One-electron photooxidation of porphyrins at low temperature

The π-cation radicals of the metalloporphyrins magnesium octaethylporphyrin (MgOEP), magnesium tetraphenylporphyrin (MgTPP), and zinc tetraphenylporphyrin (ZnTPP), as well as the free base porphyrins of tetratolylporphyrin (H₂TTP) and tetraphenylporphyrin (H₂TPP) have been formed at liquid nitrogen temperatures in a rigid matrix of alkyl chloride glasses containing CCl₄ or 1,1,2,2-tetrachloroethane (TCE), following photolysis of the porphyrins with visible light. The reaction proceeds via electron transfer from the photoexcited porphyrin to the solvent molecules; the efficiency of thie electron transfer may be qualitatively evaluated in terms of electron tunneling in the solid matrices. This is the first report of the photochemical formation of a free base porphyrin π-cation radical species.     

Induction of porphyrin synthesis in etiolated bean leaves by chelators of iron

Primary leaves of 7- to 9-day-old etiolated seedlings of Phaseolus vulgaris L. var. Red Kidney infiltrated in darkness with aqueous solutions of alpha, alpha'-dipyridyl, o-phenanthroline, pyridine-2-aldoxime, pyridine-2-aldehyde, 8-hydroxyquinoline, or picolinic acid synthesize large amounts of magnesium protoporphyrin monomethyl ester and lesser amounts of magnesium protoporphyrin, protoporphyrin, and protochlorophyllide. Pigment formation proceeds in a linear manner for up to 21 hours after vacuum infiltration with 10 mm alpha, alpha'-dipyridyl. Etiolated tissues of Zea mays L., Cucumis sativus L., and Pisum sativum L. respond in the same way to dipyridyl treatment. Compounds active in eliciting this response are aromatic heterocyclic nitrogenous bases which also act as bidentate chelators and form extremely stable complexes with iron; other metal ion chelators, such as ethylenediaminetetraacetic acid, salicylaldoxime, and sodium diethyldithiocarbamate, do not elicit any pigment synthesis. The ferrous, ferric, cobaltous, and zinc chelates of alpha, alpha'-dipyridyl are similarly ineffective. If levulinic acid is supplied to etiolated bean leaves together with alpha, alpha'-dipyridyl, porphyrin production is inhibited and delta-aminolevulinic acid accumulates in the tissue. Synthesis of porphyrins proceeds in the presence of 450 micrograms per milliliter chloramphenicol or 50 micrograms per milliliter cycloheximide with only partial diminution. We propose that heme or an iron-protein complex blocks the action of the enzyme(s) governing the synthesis of delta-aminolevulinic acid in etiolated leaves in the dark and that iron chelators antagonize this inhibition, leading to the biosynthesis of delta-aminolevulinic acid and porphyrins.     

Mesosomal structures and antimicrobial activity induced by hemin oxidation or porphyrin photodynamic sensitization inStaphylococci

The porphyrin-dependent inactivation ofStaphyloccocus aureus and the induction of mesosomal structures are described. The antimicrobial activity of different photoactivated porphyrins was compared with the dark cytotoxic effect of hemin. Deuteroporphyrin, protoporphyrin, hematoporphyrin, and hematoporphyrin derivative (Hpd) markedly reduced cell growth upon irradiation with light. Photofrin II, the polymerized fraction of Hpd, and other synthetic porphyrins had no effect on staphylococcal growth. Hemin immediately inhibited cell viability in the dark and induced the development of an irregular cell wall, analyzed by scanning electron microscopy (SEM). Inside the cytoplasm a multivesicular mesosome was formed near the septum, as detected by transmission electron microscopy (TEM). The mesosomal volume and its frequency in the cells was increased in a time-dependent manner. The mesosome appearance was not related to fixation by glutaraldehyde or to post-fixation by osmium tetroxide. Glycosyl moieties stained by ruthenium red revealed the formation of membrane-like structures in the mesosome. It is concluded that oxygen-dependent reactions potentiated by porphyrins may induce disturbances in the synthesis of staphylococcal membrane and cell wall, revealed as mesosomes.     

