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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.

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.

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.

Chromatophores from the photosynthetic bacterium Rhodospirillum rubrum contain a membrane-bound transhydrogenase catalyzing the transfer of a hydride ion between NADH and NADP⁺. The reverse reaction, i.e. reduction of NAD⁺ by NADPH, can furnish sufficient energy (ΔμH⁺) to drive the phosphorylation of inorganic orthophosphate (P(i)) to pyrophosphate (PP(i)). The rate of PP(i), synthesis is 50 nmol PP(i) formed/min per μmol Bchl which is 5% of the rate of light-induced PP(i), synthesis. PP(i), synthesis is inhibited by both the H+-PPase inhibitor fluoride and the specific transhydrogenase inhibitor palmitoyl-CoA. The effects of both DCCD and uncouplers on the system provide additional evidence that the ΔμH⁺ generated by the reverse transhydrogenase reaction drives PP(i), synthesis. The rate of PP(i), synthesis can be partially inhibited by the addition of NADP⁺, a substrate of the forward energy-consuming reaction. The ΔμH⁺ generated can also be used to drive ATP synthesis by the H⁺-ATPase, but at a lower rate than the PP(i), synthesis.

Immunological cross-reactivity among three types of inorganic pyrophosphatases, that is, the proton pumping inorganic pyrophosphate synthase (H(+)-PPi synthase) and the soluble inorganic pyrophosphatase, both from Rhodospirillum rubrum, and the vacuolar membrane inorganic pyrophosphatase (H(+)-PPase) from mung bean (Vigna radiata), were examined by means of immunoblot analyses. Antibodies raised against the mung bean H(+)-PPase cross-reacted with the H(+)-PPi synthase from R. rubrum but not with the soluble PPase from R. rubrum. N,N'-dicyclohexylcarbodiimide (DCCD), which inhibits both synthesis and hydrolysis of PPi catalysed by purified and chromatophore H(+)-PPi synthase, binds to the enzyme as shown by fluorography of [14C]DCCD labelling. These results suggest that the R. rubrum H(+)-PPase share close structural similarities with the vacuolar H(+)-PPase from Mung bean.

The steady-state membrane potentials generated by light, PPi, ATP or the reverse transhydrogenase reaction were studied in chromatophores from two different phototrophic bacteria, Rhodospirillum rubrum and Rhodopseudomonas viridis. The membrane potentials generated by the different energy-linked reactions were evaluated by a tetraphenylboron (TPB−) ion-selective electrode. The Δψ generated by light was estimated to be 110 mV and 50 mV in R. rubrum and Rps. viridis chromatophores, respectively. In the dark, PPi, ATP and reversed transhydrogenase generated membrane potentials in R. rubrum and Rps. viridis chromatophores 50, 60 and 35 mV, and 14, 35 and 25 mV, respectively. The effect of magnesium ion on the membrane potential generated by different energy-linked reactions was also studied. The Δψ induced by different energy-generating reactions in R. rubrum and Rps. viridis chromatophores and the possible relationship to the chromatophore structures are discussed.

The unique family of membrane-bound proton-pumping inorganic pyrophosphatases, involving pyrophosphate as the alternative to ATP, was investigated by characterizing 166 members of the UniProtKB/Swiss-Prot + UniProtKB/TrEMBL databases and available completed genomes, using sequence comparisons and a hidden Markov model based upon a conserved 57-residue region in the loop between transmembrane segments 5 and 6. The hidden Markov model was also used to search the approximately one million sequences recently reported from a large-scale sequencing project of organisms in the Sargasso Sea, resulting in additional 164 partial pyrophosphatase sequences. The strongly conserved 57-residue region was found to contain two nonapeptidyl sequences, mainly consisting of the four 'very early' proteinaceous amino acid residues Gly, Ala, Val and Asp, compatible with an ancient origin of the inorganic pyrophosphatases. The nonapeptide patterns have charged amino acid residues at positions 1, 5 and 9, are apparent binding sites for the substrate and parts of the active site, and were shown to be so specific for these enzymes that they can be used for functional assignments of unannotated genomes.

