Margareta Baltscheffsky

H+ -PPases: a tightly membrane-bound family.

The earliest known H+‐PPase (proton‐pumping inorganic pyrophosphatase), the integrally membrane‐bound H+‐PPi synthase (proton‐pumping inorganic pyrophosphate synthase) from Rhodospirillum rubrum, is still the only alternative to H+‐ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+‐PPase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton‐pumping enzymes. The bacterial H+‐PPi synthase and two algal vacuolar H+‐PPases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+‐PPases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+‐PPases are reviewed and compared with H+‐ATPases and soluble PPases.

H+-PPases: yesterday, today and tomorrow.

Suggestions by Calvin about a role of inorganic pyrophosphate (PPi) in early photosynthesis and by Lipmann that PPi may have been the original energy‐rich phosphate donor in biological energy conversion, were followed in the mid‐1960s by experimental results with isolated chromatophore membranes from the purple photosynthetic bacterium Rhodospirillum rubrum. PPi was shown to be hydrolysed in an uncoupler stimulated reaction by a membrane‐bound inorganic pyrophosphatase (PPase), to be formed at the expense of light energy in photophosphorylation and to be utilized as an energy donor for various energy‐requiring reactions, as a first known alternative to ATP. This direct link between PPi and photosynthesis led to increasing attention concerning the role of PPi in both early and present biological energy transfer. In the 1970s, the PPase was shown to be a proton pump and to be present also in higher plants. In the 1990s, sequences of H+‐PPase genes were obtained from plants, protists, bacteria and archaea and two classes of H+‐PPases differing in K+ sensitivity were established. Over 200 H+‐PPase sequences have now been determined. Recent biochemical and biophysical results have led to new progress and questions regarding the H+‐PPase family, as well as the families of soluble PPases and the inorganic polyphosphatases, which hydrolyse inorganic linear high‐molecular‐weight polyphosphates (HMW‐polyP). Here we will focus attention on the H+‐PPases, their evolution and putative active site motifs, response to monovalent cations, genetic regulation and some very recent results, based on new methods for obtaining large quantities of purified protein, about their tertiary and quaternary structures. IUBMB Life, 59: 76‐83, 2007

Energy conversion-linked changes of carotenoid absorbance in Rhodospirillum rubrum chromatophores.

Abstract In a study of the dark reactions of energy conversion in chromatophores from Rhodospirillum rubrum both ATP and inorganic pyrophosphate (PPi) have earlier been found by us to serve as energy donors for “reversed” energy transfer and electron transport causing reversible changes in the redox-state of endogenous cytochromes. Concomitant with these reactions there was found to occur a reversible shift in carotenoid absorbance. This shift is energy dependent in a very similar way as the cytochrome reactions as judged from results obtained with uncoupling agents and inhibitors of electron transport coupled energy transfer. With respect to both direction and extent, the ATP- or PPi-induced shift in carotenoid absorbance resembles the light-induced shift, which, however, occurs at a more than 105 times faster rate. On the other hand, the kinetics for the PPi-induced carotenoid shift has been found to resemble the kinetics of the PPi-induced reduction of endogenous b-type cytochrome. On the basis of these characteristics of the carotenoid responses, as compared to those of endogenous cytochrome, a metabolic link between an energy-rich intermediate of the electron transport coupled energy transfer pathway is postulated to exist and a scheme for the suggested connections between energy sources, chlorophyll, and cytochromes in relation to carotenoid is given.

A pyrophosphate synthase gene: molecular cloning and sequencing of the cDNA encoding the inorganic pyrophosphate synthase from Rhodospirillum rubrum.

The integrally membrane-bound, proton-pumping inorganic pyrophosphate (PPi) synthase in phototrophic bacteria is hitherto the only described alternative to the ATP synthase in biological electron transport phosphorylation. We have identified and sequenced the first gene coding for a pyrophosphate synthase. The deduced protein contains 660 amino acid residues and 15 putative membrane-spanning segments. It is homologous to the vacuolar pyrophosphatases from plants.

H+-proton-pumping inorganic pyrophosphatase: a tightly membrane-bound family

The earliest known H+‐proton‐pumping inorganic pyrophosphatase, the integrally membrane‐bound H+‐proton‐pumping inorganic pyrophosphate synthase from Rhodospirillum rubrum, is still the only alternative to H+‐ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+‐proton‐pumping inorganic pyrophosphatase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton‐pumping enzymes. The bacterial H+‐proton‐pumping inorganic pyrophosphate synthase and two algal vacuolar H+‐proton‐pumping inorganic pyrophosphatases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+‐proton‐pumping inorganic pyrophosphatases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+‐proton‐pumping inorganic pyrophosphatases are reviewed and compared with H+‐ATPases and soluble proton‐pumping inorganic pyrophosphatases.

