Search

Surface plasmons polaritons (SPP) hold great promise for the next generation of fast nanoscale optoelectronic devices, as silicon-based electronic devices approach fundamental speed and scaling limitations. However, in order to fully exploit the potential of plasmonics, devices and material systems capable of actively controlling and manipulating plasmonic response is essential. Here, we demonstrate active control of the electric field distribution of a micro antenna by coupling SPP to a photosynthetic protein with outstanding optoelectronic properties and long range and efficient exciton transfer ability. The hybrid bio-solid state active platform is able to tune and modulate the optical activity of a micro plasmonic antenna via interaction of the bioactive material with plasmon oscillations occurring in the antennae. In addition, we demonstrate that the effect of the coupling can be further enhanced and controlled by an external potential applied to the micro antenna Photosynthetic hybrid system.

The efficient optoelectronic properties of photosynthetic proteins were explored in the quest for the fabrication of novel solid biohybrid devices. As most optoelectronic devices function in a dry environment, an attempt was made to fabricate an efficient electronic junction by covalent binding of photosynthetic reaction center proteins to metals, transparent semiconductor polymers, and solid semiconductors that function in a dry environment. The primary stages of photosynthesis take place in nanometric-size protein-chlorophyll complexes. Such is photosystem I (PSI), which generates a photovoltage of 1 V. The isolated PSI generates an absorbed light-energy conversion efficiency of ∼47% (∼23% solar energy) and internal quantum efficiency of ∼100%. The robust cyanobacterial PSI was used in the fabrication of solid-state optoelectronic devices by forming oriented multilayers from genetically engineered cysteine mutants between metal and transparent conducting semiconductor electrodes. Current-voltage measurements of the cells generated diode- and photodiode-like responses in the dark and the light, respectively. The cells were stable for many months in a dry environment. The generation of photocurrent and Voc indicated the formation of good electronic coupling between PSI and the electrodes, which can serve in the fabrication of solid-state biohybrid optoelectronic devices.

In biological systems, membrane proteins play major roles in energy conversion, transport, sensing, and signal transduction. Of special interest are the photosynthetic reaction centers involved in the initial process of light energy conversion to electrical and chemical energies. The oriented binding of membrane proteins to solid surfaces is important for biotechnological applications. In some cases, novel properties are generated as a result of the interaction between proteins and solid surfaces. We developed a novel approach for the oriented tagging of membrane proteins. In this unique process, bifunctional molecules are used to chemically tag the exposed surfaces of membrane proteins at selected sides of membrane vesicles. The isolated tagged membrane proteins were self-assembled on solid surfaces, leading to the fabrication of dens-oriented layers on metal and glass surfaces, as seen from the atomic force microscopy (AFM) images. In this work, we used chromatophores and membrane vesicles containing protein chlorophyll complexes for the isolation of the bacterial reaction center and photosystem I, from photosynthetic bacteria and cyanobacteria, respectively. The oriented layers, which were fabricated on metal surfaces, were functional and generated light-induced photovoltage that was measured by the Kalvin probe apparatus. The polarity of the photovoltage depended on the orientation of proteins in the layers. Other membrane proteins can be tagged by the same method. However, we preferred the use of reaction centers because their orientation can be easily detected by the polarity of their photovoltages.

The use of dry functional protein films on solid-state platforms is essential in a variety of optoelectronic devices where aqueous environment is not favorable. The photoactive photosynthetic prote...

An extremely fast electron transfer through an electronically coupled junction between covalently bound oriented photosynthetic reaction center protein photosystem I (PS I) and n-GaAs was measured by time-resolved photoluminescence. It was found that the n-GaAs band edge luminescence intensity increased by a factor of 2, and the fast exponential decay constant was increased by a factor of 2.6 following the PS I self-assembly. We attribute this to picosecond electron transfer from the PS I to the n-GaAs surface states.