Comparative studies between the proton-pumping, membrane-bound inorganic pyrophosphatases (H(+)-PPases) from hyperthermophilic and thermophilic prokaryotes and those from mesophilic organisms can now be performed because of very recent sequence data. Typical overall factors that contribute to protein thermostability are found in H(+)-PPases from extremophiles; nevertheless, putative active site motifs of this class of enzymes may be identical over the whole range of average growth temperatures of the compared prokaryotes. Heterologous expression in yeast of H(+)-PPases from organisms spanning a wide range of thermal habitats has allowed the biochemical comparison among these proteins within the same system, ensuring that differences observed are due to intrinsic characteristics of the proteins and not to their interactions with different cellular environments. On the other hand, the availability of H(+)-PPase sequences from a variety of sources have permitted molecular phylogenetic studies of this class of proton pumps, thus providing information about their general structural and functional properties. A great step forward may be expected when one of the several groups now attempting crystallization and 3D structural determination of H(+)-PPases will be successful.

Conserved amino acid sequence motifs inenzymes often indicate involvement in the binding of metal ion(s) and/or in the binding and/or reactions of substrate(s). The four very early proteinaceous amino acids are glycine (G), alanine (A), aspartic acid (D) and valine (V) as was demonstrated with the clarification of the stepwise evolution of the genetic code (Eigen and Schuster, 1979), and in agreement with the quantities obtained in the earlier, classical work (Miller, 1953 and on) showing proteinaceous amino acid production under possible prebiotic conditions. Aspartic acid stands out as the unique very early amino acid containing an additional, highly reactive free charge, suitable i.a. for cation binding. Active site motifs with a very high content of any or all of these four amino acids may well be of early evolutionary significance.

The membrane-bound proton pumping inorganic pyrophosphate synthase/pyrophosphatase (H+-PPi synthase/H+-PPase) from the photosynthetic bacterium Rhodospirillum rubrum was functionally expressed in Escherichia coli C43(DE3) cells. Based on a new topology model of the enzyme, charged residues predicted to be located near or within the membrane were selected for site-directed mutagenesis. Several of these mutations resulted in an almost complete inactivation of the enzyme. Four mutated residues appear to show a selective impairment of proton translocation and are thus likely to be involved in coupling pyrophosphate hydrolysis with electrogenic proton pumping. Two of these mutations, R176K and E584D, caused increased tolerance to salt. In addition, the former mutation caused an increased Km of one order of magnitude for the hydrolysis reaction. These results and their possible implications for the enzyme function are discussed.

In this session on “Life without starlight” it may be useful to describe photosynthetic organisms with their light-induced transformation, conversion and conservation of energy as “life with starlight”. To what extent could photophosphorylation, this fundamental process of energy transfer in photosynthesis, have originated and evolved in discrete molecular steps in the absence of light? An answer may be sought in the first and so far only known alternative biological photophosphorylation system, where the comparatively “primitive” energy donor inorganic pyrophosphate (PPi) is produced (H. Baltscheffsky et al., 1966) instead of the more “complex” adenosine triphosphate (ATP), the well known central energy donor and “energy currency” of all now living cells. The gene for PPi synthase, which is the enzyme involved in the photosynthetic formation of PPi in chromatophores from the photosynthetic bacterium Rhodospirillum rubrum, has been cloned (M. Baltscheffsky et al., 1998) and the primary structure and some other properties of this first bacterial representative of the membrane-bound family of proton-pumping inorganic pyrophosphatases (H+-PPases) has been deduced (M. Baltscheffsky et al., 1999). In the loop between the fifth and sixth putative transmembrane segments, in what appears to be a pyrophosphate binding region and the active site for the phosphorylation reaction, three different “primitive” tetrapeptidyl motifs have been located in two nonapeptidyl sequences. Their high content of the four “very early” proteinaceous amino acids glycine, alanine, valine and aspartic acid (G, A, V and D, respectively) and their very regular arrangement of amino acids with charged side groups, would seem to be of particular evolutionary significance. Our first tentative model of the active site has resulted in a structure, which may be useful for attempts to extrapolate back to the hither toelusive molecular origin and evolution of the metabolism of phosphate compounds, specially those, which are directly involved in bioenergy conversion.

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.

The inorganic pyrophosphate synthase (PPi synthase, PPase) in Rhodospirillum rubrumwas first detected in our laboratory as a firmly membrane-bound pyrophosphatase activity, uncoupler stimulated, in the mid-sixties [1]. A few years later H. Baltscheffsky and von Stedingk together with Heldt and Klingenberg [2,3] found that, in the absence of added nucleotides, photophosphorylation yielded inorganic pyrophosphate as end product. Furthermore, this synthesis of PPi is not inhibited, but rather stimulated by the addition of oligomycin. We also found that addition, in the dark, of PPi in the presence of Mg++ ions elicited energy requiring redox changes of cytochromes [4]. Simultaneously, Keister reported that it was possible to drive the energy requiring transhydrogenase reaction in R. rubrumchromatophores [5] by addition of PPi and Mg++. Johansson established the identity of separate enzyme catalysing the PPi linked reactions in R. rubrumchromatophores by finding that antibodies against the ATPase did not impair the PPase [6]. Keister and Minton also showed that it was possible to obtain ATP synthesis in the dark with PPi as the energy source, in R. rubrumchromatophores [7]. The capability to transport protons was established by Moyle et al. [8], who found that one proton was translocated across the chromatophore membrane per PPi hydrolysed.

In our universe, life may exist on many planets, more or less similar to Tellus, the Earth. As the only life we know is life on Earth, our ideas about life in the universe are naturally based on what we know about this life and its various aspects, such as its origin from an energetic point of view.

A 1.2 kb long DNA segment from Rhodospirillum rubrum has been sequenced (EMBL/GenBank accession number: U41280). This DNA segment includes the first sequenced gene for a putative 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, termed hmgL, from a photosynthetic organism. The sequenced segment also contains a ribosome-binding site and two clusters of possible-35 and -10 promotor sequences preceding the hmgL gene. Translation of the gene would yield a 303 amino-acid-long protein with a calculated molecular weight of 31.1 kDa. This protein shows 55-60% identity and approx. 75% similarity, including conservative substitutions, with the three eukaryotic and the single prokaryotic HMG-CoA lyases which previously have been sequenced. The R. rubrum enzyme showed stronger homology to the chicken HMG-CoA lyase than to the other bacterial protein. Significant sequence similarity was also found with homocitrate synthases from nitrogen-fixing prokaryotes. In contrast to the other sequenced prokaryotic HMG-CoA lyase (from Pseudomonas mevalonii), the R. rubrum hmgL does not seem to appear in an operon together with a HMG-CoA reductase. The hmgL gene was transcribed in photosynthetically grown cells as judged by amplification of cDNAs synthesised from DNA-free total RNA. In addition, HMG-CoA lyase activity was found in R. rubrum cells grown anaerobically in the light with leucine as the carbon source.

Many of the chemical reactions on the early Earth which supposedly led to the origin of life may be assumed to have been made possible by energetic coupling between energy liberating and energy requiring reactions. Among the plausible chemical energy sources for the coupling are energy-rich compounds known to have central metabolic functions in living cells, such as, for example, inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP) [1,2], high-molecular-weight inorganic polyphosphates [3,4] and organic thioesters [5,6].

This minireview in memory of Daniel I. Arnon, pioneer in photosynthesis research, concerns properties of the first and still only known alternative photophosphorylation system, with respect to the primary phosphorylated end product formed. The alternative to adenosine triphosphate (ATP), inorganic pyrophosphate (PPi), was produced in light, in chromatophores from the photosynthetic bacterium Rhodospirillum rubrum, when no adenosine diphosphate (ADP) had been added to the reaction mixture (Baltscheffsky H et al. (1966) Science 153: 1120–1122). This production of PPi and its capability to drive energy requiring reactions depend on the activity of a membrane bound inorganic pyrophosphatase (PPase) (Baltscheffsky M et al. (1966) Brookhaven Symposia in Biology, No. 19, pp 246–253); (Baltscheffsky M (1967) Nature 216: 241–243), which pumps protons (Moyle J et al. (1972) FEBS Lett 23: 233–236). Both enzyme and substrate in the PPase (PPi synthase) are much less complex than in the case of the corresponding adenosine triphosphatase (ATPase, ATP synthase). Whereas an artificially induced proton gradient alone can drive the synthesis of PPi, both a proton gradient and a membrane potential are required for obtaining ATP. The photobacterial, integrally membrane bound PPi synthase shows immunological cross reaction with membrane bound PPases from plant vacuoles (Nore BF et al. (1991) Biochem Biophys Res Commun 181: 962–967). With antibodies against the purified PPi synthase clones of its gene have been obtained and are currently being sequenced. Further structural information about the PPi synthase may serve to elucidate also fundamental mechanisms of electron transport coupled phosphorylation. The existence of the PPi synthase is in line with the assumption that PPi may have preceded ATP as energy carrier between energy yielding and energy requiring reactions.

