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.     

Frontiers in ATP synthase research: Understanding the relationship between subunit movements and ATP Synthesis

How biological systems make ATP has intrigued many scientists for well over half the 20th century, and because of the importance and complexity of the problem it seems likely to continue to be a source of fascination to both senior and younger investigators well into the 21st century. Scientific battles fought to unravel the vast secrets by which ATP synthases work have been fierce, and great victories have been short-lived, tempered with the realization that more structures are needed, additional subunits remain to be conquered, and that during ATP synthesis, not one, but several subunits may undergo either significant conformational changes, repositioning, or perhaps even physical rotation similar to bacterial flagella^(1,2). In this introductory article, the author briefly summarizes our current knowledge about the complex substructure of ATP synthases, what we have learned from X-ray crystallography of the F₁ unit, and current evidence for subunit movements.     

The rotary mechanism of the ATP synthase

The F₀F₁ ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous F₀ sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium.     

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.     

Molecular basis of diseases caused by the mtDNA mutation m.8969G>A in the subunit a of ATP synthase

The ATP synthase which provides aerobic eukaryotes with ATP, organizes into a membrane-extrinsic catalytic domain, where ATP is generated, and a membrane-embedded F_O domain that shuttles protons across the membrane. We previously identified a mutation in the mitochondrial MT-ATP6 gene (m.8969G>A) in a 14-year-old Chinese female who developed an isolated nephropathy followed by brain and muscle problems. This mutation replaces a highly conserved serine residue into asparagine at amino acid position 148 of the membrane-embedded subunit a of ATP synthase. We showed that an equivalent of this mutation in yeast (aS₁₇₅N) prevents F_O-mediated proton translocation. Herein we identified four first-site intragenic suppressors (aN₁₇₅D, aN₁₇₅K, aN₁₇₅I, and aN₁₇₅T), which, in light of a recently published atomic structure of yeast F_O indicates that the detrimental consequences of the original mutation result from the establishment of hydrogen bonds between aN₁₇₅ and a nearby glutamate residue (aE₁₇₂) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS₁₇₅N mutation can be suppressed by second-site suppressors (aP₁₂S, aI₁₇₁F, aI₁₇₁N, aI₂₃₉F, and aI₂₀₀M), of which some are very distantly located (by 20–30?Å) from the original mutation. The possibility to compensate through long-range effects the aS₁₇₅N mutation is an interesting observation that holds promise for the development of therapeutic molecules.     

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.     

Aconitase and ATP synthase are targets of malondialdehyde modification and undergo an age-related decrease in activity in mouse heart mitochondria

The main purpose of this study was to identify mitochondrial proteins that exhibit post-translational oxidative modifications during the aging process and to determine the resulting functional alterations. Proteins forming adducts with malondialdehyde (MDA), a product of lipid peroxidation, were identified by immunodetection in mitochondria isolated from heart and hind leg skeletal muscle of 6-, 16-, and 24-month-old mice. Aconitase, very long chain acyl coenzyme A dehydrogenase, ATP synthase, and alpha-ketoglutarate dehydrogenase were detected as putative targets of oxidative modification by MDA. Aconitase and ATP synthase from heart exhibited significant decreases in activity with age. Very long chain acyl coenzyme A dehydrogenase and alpha-ketoglutarate dehydrogenase activities were unaffected during aging in both heart and skeletal muscle. This suggests that the presence of a post-translational oxidative modification in a protein does not a priori reflect an alteration in activity. The biological consequences of an age-related decrease in aconitase and ATP synthase activities may contribute to the decline in mitochondrial bioenergetics evident during aging.     

pH homeostasis and ATP synthesis: studies of two processes that necessitate inward proton translocation in extremely alkaliphilic Bacillus species

Alkaliphilic Bacillus species that are isolated from nonmarine, moderate salt, and moderate temperature environments offer the opportunity to explore strategies that have developed for solving the energetic challenges of aerobic growth at pH values between 10 and 11. Such bacteria share many structural, metabolic, genomic, and regulatory features with nonextremophilic species such as Bacillus subtilis. Comparative studies can therefore illuminate the specific features of gene organization and special features of gene products that are homologs of those found in non-extremophiles, and potentially identify novel gene products of importance in alkaliphily. We have focused our studies on the facultative alkaliphile Bacillus firmus OF4, which is routinely grown on malate-containing medium at either pH 7.5 or 10.5. Current work is directed toward clarification of the characteristics and energetics of membrane-associated proteins that must catalyze inward proton movements. One group of such proteins are the Na⁺/H⁺ antiporters that enable cells to adapt to a sudden upward shift in pH and to maintain a cytoplasmic pH that is 2–2.3 units below the external pH in the most alkaline range of pH for growth. Another is the proton-translocating ATP synthase that catalyzes robust production of ATP under conditions in which the external proton concentration and the bulk chemiosmotic driving force are low. Three gene loci that are candidates for Na⁺/H⁺ antiporter encoding genes with roles in Na⁺- dependent pH homeostasis have been identified. All of them have homologs in B. subtilis, in which pH homeostasis can be carried out with either K⁺ or Na⁺. The physiological importance of one of the B. firmus OF4 loci, nhaC, has been studied by targeted gene disruption, and the same approach is being extended to the others. The atp genes that encode the alkaliphile's F₁F_O-ATP synthase are found to have interesting motifs in areas of putative importance for proton translocation. As an initial step in studies that will probe the importance and possible roles of these motifs, the entire atp operon from B. firmus OF4 has been cloned and functionally expressed in an Escherichia coli mutant that has a full deletion of its atp genes. The transformant does not exhibit growth on succinate, but shows reproducible, modest increases in the aerobic growth yields on glucose as well as membrane ATPase activity that exhibits characteristics of the alkaliphile enzyme. Received: January 22, 1998 / Accepted: February 16, 1998     