The electronic coupling between the photoactive proteins and semiconductors can be used for fabrication of a hybrid biosolid-state electrooptical devices. The robust cyanbacterial nanosized protein-chlorophyll complex photosystem I (PS I) can generate a photovoltage of 1 V with a quantum efficiency of ∼1 and can be used as a phototransistor gate. A functional dry-oriented junction was fabricated by covalently binding genetically engineered cysteine mutants of PS I to a chemisorbed small connecting molecules on the GaAs surface. Kelvin probe force microscopy measurements showed an induced photovoltage of 0.3 and -0.47 V in PS I-coated p- and n-type GaAs, respectively. The photovoltage resulted from an opposite direction of charge transfer between PS I and the semiconductors due to a difference of almost -0.8 eV in the Fermi level energy of the p- and n-GaAs, thus providing direct evidence of an electronically coupled junction useable as a photosensor.

Fabrication of serially-oriented multilayers of photosynthetic reaction center photosystem I (PS I) was mediated by the photo-catalytic specificity that reduced Pt ions to metal patches on the reducing side of PSI forming junctions with the oxidizing end of the proteins through Pt-sulfide bond of genetically-engineered cysteine mutants. The dry multilayers can be utilized in hybrid bio-solid-state electronic devices in which an increase in photo-voltage, resulting from the larger absorption cross-section and the serial-arrangement of PS I, is required. PS I is a transmembrane multisubunit protein-chlorophyll complex that mediates vectorial light-induced electron transfer. The nano-size dimension, an absorbed light energy yield of approximately 47% (or ca. 23% of solar radiation) and a photovoltage of 1 V with quantum efficiency of almost 1, make the reaction center a promising unit for applications in molecular nano-electronics. The robust PS I used in these experiments, that was isolated from the thylakoid membranes of cyanobacteria, is sufficiently stable to be used in hybrid solid-state electronic device. The dry PS I monolayer was shown earlier to remain stable for more than three months and it stayed active for over one year in the present experiments. The structural stability is due to hydrophobic interactions that integrates 96 chlorophyll and 22 carotenoid pigment molecules and the trans membrane helixes of the core subunits. The light-induced electron transfer at cryogenic temperatures is an indication of little structural motions during function. We have fabricated self-assembled oriented monolayers by the formation of direct sulfide bonds between unique cysteine mutants of PS I from the cyanobacteria and the metal surface which generated, a photovoltage of 0.45 V under a dry environment. In earlier works, only indirect adsorption of single plant PS I molecules and binding of bacterial reaction center monolayers were functioning in such an environment. Although a Schottky junction with PS I monolayer provides electronic coupling with unique photovoltaic properties, oriented multilayers can be advantageous when a larger light absorption cross section and enhanced photovoltage values are desired. As an efficient oriented multilayer, the PS I complexes need to be physically and electronically coupled and organized in a serial fashion. The use of the unique specificity of a photo-catalytic protein with redox potential of –0.53 V enabled the reduction of Pt ions and deposition of metallic platinum at the reducing end of PS I (Fig. 1a and b). The metC O M M U N IC A IO N

The photosystem I (PS I) reaction center is a chlorophyll protein complex located in thylakoid membranes of chloroplasts and cyanobacteria. PS I mediates a light-induced electron transfer through a serial of redox reactions. It is intriguing to incorporate the PS I into optoelectronic circuits, since the PS I exhibits outstanding optoelectronic properties found only in the photosynthetic systems. The quantum yield for absorbing a photon within the whole complex is determined to be close to 100 %, while the energy yield for the process is approximately 58 %. The nanoscale dimension and the generation of 1 V photovoltage further makes the PS I reaction center a promising unit for applications in molecular optoelectronics. Utilizing a unique cysteine (Cys) mutation at the end of PS I, we demonstrate a four-step chemical procedure based on carbodiimide chemistry for covalent binding of PS I proteins to carbon nanotubes (CNTs). The method allows studying hybrid nanosystems for the construction of optoelectronic devices based on PSI-CNTs heterostructures. Three variations in the design of PSI-CNT hybrid structures are presented which allow exploiting the potential of PS I as an integrated part of CNT nanodevice for optoelectronic applications. Recently, we have demonstrated the possibility to covalently bind PS I directly to gold surfaces and indirectly via a small linker molecule to GaAs surfaces. To this end, amino acids in the extra membrane loops of the PS I facing the cytoplasmic side of the bacterial membrane (oxidizing side) were mutated to cysteines (Cys) enabling the formation of covalent bonds with a metal surface or a chemically functionalized GaAs surface. The Cys located at extra membranal loops of the protein do not have steric hindrance, when placed on a solid surface e.g. of a gold electrode or CNTs as shown here. The mutations D235C/Y634C were selected near the special chlorophyll pair P700 to allow close proximity between the reaction center and the CNTs. As depicted by white arrows in Figure 1, here we utilize a PS I with two mutants on the oxidizing side of the PS I. This single sided mutant ensures a high outcome of our chemical self-assembly procedure. For a variation of our chemical scheme we also use bipolar (BM) mu

