMs. Here we discuss the known PTMs in the heart and other mammalian tissues and their implication to function and regulation of the ATP synthase.     

Toward an Adequate Scheme for the ATP Synthase Catalysis

The suggestions from the author's group over the past 25 years for how steps in catalysis by ATP synthase occur are reviewed. Whether rapid ATP hydrolysis requires the binding of ATP to a second site (bi-site activation) or to a second and third site (tri-site activation) is considered. Present evidence is regarded as strongly favoring bi-site activation. Presence of nucleotides at three sites during rapid ATP hydrolysis can be largely accounted for by the retention of ADP formed and/or by the rebinding of ADP formed. Menz, Leslie and Walker ((2001) FEBS Lett., 494, 11-14) recently attained an X-ray structure of a partially closed enzyme form that binds ADP better than ATP. This accomplishment and other considerations form the base for a revised reaction sequence. Three types of catalytic sites are suggested, similar to those proposed before the X-ray data became available. During net ATP synthesis a partially closed site readily binds ADP and P_i but not ATP. At a closed site, tightly bound ADP and P_i are reversibly converted to tightly bound ATP. ATP is released from a partially closed site that can readily bind ATP or ADP. ATP hydrolysis when protonmotive force is low or lacking occurs simply by reversal of all steps with the opposite rotation of the subunit. Each type of site can exist in various conformations or forms as they are interconverted during a 120° rotation. The conformational changes with the ATP synthase, including the vital change when bound ADP and P_i are converted to bound ATP, are correlated with those that occur in enzyme catalysis in general, as illustrated by recent studies of Rose with fumarase. The _B structure of Walker's group is regarded as an unlikely, or only quite transient, intermediate. Other X-ray structures are regarded as closely resembling but not identical with certain forms participating in catalysis. Correlation of the suggested reaction scheme with other present information is considered.     

ATP synthase in mycobacteria: Special features and implications for a function as drug target

ATP synthase is a ubiquitous enzyme that is largely conserved across the kingdoms of life. This conservation is in accordance with its central role in chemiosmotic energy conversion, a pathway utilized by far by most living cells. On the other hand, in particular pathogenic bacteria whilst employing ATP synthase have to deal with energetically unfavorable conditions such as low oxygen tensions in the human host, e.g. Mycobacterium tuberculosis can survive in human macrophages for an extended time. It is well conceivable that such ATP synthases may carry idiosyncratic features that contribute to efficient ATP production. In this review genetic and biochemical data on mycobacterial ATP synthase are discussed in terms of rotary catalysis, stator composition, and regulation of activity. ATP synthase in mycobacteria is of particular interest as this enzyme has been validated as a target for promising new antibacterial drugs. A deeper understanding of the working of mycobacterial ATP synthase and its atypical features can provide insight in adaptations of bacterial energy metabolism. Moreover, pinpointing and understanding critical differences as compared with human ATP synthase may provide input for the design and development of selective ATP synthase inhibitors as antibacterials. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

The mitochondrial ATP synthase ofTrypanosoma brucei: Structure and regulation

The structure and regulation of theTrypanosoma brucei mitochondrial ATP synthase is reviewed. This enzyme complex which catalyzes the synthesis and hydrolysis of ATP within the mitochondrion is a multisubunit complex which is regulated in several ways. Several lines of evidence have shown that the ATP synthase is regulated through the life cycle ofTrypanosoma brucei. The enzyme complex is present at maximal levels in the procyclic form where mitochondrial activity is the highest and cytochromes and Kreb's cycle components are present. The levels of the ATP synthase are decreased in the bloodstream forms where the levels of the mitochondrial cytochromes are absent or substantially decreased. In recent preliminary work we have shown the presence of an ATP synthase inhibitor peptide which may indicate an additional level of complexity to the regulation.     

Conformational movements and cooperativity upon amino acid,ATP and tRNA binding in threonyl-tRNA synthetase

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8A at some C(alpha) positions, of a strand-loop-strand region of the core beta-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.     

Changes of mitochondrial cytochrome c oxidase and FoF1 ATP synthase subunits in rat cerebral cortex during aging

The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits , and of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for ATP synthase, while the and subunits increased progressively up to 26 months.     

ATP synthesis driven by proton transport in F1F0-ATP synthase

Topical questions in ATP synthase research are: (1) how do protons cause subunit rotation and how does rotation generate ATP synthesis from ADP+Pi? (2) How does hydrolysis of ATP generate subunit rotation and how does rotation bring about uphill transport of protons? The finding that ATP synthase is not just an enzyme but rather a unique nanomotor is attracting a diverse group of researchers keen to find answers. Here we review the most recent work on rapidly developing areas within the field and present proposals for enzymatic and mechanoenzymatic mechanisms.     

The b Subunit of Escherichia coli ATP Synthase

The b subunit of ATP synthase is a major component of the second stalk connecting the F₁and F₀ sectors of the enzyme and is essential for normal assembly and function. The156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensivelythrough mutagenesis, deletion analysis, and biophysical characterization. The two copies ofb exist as a highly extended, helical dimer extending from the membrane to near the top ofF₁, where they interact with the subunit. The sequence has been divided into four domains:the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, andthe C-terminal -binding domain. The dimerization domain, contained within residues 60–122,has many properties of a coiled-coil, while the -binding domain is more globular. Sites ofcrosslinking between b and the a, , , and subunits of ATP synthase have been identified,and the functional significance of these interactions is under investigation. The b dimer mayserve as an elastic element during rotational catalysis in the enzyme, but also directly influencesthe catalytic sites, suggesting a more active role in coupling.     

Analysis of a conserved arginine R281L in catalysis in Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase is essential for the catalytic transformation of a linear tripeptide substrate δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine to isopenicillin N in the biosynthesis of β-lactam antibiotics. The recent Aspergillus nidulans isopenicillin N synthase crystal structure proposed that a conserved arginine, R279, has a role in substrate binding. This study, the first site-directed mutagenesis experiment on arginine in isopenicillin N synthase, was carried out to ascertain the role of the similarly conserved and corresponding arginine residue R281 on catalysis in the fungal Cephalosporium acremonium isopenicillin N synthase. Replacement of the arginine residue with leucine to generate the mutant R281L Cephalosporium isopenicillin N synthase resulted in undetectable activity as shown by enzyme bioassays. It is possible that the mutant's substrate binding capability was eliminated, thus preventing the catalytic reaction. Further investigation into the corresponding arginine residues in isopenicillin N synthase of other species is warranted.     

The rotary mechanism of ATP synthase

Since the chemiosmotic theory was proposed by Peter Mitchell in the 1960s, a major objective has been to elucidate the mechanism of coupling of the transmembrane proton motive force, created by respiration or photosynthesis, to the synthesis of ATP from ADP and inorganic phosphate. Recently, significant progress has been made towards establishing the complete structure of ATP synthase and revealing its mechanism. The X-ray structure of the F(1) catalytic domain has been completed and an electron density map of the F(1)-c(10) subcomplex has provided a glimpse of the motor in the membrane domain. Direct microscopic observation of rotation has been extended to F(1)-ATPase and F(1)F(o)-ATPase complexes.