Characterization of cardiac sarcoplasmic reticulum ATP-ADP phosphate exchange and phosphorylation of the calcium transport adenosine triphosphatase.

1. The terminal phosphate of (gamma-32P)ATP is rapidly incorporated into cardiac sarcoplasmic reticulum membranes (0.7--1.3 mumol/g protein) in the presence of calcium and magnesium. Cardiac sarcoplasmic reticulum membranes catalize an ATP-ADP phosphate exchange in the presence of calcium and magnesium. 2. Half-maximum activation of the phosphoprotein formation and ATP-ADP phosphate exchange is reached at an ionized calcium concentration of about 0.3 muM. The Hill coefficients are 1.3. 3. Transphosphorylation and ATP-ADP phosphate exchange require magnesium and are maximally activated at magnesium concentrations close to or equal to the ATP concentration. 4. The phosphoprotein level is reduced to about 45% at an ADP/ATP ratio of 0.1. The rate of calcium-dependent ATP splitting declines, whilst the rate of the calcium-dependent ATP-ADP phosphate exchange increases when the ADP/ATP ratio is varied from 0.1 to 1. The sum of both, the rate of ATP splitting and the rate of ADP-ATP phosphate exchange remains constant. 5. Phosphoprotein formation and ATP-ADP phosphate exchange are not affected by azide, dinitrophenol, dicyclohexyl carbodiimide and oubain, whilst both activities are reduced by blockade of -SH groups localized on the outside of the sarcoplasmic reticulum membrane. 6. The isolated phosphoprotein is acid stable. The trichloroacetic acid denatured 32P-labelled membrane complex is dephosphorylated by hydroxylamine, which might indicate that the phosphorylated protein is an acyl-phosphate. 7. Polyacrylamide gel elctrophoresis (performed with phenol/acetic acid/water) of phosphorylated sarcoplasmic reticulum fractions demonstrates that the 32P-incorporation occurs into a protein of about 100000 molecular weight. 8. It is suggested that the phosphoprotein represents a phosphorylated intermediate of the calcium-dependent ATPase which formation occurs as an early step in the reaction sequence of calcium translocation by cardiac sarcoplasmic reticulum similar as in skeletal muscle.     

Magnesium magnetic isotope effect: a key towards mechanochemistry of phosphorylating enzymes as molecular machines

A discovery of the huge magnesium isotope effect in enzymatic ATP synthesis provides a new insight into mechanochemistry of enzymes as the molecular machines. It has been found that the catalytic activity values of ATPase, creatine kinase and phosphoglycerate kinase are 2 to 4-fold higher once their active sites contain magnetic (25Mg) not spinless, non-magnetic (24Mg, 26Mg), magnesium cation isotopes. This clearly proves that the ATP synthesis is a spin-selective process involving Mg2+ as the electron accepting reagent. The formation of ATP takes place in an ion-radical pair resulted by two partners, ATP oxyradical and Mg+. The magnesium bivalent cation is a key player in this process, this ion transforms the protein molecule mechanics into a mere chemistry. This ion is a most critical detail of structure of the magnesium dependent phosphorylation enzymes as the mechanochemical molecular machines.     

Porphyrin-proteinoid complexes as models of prebiotic photosensitizers

Photochemical activity as well as some spectral properties of various porphyrins and their model complexes with proteinoids were studied. Photochemical activity increased from the less advanced biosynthetic chlorophyll precursors to the more advanced compounds. The significant increase was detected as a result of the formation of complexes of phytol-containing porphyrins with proteinoids, which brings about the disaggregation of the pigment clusters formed in the aqueous environment. Hemin is photochemically inactive, either alone or in complex with proteinoid. Although hemoproteinoid was demonstrated to be able to catalyse photochemical electron transfer, its activity is about 20 times lower when compared with the chlorophyll a-proteinoid complex. Experimental results are discussed in context of the early evolution of photosynthesis.     