Photosystem I (PS I) is a transmembranal multisubunit complex that mediates light-induced electron transfer from plactocyanine to ferredoxin. The electron transfer proceeds from an excited chlorophyll a dimer (P700) through a chlorophyll a (A0), a phylloquinone (A1), and a [4Fe-4S] iron-sulfur cluster FX, all located on the core subunits PsaA and PsaB, to iron-sulfur clusters FA and FB, located on subunit PsaC. Earlier, it was attempted to determine the function of FX in the absence of FA/B mainly by chemical dissociation of subunit PsaC. However, not all PsaC subunits could be removed from the PS I preparations by this procedure without partially damaging FX. We therefore removed subunit PsaC by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp. PCC 6803. Cells could not grow under photosynthetic conditions when subunit PsaC was deleted, yet the PsaC-deficient mutant cells grew under heterotrophic conditions and assembled the core subunits of PS I in which light-induced electron transfer from P700 to A1 occurred. The photoreduction of FX was largely inhibited, as seen from direct measurement of the extent of electron transfer from A1 to FX. From the crystal structure it can be seen that the removal of subunits PsaC, PsaD, and PsaE in the PsaC-deficient mutant resulted in the braking of salt bridges between these subunits and PsaB and PsaA and the formation of a net of two negative surface charges on PsaA/B. The potential induced on FX by these surface charges is proposed to inhibit electron transport from the quinone. In the complete PS I complex, replacement of a cysteine ligand of FX by serine in site-directed mutation C565S/D566E in subunit PsaB caused an approximately 10-fold slow down of electron transfer from the quinone to FX without much affecting the extent of this electron transfer compared with wild type. Based on these and other results, we propose that FX might have a major role in controlling electron transfer through PS I.

Intra-subunit interactions in the environment of the iron-sulfur cluster F(X) in Photosystem I (PS I) of Synechocystis sp. PCC 6803 were studied by site-directed and second site suppressor mutations. In subunit PsaB, the cysteine ligand (C565) of F(X) and a conserved aspartate (D566) adjacent to C565 were modified. The resulting mutants D566E, C556S/D566E, C556H/D566E and C565H/D566E did not assemble PS I in the thylakoids of the cyanobacterium. Yet, this is the first report of cells of the second site-suppressor mutant (D566E/L416P) and of second site-directed mutant (C565S/D566E) in PsaB that could grow autotrophically in light and were found to assemble a stable functional PS I containing all three iron-sulfur centers, F(X) and F(A/B). The newly resolved structure of PS I (PDB 1JB0) was used to interpret the functional interactions among the amino acid residues. It is suggested that the stability of F(X) is supported by a salt bridge formed between D566, which is adjacent to the cysteine ligand C565 of the iron-sulfur cluster located on loop hi, and R703 located at the start of loop jk. Hydrogen bond between R703 and D571 at the start of loop hi further stabilizes the arginine. Lengthening of the side by 1.2 A chain in mutation D566E caused destabilization of F(X). The extended side-chain was compensated for by the Fe-O, which is 0.3 A shorter than the Fe-S bond resulting in stabilization of the F(X) in the double mutations C565S/D566E. The suppressor mutation D566E/L416P allowed greater freedom for the salt bridge E566-R703, thus relieving the pressure introduced by the D566E replacement and enabling the formation of F(X). F(X) and R703 are therefore stabilized through short- and long-range interactions of the inter-helical loops between h-i, j-k and f-g, respectively.

