Stochastic rotational catalysis of proton pumping F-ATPase

F-ATPases synthesize ATP from ADP and phosphate coupled with an electrochemical proton gradient in bacterial or mitochondrial membranes and can hydrolyse ATP to form the gradient. F-ATPases consist of a catalytic F1 and proton channel F0 formed from the alpha3beta3gammadelta and ab2c10 subunit complexes, respectively. The rotation of gammaepsilonc10 couples catalyses and proton transport. Consistent with the threefold symmetry of the alpha3beta3 catalytic hexamer, 120 degrees stepped revolution has been observed, each step being divided into two substeps. The ATP-dependent revolution exhibited stochastic fluctuation and was driven by conformation transmission of the beta subunit (phosphate-binding P-loop/alpha-helix B/loop/beta-sheet4). Recent results regarding mechanically driven ATP synthesis finally proved the role of rotation in energy coupling.     

Stochastic proton pumping ATPases: from single molecules to diverse physiological roles

We discuss the most recent reports on two proton pumps, F-ATPase (ATP synthase) and V-ATPase (endomembrane proton pump). They are formed from similar extrinsic (F1 or V1) and intrinsic (Fo or Vo) membrane sectors, and couple chemistry and proton transport through subunit rotation for apparently different physiological roles. Emphasis is placed on the stochastic rotational catalysis of F-ATPase and isoforms of V-ATPase.     

Evolution of proton pumping ATPases: Rooting the tree of life

Proton pumping ATPases are found in all groups of present day organisms. The F-ATPases of eubacteria, mitochondria and chloroplasts also function as ATP synthases, i.e., they catalyze the final step that transforms the energy available from reduction/oxidation reactions (e.g., in photosynthesis) into ATP, the usual energy currency of modern cells. The primary structure of these ATPases/ATP synthases was found to be much more conserved between different groups of bacteria than other parts of the photosynthetic machinery, e.g., reaction center proteins and redox carrier complexes.These F-ATPases and the vacuolar type ATPase, which is found on many of the endomembranes of eukaryotic cells, were shown to be homologous to each other; i.e., these two groups of ATPases evolved from the same enzyme present in the common ancestor. (The term eubacteria is used here to denote the phylogenetic group containing all bacteria except the archaebacteria.) Sequences obtained for the plasmamembrane ATPase of various archaebacteria revealed that this ATPase is much more similar to the eukaryotic than to the eubacterial counterpart. The eukaryotic cell of higher organisms evolved from a symbiosis between eubacteria (that evolved into mitochondria and chloroplasts) and a host organism. Using the vacuolar type ATPase as a molecular marker for the cytoplasmic component of the eukaryotic cell reveals that this host organism was a close relative of the archaebacteria.A unique feature of the evolution of the ATPases is the presence of a non-catalytic subunit that is paralogous to the catalytic subunit, i.e., the two types of subunits evolved from a common ancestral gene. Since the gene duplication that gave rise to these two types of subunits had already occurred in the last common ancestor of all living organisms, this non-catalytic subunit can be used to root the tree of life by means of an outgroup; that is, the location of the last common ancestor of the major domains of living organisms (archaebacteria, eubacteria and eukaryotes) can be located in the tree of life without assuming constant or equal rates of change in the different branches.A correlation between structure and function of ATPases has been established for present day organisms. Implications resulting from this correlation for biochemical pathways, especially photosynthesis, that were operative in the last common ancestor and preceding life forms are discussed.     

Energizing an invertebrate embryo: bafilomycin-dependent respiration and the metabolic cost of proton pumping by the V-ATPase

We examine herein the contribution of V-ATPase activity to the energy budget of aerobically developing embryos of Artemia franciscana and discuss the results in the context of quiescence under anoxia. (31)P-NMR analysis indicates that intracellular pH and NTP levels are unaffected by acute incubation of dechorionated embryos with the V-ATPase inhibitor, bafilomycin A(1). Bafilomycin A(1) also has no significant effect on oxygen consumption by isolated mitochondria. Taken together, these data indicate that bafilomycin does not affect energy-producing pathways in the developing embryo. However, the V-ATPase inhibitor exhibits a concentration-dependent inhibition of oxygen consumption in aerobic embryos. A conservative analysis of respirometric data indicates that proton pumping by the V-ATPase, and processes immediately dependent on this activity, constitutes approximately 31% of the aerobic energy budget of the preemergent embryo. Given the complete absence of detectable Na(+)K(+)-ATPase activity during the first hours of aerobic development, it is plausible that the V-ATPase is performing a role in both the acidification of intracellular compartments and the energization of plasma membranes. Importantly, the high metabolic cost associated with maintaining these diverse proton gradients requires that V-ATPase activity be downregulated under anoxia in order to attain the almost complete metabolic depression observed in the quiescent embryo.     