Model for prebiotic pyrophosphate formation: Condensation of precipitated orthophosphate at low temperature in the absence of condensing or phosphorylating agents

Summary The formation of pyrophosphate (PPi) by condensation of orthophosphate (Pi) at low temperature (37°C) in the absence of condensing or phosphorylating agents could have been an ancient process in chemical evolution. In the present investigation the synthesis of³²PPi from³²Pi was carried out at pH 8.0 and PPi was found in larger amounts in the presence of insoluble Pi (with calcium or manganese ions) than in its absence (with magnesium ions, or with no divalent cations present). After 10 days of incubation in the presence of precipitated calcium phosphate, about 1.6 nmol/ml of PPi was formed (0.057% yield relative to insoluble Pi). The hypothesis that the reaction is dependent on precipitated Pi was reinforced by the effect of adding dimethyl sulfoxide (2.1–9.9 M) in the presence of magnesium ions: the amount of magnesium phosphate precipitated in the presence of the organic solvent was proportional to the amount of PPi formed. The large and negative activation entropies found in aqueous media with calcium ions and in a medium containing 11.3 M dimethyl sulfoxide with magnesium ions suggest that the reaction was favored by a hydrophobic phenomenon at the surface of solid Pi. This reaction could serve as a model for prebiotic formation of PPi.     

Spectral and kinetic studies of phosphate and magnesium ion binding to yeast inorganic pyrophosphatase

Inorganic pyrophosphatase must bind two phosphate molecules in order to catalyze pyrophosphate synthesis. In this report it is shown that Pi causes marked effect on the absorption spectrum of baker's yeast inorganic pyrophosphatase and this effect can be used to analyze Pi binding to this enzyme. A series of absorbance versus Pi concentration curves in the presence of 0.5-20 mM free Mg2+ were obtained at pH 7.2 and computer-fitted to 19 models. The dissociation constant of magnesium phosphate (8.5 +/- 0.4 mM) used in this analysis was measured with a Mg2+-sensitive electrode. The best model implies successive binding of two magnesium phosphate molecules or random-order binding of magnesium phosphate and free phosphate molecules. The first route predominates at physiological concentrations of Mg2+. The Pi-inhibition pattern of pyrophosphate hydrolysis confirmed that Pi adds to the active site and provided further evidence for the existence of an activating Pi-binding site. The possibility is raised that the pathways of pyrophosphate synthesis and hydrolysis by inorganic pyrophosphatase may differ in the sense that the binding of the fourth metal ion/subunit may facilitate the synthesis and inhibit the hydrolysis.     

Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase

Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme-substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures.     

Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system

We developed a cell-free protein synthesis system that produces more than 1mg/ml of recombinant proteins in two hours. A basal system that supports the stable maintenance of ATP and amino acids was constructed by using high concentrations of CP (100 mM) and amino acids (3 mM). Approximately 0.6 mg/ml of protein was produced during the batch incubation of the basal system. We found that the accumulation of inorganic phosphate reduces the concentration of free magnesium ions and that there exists a critical concentration of magnesium at which the protein synthesis is halted. Based on this finding, we attempted to extend the duration of the protein synthesis by keeping the magnesium concentration sufficiently high throughout the reaction period. The protein synthesis reaction continued for at least 2 h when the reaction was repeatedly supplemented with magnesium, and approximately 1.2 mg/ml of active CAT or GFP was produced. The simple, fast, and highly productive cell-free protein synthesis system described herein should offer a versatile platform for the preparation of protein molecules in various post-genomic efforts.     