The structure of vanadate, a phosphate analogue which was suggested to function in the presence of tightly bound ADP and divalent cations as a transition state inhibitor of CF1-ATPase, was investigated by X-ray absorption spectroscopy. Analysis of the vanadium K-edge was used for determination of the structure of vanadate bound to a single site in CF1-ATPase containing a single tightly bound ADP. There was a decrease in the intensity of the 1s-3d pre-edge transition and a change in the shape of two other shoulders at the edge region upon binding of vanadate to CF1 in the presence of Mg2+ ions. The changes are due to alteration in the structure of vanadium from tetrahedral to a five-coordinated trigonal bipyramidal geometry. Comparison of the pre-edge peak intensity of ADP-vanadate complex, and model compound resolved by crystallography support the proposed structure of CF1-bound vanadate. 51V NMR measurements were used to verify the pentacoordinated structure of ADP-vanadate complex used as a model in the X-ray absorption studies. The inhibition of a single and multiple site activity by vanadate and by MgADP was measured. Vanadate inhibition of CF1-ATPase activity decreased more than 90 fold in the presence of MgADP. A differential specificity of the inhibition in single and multiple mode of activity was observed. It is suggested that ADP-vanadate binds to the active sites of the enzyme as a pentacoordinated vanadium having approximate trigonal bipyramidal geometry. This structure is analogous to the proposed transition state of the phosphate during the synthesis and the hydrolysis of ATP by CF1.

X-ray absorption spectra of photosystem I core protein containing F5 , the low potential iron—sulfur cluster was measured. X-ray absorption of the iron K-edge was obtained in the reduced and oxidized forms of F 5 . An edge shift of 0.5 eV to lower energy and a change in the X-ray absorption near edge structure was observed. Some of the alterations in edge shape of F. are interpreted to indicate structural changes of the iron—sulfur cluster in F. upon reduction. PACS numbers: 78.70.Dm

Photosystem I (PSI) is one of the most studied electron transfer (ET) systems in nature; it is found in plants, algae, and bacteria. The effect of the system structure and its electronic properties on the electron transfer rate and yield was investigated for years in details. In this work we show that not only those system properties affect the ET efficiency, but also the electrons’ spin. Using a newly developed spintronic device and a technique which enables control over the orientation of the PSI monolayer relative to the device (silver) surface, it was possible to evaluate the degree and direction of the spin polarization in ET in PSI. We find high-spin selectivity throughout the entire ET path and establish that the spins of the electrons being transferred are aligned parallel to their momenta. The spin selectivity peaks at 300 K and vanishes at temperatures below about 150 K. A mechanism is suggested in which the chiral structure of the protein complex plays an important role in determining the high-spin selectivity and its temperature dependence. Our observation of high light induced spin dependent ET in PSI introduces the possibility that spin may play an important role in ET in biology.

The activity of CF1-ATPase was inhibited by vanadate in an allosteric manner with respect to CaATP as substrate. The cooperative interaction was enhanced by preincubation of the enzyme in the presence of ADP and Ca2+ ions and of free divalent metal ions during assay of the activity. The strongest cooperative interaction with a Hill coefficient of 5.3 +/- 0.1 was found when the reaction was stopped after 30 s, before steady state was reached. Under these conditions, the concentration of an exchangeable ADP, tightly bound to one of the active sites on the enzyme, was shown to be the highest. A Ki of 12.4 +/- 1.2 microM for vanadate inhibition was determined under these conditions. Direct measurements with the aid of 51V NMR indicated that vanadate binds to CF1 in the presence of Ca2+ and ADP in a positive cooperative manner with a Hill coefficient of 2.3 +/- 0.2 and an average Kd of 0.3 +/- 0.04 nM. It was suggested that a formation of pentacovalent vanadyl-ADP at the active site caused the inhibition. Vanadyl-ADP was suggested to be a strong inhibitor, being an analogue of a pentacovalent phosphoryl-ADP, which is proposed to be the transition state intermediate of CF1.

Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, a quinone and a [4Fe-4S] iron-sulfur cluster F(X), located on the core subunits PsaA/B to iron-sulfur clusters F(A/B) on subunit PsaC. Structure function relations in the native and in the mutant (psaB-C565S/D566E) of the cysteine ligand of F(X) cluster were studied by X-ray absorption spectroscopy (EXAFS) and transient spectroscopy. The structure of F(X) was determined in PS I lacking clusters F(A/B) by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp PCC 6803. PsaC-deficient mutant cells assembled the core subunits of PS I which mediated electron transfer mostly to the phylloquinone. EXAFS analysis of the iron resolved a [4Fe-4S] cluster in the native PsaC-deficient PS I. Each iron had 4 sulfur and 3 iron atoms in the first and second shells with average Fe-S and Fe-Fe distances of 2.27 A and 2.69 A, respectively. In the C565S/D566E serine mutant, one of the irons of the cluster was ligated to three oxygen atoms with Fe-O distance of 1.81 A. The possibility that the structural changes induced an increase in the reorganization energy that consequently decreased the rate of electron transfer from the phylloquinone to F(X) is discussed.

On p. 3901, Shachar Richter, Alexander Holleitner, and co-workers report on a novel hybrid system, fabricated by covalent binding of photosynthetic reaction center protein photosystem I (PS I) to carbon nanotubes (CNTs) by using carbodiimide chemistry. The versatile chemical modification schemes allow contacting of single PS I protein to two carbon nanotubes, the formation of CNT–PS I–CNT junctions, and electrical contact of carbon nanotubes to gold electrodes via covalent binding to the photosystem. Such nanostructures can pave the way for the construction of electronic circuits for nano-optoelectronic applications.

The photoelectronic nature of a dried photosystem I protein attached to a metal surface is studied using various spectroscopic techniques. The proteins are found to be optically active after the chemical adsorption. In addition, energy-resolved photoelectronic measurements indicate that the interaction of photosystem I with the metal surface leads to new molecule/substrate states, yielding energy states different from those of the individual components. Such interactions increase the spectral-response range beyond the absorption spectrum of photosystem I and are expected to improve the energy-conversion efficiency of devices based on this system.

The photosystem I (PS I) complex consists of at least 11 polypeptides in cyanobacteria. The heterodimeric PsaA and PsaB subunits contain P700, which undergoes light-induced charge separation and transfers electrons through sequential carriers Ao, AI and F, and the final acceptors FA and Fg which are located on PsaC. P700, Ao, AI, Fx, FA and Fg are a chlorophyll a dimer, a chlorophyll a monomer, a phylloquinone and three [4Fe-4S] iron sulfur centers, respectively (1). The crystalline structure of PS I resolved to 4A (2) reveals 11 helixes in each of the heterodimeric PsaA and PsaB, and F, is located at the interface of PsaC. Fx was predicted to be bound by two interhelical loops, each belonging to one of the subunits PasA and PasB. It was suggested that the cysteine residues in the two heterodimers located in the sequence CDGPGRGGTC bind F. The two cysteines C556 and C565 in PsaB of Synechocystis sp PCC 6803 are probably ligands of Fx as was shown by mutations (3). In this work the structure-function role of Fx in PS I was further tested by site directed and by suppressor mutations in psaB.