Events in proton pumping by bacteriorhodopsin.

The short-circuit photoresponse of a bacteriorhodopsin-based photoactive membrane is studied. The membrane is formed by first coating a Teflon membrane with lipid and then fusing bacteriorhodopsin vesicles to it. An incandescent light source was used to obtain the rise time of the photocurrent in response to a step-function illumination. A fast response, less than 1 ms, characterizes the initial rise and decay of the photocurrent. The trailing edge of the rise and trailing edge of the decay each exhibit different time constants both greater than 1 ms. These slower components show a sensitivity to membrane charging, the presence of diethylether in the bathing solution, and the presence of a charged cation complex in the lipid region. The photoresponse is not analyzed by means of the usual equivalent electrical circuit, but rather in terms of image charges in the conducting electrolyte bathing the membrane. Further experiments using a pulsed laser (pulse width less than 1 microseconds) resolve at least three time constants in the photoresponse: 0.057 ms, 1.06 ms, and 13 ms. Three distinct charge displacements (4.4, 7.5, and 33.1 A) are derived from the data, each corresponding to one of the above time constants.     

Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology

The acidity of intracellular compartments and the extracellular environment is crucial to various cellular processes, including membrane trafficking, protein degradation, bone resorption and sperm maturation. At the heart of regulating acidity are the vacuolar (V-)ATPases--large, multisubunit complexes that function as ATP-driven proton pumps. Their activity is controlled by regulating the assembly of the V-ATPase complex or by the dynamic regulation of V-ATPase expression on membrane surfaces. The V-ATPases have been implicated in a number of diseases and, coupled with their complex isoform composition, represent attractive and potentially highly specific drug targets.     

KIN, a mammalian nuclear protein immunologically related to E. coli RecA protein

A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa). The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus. Its level is higher in proliferating than in quiescent cells. Cell treatment with mitomycin C increases the level of the KIN protein. We sought similar proteins in other mammalian cells. Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos.     

From "butyribacterium" to "E. coli": an essay on unity in biochemistry

New ideas in science frequently arise from neglected or distorted antecedents. This essay deals with the idea of biochemical unity, encapsulated in Jacques Monod's well-known phrase, dating from 1954: "Anything found to be true of E. coli must also be true of elephants." An earlier version of this phrase,--"From the elephant to butyric acid bacterium--it is all the same!"--was coined in 1926 by the Dutch microbiologist Albert Jan Kluyver. In that year Kluyver and his associate Hendrick Jean Louis Donker published a celebrated paper, "Unity in Biochemistry." The concept of biochemical unity had many antecedents, but these had never caught on. The Kluyver-Donker paper has often been regarded to provide a boost to biochemical and especially to microbiological thinking. Its interpretations and misinterpretations represent an encapsulated history of biochemistry. The present paper examines the history of the concept of biochemical unity from before to beyond Kluyver, investigates the two "elephant" phrases and their possible relationships, and ends with a discussion of the attractiveness of unifying ideas in science.     

Evidence for a role of a vicinal dithiol in catalysis and proton pumping in mitochondrial nicotinamide nucleotide transhydrogenase

The effect of glutathione, glutathione disulfide and the dithiol reagent phenylarsine oxide on purified soluble as well as reconstituted mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. Glutathione disulfide and phenylarsine oxide caused an inhibition of transhydrogenase, the extent of which was dependent on the presence of either of the transhydrogenase substrates. In the absence of NADPH glutathione protected partially against inactivation by glutathione disulfide and phenylarsine oxide. In the presence of NADPH glutathione also inhibited transhydrogenase. Reconstituted transhydrogenase vesicles behaved differently as compared to the soluble transhydrogenase and was partially uncoupled by GSSG. It is concluded that transhydrogenase contains a dithiol that is essential for catalysis as well as for proton translocation.     

Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E. coli transhydrogenase

The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha-helices, and is responsible for proton transport. In the present investigation the segment betaC260-betaS266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction. Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed. Except for betaS266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the betaC260-betaR266 region revealed an increased accessibility in the presence of NADP(H) bound to domain III; an opposite effect was observed for betaS266. A betaD213-betaR265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge. The betaS260C, betaK261C, betaA262C, betaM263, and betaN264 mutants showed a pronounced inhibition of proton-coupled reactions. Likewise, several betaR265 mutants and the D213C mutant showed inhibited proton-coupled reactions but also markedly increased values. It is concluded that the mobile hinge region betaC260-betaS266 and the betaD213-betaR265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III.     

