Reconstitution of biological molecular generators of electric current. Bacteriochlorophyll and plant chlorophyll complexes.

1. Electric generation by bacteriochlorophyll reaction center complexes from Rhodospirillum rubrum and by photosystem I complexes from pea chloroplasts has been studied. 2. The methods for the proteoliposome reconstitution from azolectin and bacteriochlorophyll- or plant chlorophyll-containing protein complexes have been elaborated. Light-dependent electric responses of the proteoliposomes were detected using (a) phenyldicarbaundecarborane anion (PCB-) probe and (b) direct measurement by a voltmeter in the proteoliposome-planar phospholipid membrane system. 3. Both PCB- and direct measurements demonstrated that bacteriochlorophyll proteoliposomes are competent in light-dependent electric generation (plus outside proteoliposomes). The photoelectric effect was shown to increase on addition of tetramethyl-p-phenylenediamine (TMPD), CoQ6, and vitamin K3, and to decrease on addition of ferricyanide, o-phenanthroline and a protonophorous uncoupler. Estimation of the photoelectromotive force of the bacteriochlorophyll proteoliposome-planar membrane system gave a value of about 0.2 V. The action spectrum of the photoelectric effect was similar to the absorption spectrum of the bacteriochlorophyll complex. 4. Reconstitution of proteoliposomes containing bacteriochlorophyll centers and bacteriorhodopsin resulted in the system generating an electric field whose direction can be changed by varying the spectral composition of the light: the red light, exciting bacteriochlorophyll, induces negative, and the green light, exciting bacteriorhodopsin, induces positive charging of the proteoliposome interior. 5. Association of isolated R. rubrum chromatophores with planar phospholipid membrane was found to give a system demonstrating light-induced electric generation as high as 215 mV in the presence of napthoquinone, TMPD (or phenazine methosulfate, PMS), and ascorbate. Under the same conditions, addition of inorganic pyrophosphate or ATP results in formation of an electric field of the same direction as that induced by light. 6. Proteoliposomes with plant chlorophyll complexes of photosystem I demonstrated light-induced PCB- responses indicating formation of the electric field with plus inside vesicles. The effect required PMS addition. A protonophorous uncoupler and o-phenanthroline were inhibitory. Electric responses in the chlorophyll proteoliposome-planar membrane system were very small (not higher than 10 mV).


Generation of a transmembrane
electric potential difference by oligomycin-sensitive ATPase complex, incorporated into spherical or planar phospholipid membrane, has been demonstrated. To this end, penetrating anion probe and direct voltmeter measurement of electric potential across phospholipid membrane were used. It was found that ATP-induced electric response is sensitive to oligomycin and protonophorous uncouplers. 2. The effect of variations in the phospholipid component of proteoliposomes on the electric generation was studied. It was revealed that the usage of mitochondrial phospholipids and phosphatidylethanolamine allows the highest values of membrane potential to be obtained in the case of ATPase proteoliposomes. In the case of cytochrome oxidase and bacteriorhodopsin proteoliposomes, phosphatidylserine was also shown to be quite suitable. Phosphatidylcholine was absolutely ineffective in all cases. 3. In proteoliposomes, containing both ATPase and bacteriorhodopsin, ATP and light induced generation of electric field of the same direction. 4. In ATPase + cytochrome oxidase proteoliposomes, ATP hydrolysis and ascorbate oxidation was found to support electric generation of the same direction if cytochrome c was inside vesicles. Oxidation via external cytochrome c resulted in formation of electric field of the direction, opposite to that induced by ATP hydrolysis. 5. The data obtained in experiments with proteoliposomes of different types are discussed. The conclusion is made that conversion of energy of different resources into electric form is a common feature of membraneous energy transducers, which is in agreement with the Mitchellian principle of cellular energetics.
In three previous papers of this series (l-3), the data on In 1971 Kagawa and Racker (5) described reconstitution of direct measurement of electric generation by bacteriorhodop-ATPase proteoliposomes catalyzing "Pi-ATP exchange which sin, chlorophyll reaction center complexes, and cytochrome proved to be sensitive to such a specific agent discharging oxidase were reported. These results seem to be sufficient to membrane potential as combination "valinomycin + nigericin conclude that the energy-supplying systems mentioned are + K+." The ATP-dependent H' uptake by the proteolipocompetent in the utilization of the corresponding energy somes was also observed (6). sources to form transmembrane electric potential.
In this work, membrane potential generation by H+-ATPase According to Mitchell's chemiosmotic theory (4), the energy-from beef heart mitochondria was studied by means of a consuming ATP synthetase reaction, localized in the same penetrating ion probe and direct voltmeter measurements in a membrane as energy-supplying mechanisms, represents rever-system "proteoliposomes-planar phospholipid membrane." sal of the ATPase reaction coupled with electrogenic uphill H+ Preliminary notes of some of these results were published transport (H+-ATPase). elsewhere (7,8).