From OCR and ECAR to energy: Perspectives on the design and interpretation of bioenergetics studies

Biological energy transduction underlies all physiological phenomena in cells. The metabolic systems that support energy transduction have been of great interest due to their association with numerous pathologies including diabetes, cancer, rare genetic diseases, and aberrant cell death. Commercially available bioenergetics technologies (e.g., extracellular flux analysis, high-resolution respirometry, fluorescent dye kits, etc.) have made practical assessment of metabolic parameters widely accessible. This has facilitated an explosion in the number of studies exploring, in particular, the biological implications of oxygen consumption rate (OCR) and substrate level phosphorylation via glycolysis (i.e., via extracellular acidification rate (ECAR)). Though these technologies have demonstrated substantial utility and broad applicability to cell biology research, they are also susceptible to historical assumptions, experimental limitations, and other caveats that have led to premature and/or erroneous interpretations. This review enumerates various important considerations for designing and interpreting cellular and mitochondrial bioenergetics experiments, some common challenges and pitfalls in data interpretation, and some potential “next steps” to be taken that can address these highlighted challenges.     

Isolation and mapping of a putative b subunit of human ATP synthase (ATP-BL) from human leukocytes.

From a human-leukocyte cDNA library, we cloned cDNA encoding a novel protein, which has a significant homology with the b subunit of ATP synthase (proton-transporting ATPase, F1F0-ATPase; EC3.6.1.34) derived from Anabaena sp. strain PCC 7120. The cDNA has an open reading frame of 1314 nucleotides corresponding to 438 amino acids. The coding sequence was 37.9% identical over 57 amino acid with b subunit of ATP synthase. The 34-amino-acid region of the predicted peptide sequence displays a coiled-coil motif that could form a complex with some other protein(s). We designated this novel gene as ATP-BL because of its homology to the b subunit of ATP synthase. The ATP-BL locus was mapped by fluorescence in situ hybridization (FISH) and radiation hybrid mapping to the q24 region of chromosome 16.     

Bioenergetics of the heart at high altitude: environmental hypoxia imposes profound transformations on the myocardial process of ATP synthesis

The low concentration of O₂ in the thin air at high altitude is undoubtedly the reason for the remarkable modifications in the structure and function of the heart, lung, and blood of humans permanently living under these conditions. The effect of natural hypoxia on the energy metabolism of the cell is however not well understood. Here we study the proces of ATP synthesis in the heart of guinea pigs native to high altitude (4500 m) as compared with those native to sea level. The following are the novel findings of this study. (1) The rates and extents of ATP synthesis in the presence of low concentrations of ADP (<30 M) are significantly higher at high altitude than at sea level. (2) The Hill coefficient, i.e. the degree of cooperativity between the three catalytic sites of the ATP synthase, is lower at high altitude (n = 1.36) than at sea level (n = 1.94). (3) Both, the affinity for ADP and the fractional occupancy of the catalytic sites by ATP, are higher at high altitude than at sea level but the P ₅₀, i.e. the concentration of ADP at which 50% of the catalytic sites are filled with ADP and/or ATP, is the same (74.7 M). (4) In the physiological range of ADP concentrations, the phosphorylation potential G _P is significantly higher at high altitude than at sea level. It is concluded that the molecular mechanism of energy transduction is profoundly modified at high altitude in order to readily and efficiently generate ATP in the presence of low concentrations of O₂ and ADP.     

Nucleotide sequences of the genes for the alpha,beta and epsilon subunits of wheat chloroplast ATP synthase

Summary The nucleotide sequences of the chloroplast genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase have been determined. Open reading frames of 1512 bp, 1494 bp and 411 bp are deduced to code for polypeptides of molecular weights 55201, 53796 and 15200, identified as the alpha, beta and epsilon subunits respectively by homology with the subunits from other sources and by amino acid sequencing of the epsilon subunit. The genes for the beta and epsilon subunits overlap by 4 bp. The gene for methionine tRNA is located 118 bp downstream from the epsilon subunit gene. Comparisons of the deduced amino acid sequences of the alpha and beta subunits with those from other species suggest regions of the proteins involved in adenine nucleotide binding.     