Selective catalysis of A.T base pair proton exchange in DNA complexes: imino proton NMR analysis.

Polyaromatic molecules with amino chain substituents, upon binding with DNA, selectively catalyze exchange of the A.T base pair protons with bulk water protons. The amine-catalyzed exchange is mediated by compounds which are A.T and G.C base sequence specific, intercalators, and outside binders. A mechanism for the selective exchange, involving transient opening and closing of individual A.T base pairs in the duplex, is discussed.     

Use of L-asparagine and N-phosphonacetyl-L-asparagine to investigate the linkage of catalysis and homotropic cooperativity in E. coli aspartate transcarbomoylase

The mechanism of domain closure and the allosteric transition of Escherichia coli aspartate transcarbamoylase (ATCase) are investigated using L-Asn, in the presence of carbamoyl phosphate (CP), and N-phosphonacetyl-L-asparagine (PASN). ATCase was found to catalyze the carbamoylation of L-Asn with a K(m) of 122 mM and a maximal velocity 10-fold lower than observed with the natural substrate, L-Asp. As opposed to L-Asp, no cooperativity was observed with respect to L-Asn. Time-resolved small-angle X-ray scattering (SAXS) and fluorescence experiments revealed that the combination of CP and L-Asn did not convert the enzyme from the T to the R state. PASN was found to be a potent inhibitor of ATCase exhibiting a K(D) of 8.8 microM. SAXS experiments showed that PASN was able to convert the entire population of molecules to the R state. Analysis of the crystal structure of the enzyme in the presence of PASN revealed that the binding of PASN was similar to that of the R-state complex of ATCase with N-phosphonaceyl-L-aspartate, another potent inhibitor of the enzyme. The linking of CP and L-Asn into one molecule, PASN, correctly orients the asparagine moiety in the active site to induce domain closure and the allosteric transition. This entropic effect allows for the high affinity binding of PASN. However, the binding of L-Asn, in the presence of a saturating concentration of CP, does not induce the closure of the two domains of the catalytic chain, nor does the enzyme undergo the transition to the high-activity high- affinity R structure. These results imply that Arg229, which interacts with the beta-carboxylate of L-Asp, plays a critical role in the orientation of L-Asp in the active site and demonstrates the requirement of the beta-carboxylate of L-Asp in the mechanism of domain closure and the allosteric transition in E. coli ATCase.     

Comparison of dissimilarity patterns of E coli, yeast and mammalian tRNAs.

A number of experimental approaches have been developed for identification of recognition (identity) sites in tRNAs. Along with them a theoretical methodology has been proposed by McClain et al that is based on concomitant analysis of all tRNA sequences from a given species. This approach allows an evaluation of nucleotide combinations present in isoacceptor tRNAs specific for the given amino acid, and not present in equivalent positions in cloverleaf structure in other tRNAs of the same organism. These elements predicted from computer analysis of the databank could be tested experimentally for their participation in forming recognition sites. The correlation between theoretical predictions and experimental data appeared promising. The aim of the present work consisted of introducing further improvements into McClain's procedure by: i), introducing into analysis a variable region in tRNAs which had not been previously considered; to accomplish this, 'normalization' of variable nucleotides was suggested, based on primary and tertiary structures of tRNAs; ii), developing a new procedure for comparison of patterns for synonymous and non-synonymous tRNAs from different organisms; iii), analysis of 3- and 4-positional contacts between tRNAs and enzymes in addition to a formerly used 2-positional model. A systematic application of McClain's procedure to mammalian, yeast and E coli tRNAs led to the following results: i), imitancy patterns for non-synonymous tRNAs of any amino acid specificity and from any organisms analysed so far overlap by no more than 30%, providing a structural basis for discrimination with high fidelity between cognate and non-cognate tRNAs; ii), the predicted identity sites are non-randomly distributed within tRNA molecules; the dominant role is ascribed to only two regions--anticodon and amino acid stem which are located far apart from one another at extremes of all tRNA molecules; iii), the imitancy patterns for synonymous tRNAs in lower (yeast) and higher (mammalian) eukaryotes are similar but not identical; iv), distribution of predicted identity sites in the cloverleaf structure in prokaryotes and eukaryotes is essentially different: in eubacterial tRNAs the major role in recognition plays anticodon and/or amino acid acceptor stem, whereas in eukaryotic (both unicellular and multicellular) tRNAs the remaining part of the molecules is also involved in recognition; v), the imitancy patterns of synonymous tRNAs from prokaryotes and eukaryotes are dissimilar, this observation leads to the prediction that the tRNA identity sites for the same amino acid in prokaryotes and eukaryotes may differ.