Publisher Summary This chapter discusses the inorganic pyrophosphate and inorganic pyrophosphatases. A number of remarkable developments have expanded the field of PP i bioenergetics in several new and also old directions. Progress has been in areas primarily concerned with other aspects of PP i metabolism, but has been of apparent bioenergetics significance. The sequence homologies between cytoplasmic PPases from archaebacteria, eubacteria and eukaryotes, and the yeast mitochondrial PPase, which appears to be energy linked, indicate conservation of early basic PPase characteristics. On the other hand, the vacuolar PPase was found to be very different. The immunological cross-reactivity between photobacterial and plant vacuolar H + -PPases would appear to allow the guess that they belong to a new, distinct family of energy linked PPases.

It has been shown for the first time that several photosystem-II thylakoid proteins and the main chlorophyll-a/b light-harvesting complex can be phosphorylated with inorganic pyrophosphate as phosphate donor. With pyrophosphate, as with ATP, the protein-kinase reaction is dependent on light or a strong reducing agent. The reaction which can be demonstrated in well-washed spinach thylakoids is dependent on electron transport and is controlled by the redox state of the plastoquinone pool. It is suggested that the pyrophosphate-dependent thylakoid protein phosphorylation is mediated by the same kinase which is responsible for the ATP-dependent protein phosphorylation. This pyrophosphate-dependent kinase activity may be derived from an evolutionary precursor from which ATP-dependent protein phosphorylation also developed.

Membrane bound inorganic pyrophosphatases (PPases) appear to play a role in energy metabolism (1,2). PPases are present in bacterial, plant and animal cells and cell organelles. Membrane bound PPases have been found in chromatophores (2), mitochondria (3) and chloroplasts (4). The membrane bound PPase from Rhodospirillum rubrum chromatophores can synthesize PPi (inorganic pyrophosphate) at the expense of light energy (1) and has been described in some detail (2). Mitochondrial membrane bound PPase has been reported to produce PPi as a result of oxidative phosphorylation (3). (Genes encoding soluble PPases of E. coli (5) and yeast (6) have been cloned and characterized). We have studied some similarities and differences between chromatophore, chloroplast and mitochondrial membrane bound PPases.

LHCII and several PSII thylakoid proteins are phosphorylated by an ATP dependent protein kinase (1,2), which is under control of the redox state of the PQ pool (3). This protein phosphorylation leads to rearrangements in the lateral organization of the thylakoid membrane (4). As a consequence, it is postulated that LHCII protein phosphorylation regulates excitation energy distribution between PSI and PSII (5); protects PSII against photoinhibition as well as regulates protein turnover (for a review, see ref. 6).

In Rhodospirillum rubrum chromatophores, diethylstilbestrol inhibits the photoinduced synthesis of ATP and PPi by the membrane-bound H+-ATPase and H+-PPase, respectively. 50% inhibition of ATP synthesis is obtained at 8 μM diethylstilbestrol in the presence of 0.13 μM BChl, while I50 for the PPi formation is 20 μM diethylstibestrol at the same chromatophore concentration. Diethylstilbestrol also inhibits the hydrolytic activity of the H+-PPase, both in the membrane-bound and in the solubilized and purified state. Inhibition to 50% is already attained at 3 μM diethylstilbestrol in chromatophores when 1 μM FCCP is present and the BChl-concentration is 0.62 μM. The hydrolysis by the solubilized enzyme has an I50 of 5 μM when 5 μg protein/ml is used. In contrast to the PPi-hydrolysis, the ATPase activity of the chromatophores shows a small activation at low diethylstilbestrol concentration and becomes inhibited at higher concentrations. Also, solubilized F0F1-ATPase is activated to a small extent by diethylstilbestrol at the concentrations tested. At low concentrations of BChl, the inhibitory action of diethylstilbestrol on ATP and PPi synthesis can be reversed by addition of bovine serum albumin. The time dependence and inhibition dependence on the energy state of the membrane and on the BChl concentration are examined for the ATP synthesis. The mechanism of inhibition by diethylstilbestrol is discussed.

Two alternative procedures for isolation of the oligomycin-sensitive ATPase complex (E. C. 3.6.1.3) from R. rubrum chromatophores are compared with respect to ease, rapidity, and yield. The inhibitory effect of oligomycin on the membrane-bound Ca2+ -ATPase activity is increased during storage of the chromatophores, whereas the effect of oligomycin on the membrane-bound Mg2+ -ATPase activity does not change within a week. Oligomycin-sensitivity of the solubilized ATPase complex depends on the isolation procedure. The enzyme complex consists of at least nine different polypeptides with the apparent molecular weights of (1) 67,000, (2) 60,000, (3) 56,000, (4) 35,000, (5) 22,000, (6) 20,000, (7) 17,000, (8) 12,000, (9) 11,000. The polypeptides 2 - 4, 7, and 8 represent subunits of coupling factor 1.