Reconstitution of ATPase proteoliposomes was carried out essentially after the procedure by Kagawa and Racker (5 The ATP addition to such a system was found to give rise to electric generation which disappeared after oligomycin treatment (Fig. 1A). The form of the voltmetermeasured electric response was very similar to that of the ATP-induced PCB-response of the proteoliposome suspension (Fig. 1B)  ATPase. Mitochondrial phospholipids and phosphatidylethanolamine were effective in all cases.
Measurements of electric resistance of the planar membranes made of different phospholipids showed that it is several times lower in the case of phosphatidylcholine than in the case of other phospholipids.
Electron microscopic study revealed only small amounts of membrane vesicles when phosphatidylcholine was used for the reconstitution. Apparently, these facts may explain why membrane potentialgenerating proteoliposomes cannot be obtained with this phospholipid. Other effects of lipid composition should be due to the specific demands of proteins on their phospholipid partners during the reconstituting or the functioning of proteoliposomes.
Proteoliposomes with ATPase and Bacteriorhodopsin or Cytochrome Oxidase-In the last series of experiments, proteoliposomes were reconstituted from the mixture containing ATPase together with bacteriorhodopsin or cytochrome oxidase. As one can see in Fig. 2, ATPase + bacteriorhodopsin proteoliposomes generate electric potential at the expense of ATP, or alternatively of light energy, the process being revealed by both PCB-and voltmeter measurements. Usually, the ATP-supported potential was lower than the light-supported one. In both cases, the electric fields were found to be of the same direction (plus inside proteoliposomes).
The study on the interaction of two types of generators was continued in the experiments with ATPase + cytochrome oxidase proteoliposomes.
In formed by the cytochrome oxidase generator was regulated by actuation of the extra-or, alternatively, intravesicular cytochrome c. As the experiments showed, the electron transport via inner cytochrome c generates the field of the same direction as ATPase does (plus inside the proteoliposomes), whereas that via external cytochrome c generates the field of the opposite direction (minus inside). These relationships are demonstrated in Fig. 3, where PCB-responses are shown. It is noteworthy that ATP-supported membrane potential collapses when external cytochrome c is actuated. Subsequent addition of cyanide abolishes the ascorbate effect, so that ATP-supported membrane potential can be observed again.
In Fig. 4 one can see PCB-response of the proteoliposomes containing cytochrome oxidase (+ cytochrome c inside) and hydrophobic proteins of ATPase. Reconstitution of the proteoliposomes with F, was not carried out. According to the data obtained, the membrane potential, generated by cytochrome oxidase, can be increased by oligomycin. This effect corresponds to the "coupling" action of oligomycin on the respiratory chain-supported membrane potential formation in the F,deficient submitochondrial particles (12 been developed. The procedure includes (a) reconstitution of the protein with phospholipid to form vesicles (proteoliposomes) according to the method of Kagawa and Racker (5), (b) association of proteoliposomes with planar membrane induced by Ca2+ ions, and (c) measurement of electric potential difference across planar phospholipid membrane by electrodes immersed into electrolyte solutions on both sides of this membrane.
Using this method, we found the electric current to be produced by light-dependent systems (bacteriochlorophyll reaction centers of Rhodospirillum rubrum chromatophores and bacteriorhodopsin from Halobacterium halobium), by the complex forming an energy coupling site of the mitochondrial respiratory chain (cytochrome c-cytochrome oxidase), and by the mitochondrial oligomycin-sensitive H+-ATPase.The good correlation of the data obtained by this method and by the synthetic penetrating ion probe is noteworthy.
These results directly confirm Mitchell's postulate (4) on electric potential generation as a process intrinsic of respiratory and photosynthetic energy coupling systems. The validity of the postulate in question was supported by several independent pieces of evidence provided by indirect methods. Nevertheless there were some grounds for skepticism left, mainly because of the failure of an attempt to measure the membrane potential in mitochondria with a voltmeter (for references, see the first paper of this series (1)). Now it seems to be high time to throw away the remaining doubts and accept the point that molecular electric generators exist in coupling membranes.
When considering energy coupling mechanisms, it is noteworthy that both energy-providing reactions of respiratory (photosynthetic) chain and H+-ATPase systems prove to be competent in electric generation. It means that H+-ATPase, if reversible, can utilize electric membrane potential energy to form ATP. Reversibility of ion-transporting ATPases, including H+-ATPase of coupling membranes, is well established (for review, see Ref. 13). So, energy-providing reactions can be coupled with phosphorylation via membrane potential as was originally postulated by Mitchell (4).
Mode of Proteoliposome Association with Planar Mem-brane-As was mentioned in the first paper of the series (I), variations in the direction of the bacteriorhodopsin-generated photo electromotive force could be observed when bacteriorhodopsin was added directly into the planar membrane-forming solution. On the other hand, this parameter was invariant when bacteriorhodopsin proteoliposomes were used. In the cases of proteoliposomes of other types, the direction of electric field was also regular. Results of measurements of the electric field direction in proteoliposomes as detected by PCB-probe and by voltmeter are summarized in Table II. It is seen that the signs of the electric potential difference on the proteoliposome-free side of the planar membrane always coincide with those in the proteoliposomal interior, as can be judged by the PCB-flux direction.
Three possible versions of the proteoliposome association with planar membrane may be considered.
1. Proteoliposomes are attached to the surface of the planar membrane. The charges, e.g. H+ ions accumulated inside these proteoliposomes, move electrophoretically from the proteoliposomal interior to the extraproteoliposomal compartments: (a) across the proteoliposomal membrane-to the same chamber which proteoliposomes were added to; or (b) across the planar membrane-to the opposite chamber. In the latter case, an electric potential difference between the two chambers separated by the planar membrane should arise (Fig. 5).
2. Attachment of proteoliposomes to the black planar membrane entails proteoliposomes opening in such a way that their membranes prove to be a part of the planar membrane.
3. The opening of the proteoliposome, attached to the thick planar membrane, results in some areas of the planar membrane surface being covered by the proteoliposomal material. Experiments confirmed the first scheme. It was found (1) that electric responses of the bacteriorhodopsin proteoliposomes, associated with planar membrane, are sensitive to gramicidin A, added in concentrations which do not reduce the planar membrane resistance in the light. This can be accounted for by the fact that gramicidin, a compound forming K+-, Na+-, and H+-permeable channels through thin (black), rather than thick, phospholipid membranes, shunts the proteoliposo-ma1 membrane whose thickness is of about 70 A according to the electron microscope data (1). As to the planar membrane, it remains unaffected, being too thick for a gramicidin channel to be organized. It is noteworthy that, under the same conditions, the decrease in the electric response of proteoliposomes caused by proton carriers such as CCCP correlates with the drop in the electric resistance of the planar membrane (1). These facts fail to be accounted for in terms of the second and the third schemes mentioned above.
The observation incompatible with the second scheme (the opening of proteoliposomes attached to the black planar membrane) was described when cytochrome oxidase proteolip-I osomes were studied. It was shown (3) that cytochrome c and ascorbate, added to the proteoliposome-free compartment, did not induce any electric response until the mixture was supplemented with a penetrating H atom carrier. This means that cytochrome oxidase originally incorporated into the proteoliposomal membrane cannot interact with cytochrome c if proteoliposomes and cytochrome c are separated by the planar membrane. So, the proteoliposomal membrane, if associated with the planar one, is still inaccessible to the solution on the opposite side of the planar membrane.
Another fact testifying against the second scheme is that not only black (bilayer) but also thick planar membranes can be Biolo&d Electric Generators: H+-ATPase 7081 used in electric generation experiments (1). As a matter of fact, most of the experiments described in these four papers were carried out in thick planar membranes since black ones treated with proteoliposomes proved to he unstable. Mechanism of Electric Generation by Proteoliposome-Plo-MT Membrane System-The schemes illustrating mechanism of electric current generation by different types of proteolipo-some8 associated with planar membrane are given in Fig. 5. It is shown that electrogenic transfer of electrons or protons across proteoliposomal membrane is carried out by a specific protein system utilizing the corresponding energy source: light (hacteriorhodopsin and chlorophyll), hydrogen donor and oxygen (cythochrome oxidase), or ATP (H'-ATPase).
In all the cases studied, formation of electrochemical H+ potential gradient (ACH) between extra-and intraproteoliposomal compart-

Eaeteriorhodopsin Bacterioehlorophyll
Cytoehromeoxidase with cytoehrome e outside Cytoehromeoxidase with eytoehrome c inside El+-AT&se Influx + Ef""X ml", lnllux + Influx + merits proves to take place. Movement of H+ ions down AfiH when it occurs through planar membrane results in an electric potential difference between two planar membrane-separated solutions. In fact, this is the value that is measured by a voltmeter.
According to the schemes, proteins localized in the region of spherical and planar membrane fusion, might, in principle, generate an electric field of the direction opposite to that produced by other membrane regions. Apparently, this does not take place since native arrangements of biological electric generators requires opposite parts of them to he on the water-lipid interphases. This requirement is not fulfilled if the protein complex is immersed into thick phospholipid membrane, as it should take place in the fusion area.
Asymmetry of Proteoliposome Reconstitution-In the case of both hacteriorhodopsin and chlorophyll, there should he reasons for the fact that the majority of bacteriorhodopsin and chlorophylls, incorporated into proteoliposomes, transfer pmteins or, respectively, electrons across the proteoliposomal membrane from outside to inside. The simplest explanation might consist in that chromophores, localized on the outer surface of the proteoliposomal membrane, are better saturated with light than the chromophores on the inner surface. However, this can hardly account for the phenomenon ohserved because the sign of the photoeffect does not depend on whether the planar membrane was illuminated from the proteoliposome or proteoliposome-free side (1,2). Apparently, the effect discussed is due to asymmetric reconstitution of the proteolipasome membrane. This may he a consequence of different factors affecting the reconstitution process. One of them may be the difference in the extra-and intraproteoliposo-Fro. 5