Self-association of calcium and magnesium complexes of dentin phosphophoryn

Self-association of rat dentin phosphophoryn in the presence of calcium and magnesium ions was examined by chemical cross-linking and electron microscopy. Highly phosphorylated phosphophoryn (HP) binds a maximum of 1.33 calcium ions or 1.07 magnesium ions per organic phosphate residue at pH 7.4-8.0. The Ca-HP complexes are predominantly linear when the calcium content of the complex is less than about 65% of the saturation level. At higher calcium levels, the protein has a folded conformation, and transient protein-protein interactions occur. The equilibrium mixture of monomers and oligomers is predominantly monomeric unless the protein is saturated with calcium. The saturated Ca-HP complex forms discrete high molecular weight particles about 25 nm in diameter. The particles are electrically neutral and generally occur in clusters. Mg-HP complexes appear predominantly linear by electron microscopy at all concentrations of bound magnesium up to about 99% of the saturation level; however, protein-protein interaction is measurable when the magnesium content is as little as 65% of the saturation level. At saturation, Mg-HP complexes form high molecular weight particles which are negatively charged. Because of the negative charge, these particles form a stable colloidal suspension and have a rather stellate configuration.     

Distribution of calcium,magnesium and inorganic phosphate in plasma of estradiol-17β treated rainbow trout

Summary Freshwater rainbow trout,Salmo gairdneri, were injected with different doses of estradiol-17 in order to induce the synthesis of a protein, regarded as identical to vitellogenin. The plasma levels of free and protein-bound calcium, magnesium and inorganic phosphate were studied in control and estradiol-17 treated fish, using an ultrafiltration method. Estradiol-17 caused a dose-dependent increase in plasma vitellogenin levels, which strongly correlated to protein-bound levels of calcium and magnesium in plasma. Calcium and magnesium were bound to vitellogenin in a ratio of 9:1, which was considerably higer than the protein-binding ratio of these ions in normal plasma (5.2:1). The dose-dependent increase in total plasma levels of calcium, magnesium and inorganic phosphate during estradiol-17 treatment was solely due to an increase in the protein-bound fraction of these ions. It is concluded that the physiologically important plasma levels of free calcium, magnesium and inorganic phosphate are effectively regulated at normal levels during vitellogenin synthesis.     

Optical absorption and fluorescence spectroscopy studies of ground state melanin-cationic porphyrins complexes.

Optical absorption and fluorescence spectroscopies were employed in the study of the interaction between synthetic L-dopa (dihydroxyphenylalanine) melanin and the cationic porphyrins tetrakis(4-N-methylpyridyl) porphyrin (TMPyP), tetrakis(4-N-benzylpyridyl)porphyrin (TBzPyP), zinc tetrakis(4-N-methylpyridyl)porphyrin (ZnTMPyP) and zinc tetrakis (4-N-benzylpyridyl)porphyrin (ZnTBzPyP). Optical absorption and fluorescence properties of the porphyrins were dependent on the symmetry of the central ring. No evidence was found for dimerization of the porphyrins in phosphate buffer, pH 7, in the concentration range between 4 x 10(-8) to 5 x 10(-5) M. Addition of L-dopa melanin red shifted the optical absorption spectra of porphyrins, concomitant to broadening and reduction in intensity of the bands. L-Dopa melanin also strongly quenched the fluorescence of the porphyrins. Time resolution of the fluorescence decay of porphyrins showed at least two lifetimes that were only slightly modified in the presence of melanin. The interaction between melanin and porphyrin resulted in the formation of non-fluorescent ground state complexes. It was found that there are two different classes of binding sites in melanin for complexation with cationic porphyrins and the values of dissociation constants are of the order of 10(-8) M. These values and the number of binding sites are dependent on the nature of the porphyrins. It was shown that the binding has electrostatic origin, but it is also affected by metal coordination and hydrophobic interaction.     

Structural requirements for porphyrin inhibition of the hemin-controlled protein kinase and maintenance of protein synthesis in reticulocytes