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.

The proton translocating membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum S1, has been solubilized with good yield from chromatophores using Triton X-100 (9–10 oxyethylene groups) in the presence of high concentrations of MgCl2 and ethyleneglycol. The enzyme has been purified 80-fold by hydroxylapatite column chromatography, to a state of near homogeneity, according to polyacrylamide-gelelectrophoresis. The enzyme appears to be a very hydrophobic integrally bound membrane protein. Phospholipids or Triton X-100 reconstitutes the enzyme activity after solubilization and purification. The purified enzyme preparation has a specific activity of 24 units. Both the purified and the chromatophore-bound enzyme are inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxo-1,3-diazol (NBF-Cl), sodium fluoride, imidodiphosphate, methylenediphosphonate and the antibiotic Dio-9 (energy-transfer inhibitor). In the solubilized state the purified enzyme is not stimulated by uncouplers or inhibited by dicyclohexylcarbodiimide in contrast to the chromatophore-bound pyrophosphatase. When reconstituted into liposomes the purified enzyme regains the stimulation by uncouplers.

The oxidation-reduction potential dependence of pyrophosphate-induced reactions in chromatophores from the blue-green mutant of Rhodospirillum rubrum has been studied. Two types of energy-requiring reactions are apparent: (1) Absorption changes resulting from bacteriochlorophyll interaction with a high energy state within the membrane. This reaction is independent of the oxidation-reduction potential poise between −475 mV and +430 mV; the reaction is attenuated from +430 mV to 530 mV as the bacteriochlorophyll is chemically bleached. (2) Oxidation-reduction reactions of cytochrome b ( λ max 562–563 nm; E m7.0 − 105 mV) and a cytochrome c ( λ max 552–553 nm; E m7.0 + 295 mV). Antimycin A abolishes the pyrophosphate-induced cytochrome oxidation-reductions but not the bacteriochlorophyll reactions, whereas uncoupler abolishes both cytochrome and bacteriochlorophyll reactions.

Menadione exerts a slight stimulation upon cyclic photosphorylation in isolated chromatophores of Rhodopirillum rubrum. This stimulation is markedly increased, both n a percent and an absolute basis, if the chromatophore preparation has been pre-aged. In contrast to phenazine methosulfate, menadione cannot by-pass the inhibitory effect caused by antimycin A upon photophosphorylation. Thus the sites of action of these two agents are not identical. The effects of added menadione are in agreement with the following hypothesis: the site of action of menadione is between a flavoprotein and a b-type cytochrome, on a rate-limiting and comparatively easily destroyed electron transport step in cyclic photophosphorylation of isolated chromatophores from Rhodospirillum rubrum.

1 A coupling factor has been purified from an acetone powder extract of Rhodospirillum rubrum chromatophores. This restores to decoupled chromatophores (extracted with respect to coupling factor) both photophosphorylation and Mg2+-dependent ATPase activities. The coupling factor had been partially purified by gel filtration through a Sepharose 6B column, and further purified by ultra-filtration and sucrose-density-gradient centrifugation. At each stage of purification, the coupling factor activity was coincident with a Ca2+-dependent ATPase activity, increasing in total activity during the purification. Mg2+, Mn2+, Co2+ or Zn2+ could not substitute for Ca2+ in activation of this soluble ATPase, but these four metals strongly inhibited the activity of the Ca2+-dependent ATPase. 2 By incubation with decoupled chromatophores, the soluble Ca2+-dependent ATPase was rebound to the membrane in the presence of either Mg2+ or Ca2+. This binding resulted in a partial masking of the Ca2+-dependent ATPase activity of the soluble enzyme. 3 The purified ATPase has a Km value of 1.2 mM for ATP. The Ki values for inhibition of the soluble Ca2+-dependent ATPase by Mg2+ (170 μM), and by ADP (42 μM) are in close agreement with the corresponding Km values for Mg2+-dependent ATPase and photophosphorylation, respectively, as measured with chromatophores. 4 The membrane-bound Mg2+- or Ca2+-dependent ATPase activities were inhibited by oligomycin. Azide and parahydroxymercuribenzoate inhibited both the soluble and the membrane-bound ATPase activities. Treatment with phospholipase A inhibited both the membrane-bound ATPase, but had no effect on the soluble Ca2+-dependent ATPase. 5 The sedimentation constant at 1 mg protein/ml of the extensively purified coupling factor is s20, w= 13.1 × 10−13 s, and the approximate molecular weight 350000.

The behaviour of the membrane-bound proton-translocating pyrophosphatase (H+-PPase) in Rhodospirillum rubrum chromatophores upon application of an electrochemical potential is studied. The rate constants are shown to be affected in an asymmetric fashion. The forward rate constant (PPi synthesis) is shown to be at least 45-times larger during illumination than when there is no proton-motive force. The hydrolysis rate is increased maximally 8-times when the potential is dissipated. The effect of the electrical field gradient is thus mainly to increase the forward rate of the reaction. The H+-PPase also seems to be a functionally simpler enzyme than the H+-ATPase, lacking the hydrolysis activation step during energization found in the latter.

The membrane-bound proton-pumping inorganic pyrophosphatase from Rhodospirillum rubrum was heterologously expressed in Escherichia coli C43(DE3) cells and was inhibited by 4-bromophenacyl bromide (BPB), N , N ′-dicyclohexylcarbodiimid (DCCD), diethyl pyrocarbonate (DEPC) and fluorescein 5′-isothiocyanate (FITC). In each case, the enzyme activity was rather well protected against inhibitory action by the substrate Mg 2 PPi. Site-directed mutagenesis was employed in attempts to identify target residues for these inhibitors. D217 and K469 appear to be the prime targets for DCCD and FITC, respectively, and may thus be involved in substrate binding. No major effect on enzyme activities was seen when any one of the four histidine residues present in the enzyme were substituted. Nevertheless, a mutant with all of the four charged histidine residues replaced retained only less than 10% of the hydrolysis and proton-pumping activities. Substitution of D217 with A or H yielded an enzyme with at least an order of magnitude lower hydrolysis activity. In contrast with the wild-type, these variants showed higher hydrolysis rates at lower concentrations of Mg 2+ , possibly reflecting a change in substrate preference from Mg 2 PPi to MgPPi. BPB is a H + -pyrophosphatase inhibitor that apparently has not been used previously as an inhibitor of these enzymes.

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

It is possible to obtain synthesis of PP i by artifical ion potentials in Rhodospirillum rubrum chromatophores. PP i can be formed by K + -diffusion gradients (Δψ), H + gradients (ΔpH) or a combination of both. In contrast, ATP can only be synthesized by imposed Δψ or Δψ+ΔpH. For ATP formation there is also a threshold value of K + concentration below which synthesis of ATP is not possible. Such a threshold is not found for PP i formation. Both PP i and ATP syntheses are abolished by addition of FCCP or nigericin and only marginally affected by electron transport inhibitors. The synthesis of PP i can be monitored for several minutes before it ceases, while ATP production stops within 30 s. As a result the maximal yield of PP i is 200 nmol PP i /μmol BChl, while that of ATP is no more than 25 nmol ATP/μmol BChl. The initial rates of syntheses were 0.50 μmol PP i /μmol BChl per min and 2.0 μmol ATP/μmol per min, respectively. These rates are approx. 50 and 20% of the respective photophosphorylation rates under saturating illumination.

PP 1 driven ATP synthesis has been reconstituted in a liposomal system containing the membrane-bound energy-linked PP 1 ase and coupling factor complex, both highly purified from Rhodospirillum rubrum . This energy converting model system was made by mixing both enzyme preparations with an aqueous suspension of sonicated soybean phospholipids and subjecting to a freeze—thaw procedure. In the presence of ADP, Mg 2+ , P 1 and PP 1 the system catalyzed phosphorylation by up to 25 nmol ATP formed X mg protein −1 X min −1 , at 20°C, which was sensitive to uncouplers and inhibitors of phosphorylation such as oligomycin, efrapeptin and N,N ′-dicyclohexylcarbodiimide.

The effect of the electrical potential on the H+-ATPase of Rhodospirillum rubrum is examined. It is shown that the forward reaction rate (ATP synthesis) is increased by a factor of 10 during illumination while the reversed rate is only slightly decreased. This indicates that the electrical potential across the membrane affects the rate constants mainly by increasing the forward rate constants rather than decreasing the reversed rate constants in order to go from net hydrolysis to net synthesis.