The essential role of sodium bioenergetics and ATP homeostasis in the developmental transitions of a cyanobacterium

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Binding affinities and protein ligand complex geometries of nucleotides at the F(1) part of the mitochondrial ATP synthase obtained by ligand docking calculations

F₀F₁ ATP synthases utilize a transmembrane electrochemical potential difference to synthesize ATP from ADP and phosphate. In this work, the binding modes of ADP, ATP and ATP analogues to the catalytic sites of the F₁ part of the mitochondrial ATP synthase were investigated with ligand docking calculations. Binding geometries of ATP and ADP at the three catalytic sites agree with X-ray crystal data; their binding free energies suggest an assignment to the ‘tight’, ‘open’ and ‘loose’ states. The rates of multi-site hydrolysis for two fluorescent ATP derivatives were measured using a fluorescence assay. Reduced hydrolysis rates compared to ATP can be explained by the ligand docking calculations.     

Molecular evolution of the modulator of chloroplast ATP synthase: origin of the conformational change dependent regulation

Chloroplast ATP synthase synthesizes ATP by utilizing a proton gradient as an energy supply, which is generated by photosynthetic electron transport. The activity of the chloroplast ATP synthase is regulated in several specific ways to avoid futile hydrolysis of ATP under various physiological conditions. Several regulatory signals such as Delta mu H(+), tight binding of ADP and its release, thiol modulation, and inhibition by the intrinsic inhibitory subunit epsilon are sensed by this complex. In this review, we describe the function of two regulatory subunits, gamma and epsilon, of ATP synthase based on their possible conformational changes and discuss the evolutionary origin of these regulation systems.     

Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene

Summary An allele (oliC31) of the A. nidulans oliC gene has been cloned using homology with the equivalent gene from N. crassa. OliC31 codes for an oligomycin-resistant, triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. Direct selection for oligomycin-resistance was possible following transformation of A. nidulans with the oliC31 gene. The phenotypes of transformants cultured in the presence of oligomycin were indicative of the position of integration of the transforming plasmid within the genome. Subsequent recombination events involving the integrated oliC31 gene were also apparent from altered levels of resistance to oligomycin or triethyltin. This gene should prove useful as a marker for transformation of strains lacking auxotrophic lesions and in gene replacement or disruption experiments.     

ATP synthase from Saccharomyces cerevisiae: location of subunit h in the peripheral stalk region

Subunit h is a component of the peripheral stalk region of ATP synthase from Saccharomyces cerevisiae. It is weakly homologous to subunit F6 in the bovine enzyme, and F6 can replace the function of subunit h in a yeast strain from which the gene for subunit h has been deleted. The removal of subunit h (or F6) uncouples ATP synthesis from the proton motive force. A biotinylation signal has been introduced following the C terminus of subunit h. It becomes biotinylated in vivo, and allows avidin to be bound quantitatively to the purified enzyme complex in vitro. By electron microscopy of the ATP synthase-avidin complex in negative stain and by subsequent image analysis, the C terminus of subunit h has been located in a region of the peripheral stalk that is close to the Fo membrane domain of ATP synthase. Models of the peripheral stalk are proposed that are consistent with this location and with reconstitution experiments conducted with isolated peripheral stalk subunits.     

ATP synthase from Saccharomyces cerevisiae: location of the OSCP subunit in the peripheral stalk region

A biotinylation signal has been fused to the C terminus of the oligomycin sensitivity conferral protein (OSCP) of the ATP synthase complex from Saccharomyces cerevisiae. The signal is biotinylated in vivo and the biotinylated complex binds avidin in vitro. By electron microscopy of negatively stained particles of the ATP synthase-avidin complex, the bound avidin has been localised close to the F(1) domain. The images were subjected to multi-reference alignment and classification. Because of the presence of a flexible linker between the OSCP and the biotinylation signal, the class-averages differ in the position of the avidin relative to the F(1) domain. These positions lie on an arc, and its centre indicates the position of the C terminus of the OSCP on the surface of the F(1) domain. Since the N-terminal region of the OSCP is known to interact with the N-terminal regions of alpha-subunits, which are on top of the F(1) domain distal from the F(o) membrane domain, the OSCP extends almost 10nm along the surface of F(1) down towards F(o) where it interacts with the C terminus of the b subunit, which extends up from F(o). The labelling technique has also allowed a reliable 2D projection map to be developed for the intact ATP synthase from S.cerevisiae. The map reveals a marked asymmetry in the F(o) part of the complex that can be attributed to subunits in the F(o) domain.