The role of hemin in the maintenance of protein synthesis in reticulocyte lysates was examined by comparing the effects of various porphyrins and metalloporphyrins on the protein kinase activity of the hemin-controlled repressor and on protein synthesis. The porphyrin requirements for maintenance of protein synthesis were relatively specific. Iron and cobalt metalloporphyrins sustained protein synthesis whereas other metalloporphyrins, metal-deficient porphyrins, and non-porphyrin precursor and degradation products of protoporphyrin IX were ineffective. These same compounds were examined for their effectiveness in inhibiting the protein kinase activity of the hemin-controlled repressor with initiation factor 2 (eIF-2). Most of the metalloporphyrins and porphyrins tested were inhibitory. The presence of the iron atom in the porphyrin was not essential for inhibition, but the maintenance of the integrity of the porphyrin ring was imperative. The porphyrins which inhibited the hemin-regulated protein kinase contained vinyl groups or ethyl groups, or were protonated in the 2- and 4-positions of the porphyrin ring, whereas those with bulky or acidic groups in these positions were ineffective. Precursor and degradation products of protoporphyrin IX and synthetic porphyrins modified at other positions had no effect on the enzyme. Both hemin and protoporphyrin IX inhibited phosphorylation of eIF-2 exogenously added to a reticulocyte lysate; however, hemin sustained protein synthesis in the lysate, whereas protoporphyrin IX did not. These results suggest that regulation of the protein kinase phosphorylating the alpha subunit of eIF-2 is not the only point at which hemin modulates protein synthesis in reticulocytes and reticulocyte lysates, since a correlation between inhibition of protein synthesis, inhibition of protein kinase activity, and phosphorylation of eIF-2 is not observed with all porphyrins.     

Features of transformation of phosphates in Rhodobacter sphaeroides chromatophores

We have found the ATP production in the Rhodobacter sphaeroides chromatophores illuminated by single short light flash, that is under conditions when the proton gradient formed as a result of electron transport after the second flash, is absent. The ATP synthesis was accompanied by the H2O2 formation. Simultaneous formation of H2O2 is indicative of the oxidative activation of phosphate during the ATP synthesis, as in the model systems with isolated chlorophyll. These data provide a theoretical background to the fitting of illumination parameters in both laboratory and industrial photobioreactors with photosynthetic bacteria used in biotechnological processes.     

Inhibition of (Na+,K+)-ATPase by magnesium ions and inorganic phosphate and release of these ligands in the cycles of ATP hydrolysis

The kinetic data of magnesium and inorganic phosphate inhibition of the (Na+,K+)-dependent ATP hydrolysis are consistent with a model where both ligands act independently and their release in the ATPase cycle is an ordered process where inorganic phosphate is released first. The effects of magnesium on the stimulation of the ATPase activity by Na+, K+ and ATP, and the inhibition of that activity by inorganic phosphate, are consistent with Mg2+ acting both as a 'product' and as a dead-end inhibitor. The dead-end Mg-enzyme complex would be produced with an enzyme form located downstream in the reaction sequence from the point where Mg2+ acts as a 'product' inhibitor. In the absence of K+, Mg2+ inhibition was reduced when either Na+ or ATP concentrations were increased well beyond those concentrations needed to saturate their high-affinity sites. This ATP effect suggests that the dead-end Mg-enzyme complex formation is affected by the speed of the E2-E1 conformational change. The present model is consistent with the formation of an Mg-phosphoenzyme complex insensitive to K+ which could become K+-sensitive in the presence of high Na+ concentrations. These Mg-enzyme complexes appear as intermediaries in the Na+-ATPase activity found in the absence of external Na+ and K+. These results can be interpreted on the basis of Mg2+ binding to a single site in the enzyme molecule. In addition, these experiments provide kinetic evidence indicating that the stimulation by external Na+ of the ATPase activity in the absence of K+ is due to a K+-like action of Na+ on the external K+ sites.     

Macroscopic and microscopic variation in recovered magnesium phosphate materials: Implications for phosphorus removal processes and product re-use

Phosphorus (P) recovery and re-use will become increasingly important for water quality protection and sustainable nutrient cycling as environmental regulations become stricter and global P reserves decline. The objective of this study was to examine and characterize several magnesium phosphates recovered from actual wastewater under field conditions. Three types of particles were examined including crystalline magnesium ammonium phosphate hexahydrate (struvite) recovered from dairy wastewater, crystalline magnesium ammonium phosphate hydrate (dittmarite) recovered from a food processing facility, and a heterogeneous product also recovered from dairy wastewater. The particles were analyzed using “wet” chemical techniques, powder X-ray diffraction (XRD), and scanning electron microscopy in conjunction with energy dispersive X-ray spectroscopy (SEM–EDS). The struvite crystals had regular and consistent shape, size, and structure, and SEM–EDS analysis clearly showed the struvite crystals as a surface precipitate on calcium phosphate seed material. In contrast, the dittmarite crystals showed no evidence of seed material, and were not regular in size or shape. The XRD analysis identified no crystalline magnesium phosphates in the heterogeneous product and indicated the presence of sand particles. However, magnesium phosphate precipitates on calcium phosphate seed material were observed in this product under SEM–EDS examination. These substantial variations in the macroscopic and microscopic characteristics of magnesium phosphates recovered under field conditions could affect their potential for beneficial re-use and underscore the need to develop recovery processes that result in a uniform, consistent product.     

6-phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism.

The inorganic pyrophosphate-requiring 6-phosphofructokinase of Entamoeba histolytica has been further investigated. The molecular weight of the enzyme is approximately 83,000 and its isoelectric point occurs at pH 5.8 to 6.0. The divalent cation requirement for reaction was explored. In the direction of fructose 6-phosphate formation half-maximal rate required 500 muM magnesium ion; in the direction of fructose bisphosphate formation 8 muM magnesium ion sufficed. ATP, PPi, polyphosphate, acetyl phosphate, or carbamyl phosphate cannot replace PPi as phosphate donor for the conversion of fructose 6-phosphate to fructose bisphosphate. In the direction of fructose 6-phosphate formation arsenate can replace orthophosphate. Isotope exchange studies indicate that little or no exchange occurs between Pi and PPi or between fructose 6-phosphate and fructose bisphosphate in the absence of a third substrate. These findings appear to rule out phosphoenzyme formation and a ping-pong reaction mechanism. PPi, Pi, and fructose bisphosphate are competitive inhibitors of fructose bisphosphate, PPi, and fructose 6-phosphate, respectively. This argues against an ordered mechanism and suggests a random mechanism. Fructose 6-phosphate and Pi were noncompetitive with respect to each other indicating the formation of a dead end complex. These product inhibition relationships are in accord with a Random Bi Bi mechanism.     

Specific Features of Phosphate Transformation on Rhodobacter sphaeroides Chromatophores

ATP production has been shown to take place on illumination of Rhodobacter sphaeroides chromatophores by a single light flash, i.e., in the absence of a proton gradient (which would form as a result of electron transport should a second flash occur). ATP synthesis was accompanied by H₂O₂ formation. Simultaneous formation of ATP and H₂O₂ is indicative of oxidative activation of phosphate during ATP synthesis, as in model systems with isolated chlorophyll. These data provide a theoretical background for selecting illumination parameters in laboratory and industrial photobioreactors used for cultivation of photosynthetic bacteria in biotechnological processes.     

Functional groups at the catalytic site of F1 adenosine triphosphatase

The protection of F1 ATPase by inorganic phosphate, ADP, ATP, and magnesium ion against inactivation by 1-fluoro-2,4-dinitrobenzene, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, respectively, has been investigated. Dissociation equilibrium constants and rate constants for the labeling reactions have been deduced from a quantitative treatment of the kinetic data. Comparison of these dissociation constants with each other and with the corresponding literature values indicates that the essential Tyr, Arg, Lys, and Glu or Asp residues are indeed located at the catalytic site of the enzyme. Examination of the rate constants for the labeling reactions in the presence of excess inorganic phosphate, ADP, ATP, or magnesium ion, respectively, suggests that the essential phenol and amino groups are located nearer to the bound inorganic phosphate or the gamma-phosphate group than to the alpha- or beta-phosphate group of the bound ATP, that the essential guanidinium group is located nearer to the alpha- or beta-phosphate group than to the gamma-phosphate group of the bound ATP or the bound inorganic phosphate, and that the essential carboxylate group is located slightly farther away but complexed with magnesium ion which it shares with the bound inorganic phosphate. A mechanism consistent with these topographical relationships is proposed for the catalytic hydrolysis and synthesis of ATP.