SENSITIVE MEASUREMENT OF FLASH INDUCED PHOTOPHOSPHORYLATION IN BACTERIAL CHROMATOPHORES BY FIREFLY LUCIFERASE

The use of firefly luciferase for measurement of electron transport-linked ATP formation in chloroplasts and mitochondria has been previously described [l-6]. Most of the observations were of a qualitative rather than quantitative nature. However, Lemasters and Hackenbrock [5,6] have described the use of firefly luciferase for quantitative, continuous measurement of oxidative phosphorylation in mitochondria and submitochondrial particles. Under the conditions employed in their experiments the bioluminescence intensity gradually decreased by end product inhibition of luciferase. This effect could be corrected for by the use of internal standards, i.e., the addition of known concentrations of ATP. In contrast to the brief flash usually obtained when mixing ATP and luciferase reagent, it was recently demonstrated that under appropriate conditions the intensity of the bioluminescence may be maintained essentially constant for several minutes using a purified luciferase reagent [7]. With this reagent any given ATP concentration up to 1 O-4 M results in a constant light emission directly proportional to the ATP concen&ation. The luciferase reagent consumes only negligable amounts of ATP and may be added to ATP converting systems using the bioluminescence intensity for continuous measurement of the ATP concentration in the system. The kinetic properties of the reagent

Reversed energy conversion reactions of bacterial photophosphorylation

Abstract The final steps of the reactions leading to the formation of inorganic pyrophosphate (PP i ) or ATP in bacterial photophosphorylation have been shown by us to be reversible to the level of electron transport in chromatophores from Rhodospirillum rubrum by addition of either of these two compounds in the dark. This reversal of photophosphorylation reactions, which is more rapid and larger with PP i than with ATP, results in a change in the redox state of at least two endogenous components of the electron transport chain, i.e. a reduction of b -type cytochrome and an oxidation of cytochrome c 2 . The half-time for the PP i -induced changes was around 0.4 sec which was one-tenth of the half-time when ATP was used as the energy donor. Some of the characteristics of the PP i -induced b -type cytochrome changes are described, such as the relationship between concentration of PP i and steady state redox level of b -type cytochrome, as well as the dependence of the reduction of this compound on concentration of Mg ++ -ion. Mn ++ , Zn ++ , and Co ++ may substitute for Mg ++ as co-factors. Ca ++ inhibits the PP i -induced changes. The sensitivity to the inhibitors antimycin A, gramicidin D, p -trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP), 2,6-dinitro-4-isooctylphenol (octyl-DNP), desaspidin and oligomycin has also been studied.

Reconstitution of highly purified proton-translocating pyrophosphatase from Rhodospirillum rubrum

Membrane-bound PPase is of interest as it functions in various organisms as a coupling factor between electron transport and PPi synthesis and is involved in the energy transduction pathway as an independent alternative to the ATPase system [1-4]. PPi hydrolysis in chromatophores of Rhodospirillum rubrum caused a change in the fluorescence of added 8-anilino-naphtalene-l-sulfonic acid [5], an uptake of phenyl dicarbaundecaborane anion [6] and a pH change of the outer medium [7]. Therefore, it was concluded that chromatophore PPase translocates protons across the membrane coupled to PPi hydrolysis, apparently in a manner corresponding to that of the chromatophore ATPase. Further investigation of membrane-bound PPase has been impeded by the lack of an efficient method of isolation. Although a method of purifying the PPase has been reported [8], only recently has a method of obtaining a highly purified mem-

On the subunit composition of the coupling factor (ATPase) from Rhodospirillum rubrum

Coupling factor proteins (Fr , CFr , ATPase), purified to homogeneity from mitochondria, chloroplasts and aerobic bacteria can be dissociated in sodium dodecylsulfate (SDS) into subunits of different molecular weights. From all these sources, five or more subunits of different size have been detected [l-S] . This paper presents data, which demonstrate the existence of five different subunit sizes of polypeptides present in the coupling factor protein puritied to apparent homogeneity from the photosynthetic bacterium Rhodospirillum rubrum [6,7] . This coupling factor shows a Ca2+-dependent ATPase activity, which is stimulated during purification [6]. Evidence for partial dissociation of the enzyme as a result of treatment with LiCl is also presented.

Studies on photosynthetic inorganic pyrophosphate formation in Rhodospirillum rubrum chromatophores

Photosynthetic formation of inorganic pyrophosphate (PP(i)) in Rhodospirillum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PP(i) synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PP(i) synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PP(i) synthesis much less. Even at low rates of electron transport a significant rate of PP(i) synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PPI synthesis is inhibited. It is shown that PP(i) synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions.