PhotosynthesisResearch 46:87-91, 1995. (~) 1995KluwerAcademicPublishers. Printedin the Netherlands. Minireview
Alternative photophosphorylation, inorganic pyrophosphate synthase and inorganic pyrophosphate Margareta Baltscheffsky & Herrick Baltscheffsky Department of Biochemistry, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden Received 16 March 1995;acceptedin revisedform5 May 1995 Key words: bioenergetics, photosynthesis, chromatophores, energy coupling, evolution
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 e t 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. Introduction
This minireview is written in memory of Daniel (Dan) I. Arnon, pioneer in several areas of photosynthesis research. His discovery, with Bob Whatley and Mary Belle Allen, of photophosphorylation (or photosynthetic phosphorylation, as they first called it) is a landmark in 20th century science. Their first papers on photophosphorylation in isolated spinach chloroplasts were published in 1954 (Arnon et al. 1954a, b), which was also the year of the subsequent finding by Albert Frenkel of photophosphorylation in chromatophores isolated from the pur-
pie, non-sulfur photosynthetic bacterium Rhodospirillum rubrum (Frenkel 1954). It has long been known that in photophosphorylation, the absorption of light by chlorophyll initiates a chain of oxidation-reduction reactions which, similarly as in mitochondrial oxidative phosphorylation, can drive ion movements over the subcellular membranes on which these energy conversion systems are located, giving ATP as the primary end product of the phosphorylation reactions. However, as was found with isolated chromatophores from R. rubrum, (H Baltscheffsky et al. 1966), there exists an alternative end product of photophosphorylation, inorganic pyrophosphate (PPi). This light induced formation of PPi was abolished by
88 uncouplers whereas oligomycin, the inhibitor of ATP formation, stimulated PPi formation (H Baltscheffsky and von Stedingk 1966a), indicating a light induced and electron transport coupled production of PPi with a phosphorylation system different from that involved in ATP formation. The first direct demonstration of light induced proton movement at the prokaryote level, with this chromatophore system (H Baltscheffsky and von Stedingk 1966b), established the existence of a thus far unique biological system with three distinct levels of electron transport coupled energy transfer: Conditions
Energy transfer reaction
1. No Pi or ADP added
2. Pi but no ADP added
Formation of PPi
3. Both Pi and ADP added
Formation of ATP (and PPi)
Several review articles have treated various aspects of the progress with the photophosphorylation system involving a PPase (PPi synthase) for the production and utilization of PPi (more correctly MgPPi, as magnesium ion is obligatory in the reactions catalyzed by PPase), the alternative carrier of biologically useful chemical energy (M Baltscheffsky and Nyrtn 1984; M Baltscheffsky and H Baltscheffsky 1992), and possible evolutionary implications have also been discussed in some detail (H Baltscheffsky 1971, 1993; H Baltscheffsky and M Baltscheffsky 1994). Thus, with reference to these earlier papers, only certain selected aspects of the system will be covered in this minireview.
The synthesis of PPi The suggestion that the pathway to PPi was catalysed by the PPase bound to chromatophore membranes (Baltscheffsky M e t al. 1966; Baltscheffsky M 1967) was soon substantiated by the demonstration that LiC1 treated R. rubrum chromatophores, while losing all ATP linked reactions, retained the full capacity to synthesise PPi (Fisher and Guillory 1969). They studied the light-induced PPi synthesis using a trapping system. (Guillory and Fisher 1972) and found the synthesis of PPi to be maximally 25% of the ATP synthesis. They also found that the light saturation for PPi synthesis was considerably lower than for ATP synthesis and pointed out that at low light intensities, conditions under which the organism may normally be found in
nature, the rate of PPi synthesis is almost the same as the rate of ATP synthesis. This finding was confirmed and extended (Nyrtn et al. 1986) in experiments at very low light intensities (0.3--0.4 W/m 2) showing that the rate of PPi synthesis was twice that of ATP synthesis. With a sensitive measuring system allowing continous monitoring it was also found that the effect of both electron transport inhibitors and uncouplers is less on the PPi synthesis than on ATP synthesis. This has been interpreted as a result of the lower energy requirement for PPi synthesis and is in line with the results from experiments with artificial proton gradients and diffusion potentials (Strid et al. 1987a, b). With an acid-base transition as the only energy source for PPi and ATP synthesis profound differences were obvious. Whereas no ATP synthesis occurred unless the acid-base transition was complemented with a diffusion potential of K + in the presence of valinomycin, as had already been shown (Leiser and GrometElhanan 1974), the PPi synthesis could be supported by a proton gradient alone (Strid et al. 1987a,b). If a diffusion potential was the sole driving force for ATP synthesis, there appeared a threshold below which no ATP was formed. No such threshold was found for PPi synthesis. The initial rate for PPi synthesis was only 25% of that for ATP synthesis, but the total yield of PPi was ten times that of ATP. A lower rate for PPi synthesis than for ATP synthesis is also seen under continuous, saturating illumination (Nyrtn et al. 1986). Under these conditions, by applying specific inhibitors of each of the two pathways, it was possible to measure either one separately or, in the absence of inhibitors to demonstrate that both ATP and PPi synthesis occur simultaneously, and thus compete for the available proton motive force. As the earlier results discussed above indicated, the synthesis of PPi at the expense of light energy in bacterial chromatophores occurs with the integrally membrane bound PPi synthase, which resides in the same membranes as the earlier known ATP synthase, and the more recent observations corroborate the assumption that these energy conversion systems are in simultaneous operation. The fact that only a proton gradient but no membrane potential was necessary for PPi synthesis in contrast to that of ATP is another valid reason for the expression 'PPi serves as a poor man's ATP', which would seem originally to have emerged from knowledge that the amount of energy liberated from PPi is only about 50-70% of that obtainable from ATP. Recalling the observations that photosynthetic bacteria
89 are often living in muddy waters, the characteristics of PPi and of its light induced synthesis under conditions of limited energy supply, may be taken as an indication about a potential physiological use of the PPi synthase system.
The PPi synthase (PPase) Due to its extremely hydrophobic properties, as an integrally membrane bound protein, the solubilization and purification of the PPi synthase with retained activity required much time and effort (Shakhov et al. 1982; Nyr6n et al. 1984), until an easily reproducible method for a high degree of purification was obtained (Nyr6n et al. 1991). Further purification by electroelution gave a satisfactory product for antibody production (Baltscheffsky M and Sakai-Nore, unpublished). In contrast to the ATPase, which in R. rubrum has nine different subunits, the PPi synthase consists of only one polypeptide, carrying both the proton channel and the catalytic site. The PPi synthase shows immunological cross reaction with a plant vacuolar proton translocating PPase (Nore et al. 1991) isolated from mung bean (Vigna radiata). The gene for the vacuolar PPase from Arabidopsis thaliana was the first to be cloned and sequenced among membrane bound PPase genes (Sarafian et al. 1992), and antibodies against selected parts of that sequence also cross react with the isolated R. rubrum enzyme. Attempts to establish the degree of structural similarity between PPi synthases and vacuolar PPases are in progress, but will have to await the completed sequence of the R. rubrum enzyme.
PPi and early evolution Fritz Lipmann, the discoverer of the central role of ATP in cellular energetics, pointed out that in connection with the origin and early evolution of life, the 'generation of the phosphate group potential might have originated with inorganic pyrophosphate as the primitive group carrier' (Lipmann 1965), and that PPi had been found to substitute for guanosine triphosphate (GTP) as donor of energy-rich phosphate in the formation of phosphoenolpyruvate and CO2 from oxaloacetate (Siu and Wood 1962). The discovery of PPi formation in photophosphorylation and the ensuing development made it seem natural to speculate further about the pos-
sible role of this inorganic energy carrier in the origin and early evolution of life on Earth (Baltscheffsky H 1967, 1971, 1993). In a prenucleotide world preceding the suggested, early RNA world (Gilbert 1986), PPi (Baitscheffsky H 1971) and also high-molecular-weight inorganic polyphosphates (Kulaev 1979) have emerged as plausible early energy and phosphate donors. Both the biochemical results discussed above and other findings, such as the occurrence of PPi as a mineral (Rose et al. 1988) and the formation of PPi during cooling of hot volcanic magma (Yamagata et al. 1991), underline the possibility that PPi may have preceded ATP as the primary energy carrier between energy yielding and energy consuming reactions. Some recent results appear particularly pertinent with respect to the question of whether a still traceable pathway of stepwise evolution may exist (H Baltscheffsky 1993) between the enzymes catalyzing the reactions of PPi (or, to broaden the question: inorganic polyphosphates!) on the one hand and those of nucleotides on the other. A novel exopolyphosphatase ofEscherichia coli (Akiyama et al. 1993) was recently found to be homologous with guanosine pentaphosphate phosphohydrolase (Reizer et al. 1993) and about simultaneously a second exopolyphosphatase from a mutant E. coli strain lacking the principal one was shown to be identical with the guanosine pentaphosphate hydrolyzing enzyme (Keasling et ai. 1993). They all belong to the sugar kinase/actin/hsp70 superfamily (Reizer et al. 1993). Thus, the original question of whether an early evolutionary pathway from PPi metabolism to ATP metabolism, possibly over enzymes which can metabolize both PPi and ATP, can still be traced (Baltscheffsky H 1993), has been illuminated already in an affirmative direction for another, structurally closely related energy transferring member of the inorganic phosphate family, PPi being substituted by high-molecular weight, straight-chain inorganic polyphosphates. The uncomplicated structure of PPi, as compared with that of ATP, restricts the inorganic molecule with respect to its functions to energy and phosphate transfer, whereas ATP with its much more complex structure has the capability also to pyrophosphorylate and adenylate. A very plausible reason for a suggested early takeover from PPi by ATP is its adenylation function, of major importance for cellular biosynthetic systems. The questions, however, still remain, if there has existed an early 'PPi world' before the present 'ATP world' (H Baltscheffsky 1993), and if so, if traces of this
90 a s s u m e d stepwise t a k e o v e r still can be traced in protein structures o f presently living organisms.
Future directions W h e n the c l o n i n g and sequencing o f the PPi synthase gene, in a h o p e f u l l y not so distant future, has given the c o m p l e t e a m i n o acid s e q u e n c e o f the protein, s o m e i m m e d i a t e future goals are: 1. O v e r - e x p r e s s i o n o f the PPi synthase 2. Site-specific mutation studies in order to determine details about the reaction m e c h a n i s m 3. C o m p a r i s o n o f the a m i n o acid sequence with those o f v a c u o l a r m e m b r a n e - b o u n d PPases and various FoFI ATPases and other ATPases. It m a y be h o p e d that results f r o m such studies will be helpful in elucidating not only the m e c h a n i s m s i n v o l v e d in PPi synthesis but also so far hidden aspects o f the c o r r e s p o n d i n g m e c h a n i s m s for the synthesis o f A T E In addition, it m a y well be possible to find properties o f the PPi synthase structure, which m a y be o f significance to a l l o w the e n z y m e to function, in contrust to the v a c u o l a r m e m b r a n e b o u n d PPases, also in the direction o f PPi synthesis. In both these directions the photobacterial PPi synthase could thus serve as a m o d e l system for p r o v i d i n g n e w information about energy c o u p l i n g mechanisms.
Acknowledgements T h e authors gratefully a c k n o w l e d g e grants r e c e i v e d f r o m Carl T r y g g e r s Stiftelse f r r Vetenskaplig Forskning and (to M B ) f r o m the S w e d i s h Natural Science R e s e a r c h Council.
References Akiyama M, Crooke E and Kornberg A (1993) An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon. J Biol Chem 268:633-639 Arnon DI, Allen MB and Whatley FR (1954a) Photosynthesis by isolated chloroplasts. Nature 174:394-396 Amon DI, Whatley FR and Allen MB (1954b) Photosynthesis by isolated chloroplasts. II. Photosynthetic phosphorylation, the conversion of light into phosphate bond energy. J Am Chem Soc 76: 6324--6329 Baltscheffsky H (1967) Inorganic pyrophosphate and the evolution of biological energy transformation. Acta Chem Scand 21: 19731974
Baltscheffsky H (1971) Inorganic pyrophosphate and the origin and evolution of biological energy transformation. In: Buvet R and Ponnamperuma C (eds) Molecular Evolution I. Chemical Evolution and the Origin of Life, pp 466-474. North-Holland Publ, Amsterdam Baltscbeffsky H (1993) Chemical origin and early evolution of biological energy conversion. In: Ponnamperuma C and ChelaFlores J (eds) Chemical Evolution: Origin of Life, pp 13-23. A Deepak Publ, Hampton Baltscheffsky H and Baltscheffsky M (1994) Molecular origin and evolution of early biological energy conversion. In: Bengtson S (ed) Early Life on Earth, Nobel Symposium No 84, pp 81-90. Columbia UE New York Baltscheffsky H and von Stedingk L-V (1966a) Bacterial photophosphorylation in the absence of added nucleotide. A second intermediate stage of energy transfer in light-induced formation of ATE Biochem Biophys Res Commun 22:722-728 Baltscheffsky H and von Stedingk L-V (1966b) Energy transfer from two coupling sites in bacterial photophosphorylation. In: Thomas JB and Goedheer JC (eds) Currents in Photosynthesis, pp 253261. Donker Publ, Rotterdam Baltscheffsky H, von Stedingk L-V, Heldt HW and Klingenberg M (1966) Inorganic pyrophosphate: Formation in bacterial photophosphorylation. Science 153: 1120-1122 Baltscbeffsky M (1967) Inorganic pyrophosphate and ATP as energy donors in chromatophores from Rhodospirillum rubrum. Nature 216:241-243 Baltscheffsky M and Baltscheffsky H (1992) Inorganic pyrophosphate and inorganic pyrophosphatases. In: Ernster L (ed) Molecular Mechanisms in Bioenergetics, pp 331-348. Elsevier, Amsterdam Baltscheffsky M and Nyrrn P (1984) The synthesis and utilization of inorganic pyrophosphate. In: Emster L (ed) Bioenergetics. pp 187-206. Elsevier, Amsterdam Baltscheffsky M, Baltscheffsky H and yon Stedingk L-V (1966) Light-induced energy conversion and the inorganic pyrophosphatase reaction in chromatophores from Rhodospirillum rubrum. In: Brookhaven Symposia in Biology, No 19, pp 246253 Fisher RR and Guillory RJ (1969) Partial resolution of energy-linked reactions in Rhodospirillum rubrum chromatophores. FEBS Lett 3:27-30 Frenkel AW (1954) Light induced phosphorylation by cell-free preparations of photosynthetic bacteria. J Am Chem Soc 76: 5568-5569 Guillory RJ and Fisher RR (1972) Studies on the light-dependent synthesis of inorganic pyrophosphate by Rhodospirillum rubrum chromatophores. Biochem J 129:471-481 Keasling JD, Bertsch L and Komberg A (1993) Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase. Proc Natl Acad Sci USA 90:7029-7033 Kulaev IS (1979) The Biochemistry of Inorganic Polyphosphates. Wiley, New York Leiser M and Gromet-Elhanan Z (1974) Demonstration of acidbase phosphorylation in chromatophores in the presence of a K+ diffusion potential. FEBS Lett 43:267-270 Lipmann F (1965) Projecting backward from the present state of evolution of biosynthesis. In: Fox SW (ed) The Origins of Prebiological Systems and of Their Molecular Matrices, pp 259-280. Academic Press, New York Moyle J, Mitchell R and Mitchell P (1972) Proton-translocating pyrophosphatase ofRhodospirillum rubrum. FEBS Lett 23: 233236
91 Nore BE Sakai-Nore Y, Maeshima M, Baltscheffsky M and Nyr6n P (1991) Immunological cross-reactivity between protonpumping inorganic pyrophosphatase of widely phylogenic separated species. Biochem Biophys Res Commun 181: 962-967 Nyr6n P, Hajnal K and Baltscheffsky M (1984) Purification of the membrane-bound proton-translocating inorganic pyrophosphatase from Rhodospirillum rubrum. Biochim Biophys Acta 766:630-635 Nyr6n P, Nore BF and Baltscheffsky M (1986) Studies on photosynthetic inorganic pyrophosphate formation in Rhodospirillum rubrura chromatophores. Biochim Biophys Acta 851:276-282 Nyr~n P, Nore BF and Strid .~ (1991) Proton-pumping N,N~-dicyclohexylcarbodiimide-sensitive inorganic pyrophosphate synthase from Rhodospirillum rubrum: Purification, characterization, and reconstitution. Biochemistry 30:2883-2887 Reizer J, Reizer A, Saler Jr MH, Bork P and Sander C (1993) Exopolyphosphate phosphatase and guanosine pentaphosphate phosphatase belong to the sugar kinase/actin/hsp70 superfamily. TIBS 18:247-248 Rose RC, Peacor DR and Freed RL (1988) Pyrophosphate groups in the structure of canaphite, CaNa2P20(4H20: the first occurence of a condensed phosphate as a mineral. Am Mineral 73:168-171
Saxafian V, Kim Y, Poole RJ and Rea PA (1992) Molecular cloning and sequence of cDNA encoding the pyrophosphate energized vacuolar membrane proton pump (H + PPase) of Arabidopsis thaliana. Proc Natl Acad Sci USA 89:1775-1779 Shakhov YA, Nyr6n P and Baltscheffsky M (1982) Reconstitution of highly purified proton-translocating pyrophosphatase from Rhodospirillum rubrum. FEBS Lett 146:177-180 Siu PML and Wood HG (1962) Phosphoenolpyruvic carboxytransphosphorylase, a CO2 fixation enzyme from propionic acid bacteria. J Biol Chem 237:3044-3051 Strid ~, I-M Karlsson and Baltscheffsky M (1987a) ApH- and A 0-d-induced ATP and PPi synthesis in Rhodospirillum rubrum chromatophores. Acta Chem Scand B41: 116-118 Strid ,~, I-M Karlsson and Baltscheffsky M (1987b) Demonstration of ApH- and A V-induced synthesis of inorganic pyrophosphate in chromatophores from RhodospiriUum rubrum. FEBS Lett 224: 348-352 Yamagata Y, Watanabe H, Saitoh M and Namba T (1991 ) Volcanic production of polyphosphates and its relevance to prebiotic evolution. Nature 352:516-519