Inhibition of ATPase activity by vanadate, having K 1 2 of 0.5 mM, was demonstrated in the CF1-ATPase. The Ca2+-dependent ATPase activity of the isolated enzyme was inhibited in an allosteric manner by vanadate with a Hill coefficient of 3.19 ± 0.6. Vanadate also inhibited ATPase and Pi—ATP exchange activities of the chloroplast membrane-bound enzyme. Using 51V NMR it was demonstrated that ATP caused partial release of about 1.87 equivalents while ADP caused additional binding of approximately 1.46 equivalents of vanadate, when added to a solution containing CF1 equilibrated with vanadate. The relevance of these results to a possible involvement of a pentacovalent phosphate as transition state intermediate in the hydrolysis of ATP by CF1-ATPase is discussed.

Rodobacter capsulatus cells, which were cultured anaerobically in high light intensity, had fewer foldings in the cytoplasmic membrane than those which were grown in lower light intensities. Spheroplast-derived membrane fractions obtained from cells cultured under high light intensity contained a high yield of large right-side-out membrane vesicles. The right-side-out vesicles catalyzed reversible light-induced proton efflux as did intact cells. Nucleotide transport activity was also catalyzed by these membrane vesicles. This activity was indirectly monitored by measurement of photophosphorylation or hydrolysis of externally added diphospho- and triphosphonucleosides. These enzymatic activities occur inside the cytoplasmic membrane of spheroplasts and membrane vesicles and therefore require the transport of the externally added reagents. The indirect measurements of transport were complemented by the demonstration of direct uptake of radiolabeled nucleotides into the membrane vesicles. These data support the suggestion that a nucleotide transporter located in the cytoplasmic membrane of R. capsulatus bacteria mediates these activities.

The surface potential of purple membrane fragments, determined from the distribution of the aqueous free and the membrane-bound positively charged, paramagnetic, amphiphilic probe 4-(dodecyldimethylammonium)-1-oxyl-2,2,6,6-tetramethylpiperidine bromide varied almost 60 mV as a function of ionic strength and 50 mV as a function of pH of the medium. Light-induced changes in surface potential followed the changes observed in the M412 intermediate of the photocycle of bacteriorhodopsin as a function of pH, temperature, and response to antibiotics beauvericin and valinomycin. The number of induced charges per M412 appearing at the surface of purple membranes decreased from about 0.75 to 0.45 as the surface potential became more negative. The stoichiometry would be twice as large if the charge changes were localized exclusively on one side of the purple membrane. Laser flash-induced kinetics of the rise and decay of surface charge changes were slightly slower than the kinetics of the rise and decay of M412 which is associated with the reversible deprotonation of the retinal Schiff base nitrogen in the chromophore. It is suggested that the light-induced charge changes monitor a dissociable amino acid residue which may be a step in the movement of protons across the purple membrane.

— The precise molecular description of the time dependent steps in the uptake, translocation and release of protons by bacteriorhodopsin following photon absorption requires information on the time resolved changes in protonation of the side chains of specific amino acid residues and the correlation of these changes with photocycle kinetics. Thus far, the use of chemical modification to probe the role of amino acid side chains in this process has proven of value in demonstrating a role for tyrosine residues in release and uptake of protons associated with early and later stages (before and after M412 formation) of the photocycle. In addition, it has demonstrated the essential role of ionic interactions between negatively charged carboxyl groups and positively charged guanidinium groups of arginine, and amino groups of lysine. The transmembrane regulatory effect of ΔμH+ on the M412 species of the photocycle provides additional evidence for the participation of reversible protonation of amino acid side chains at the surfaces of the purple membrane in the mechanism of proton translocation. Thus, our studies relate molecular events of proton translocation to the bioenergetics of the purple membrane.

Coupling factor extracted from chromatophores of the photosynthetic bacteria Chromatium strain D was partially purified. The enzyme catalyzed ATPase activity in the presence of Ca2+ and Mg2+ ions. Higher Vapp values were obtained when the activity was measured as a function of the divalent cation-ATP complex rather than as a function of either the divalent cation or ATP because the free components competitively inhibited the activity in the presence of the cation-ATP complex. The Km values were lower than or equal to the Ki values for free ATP indicating that the cation-ATP complex is bound tighter than the free ATP to the enzyme. Based on these results a possible mode of binding of substrate to the active site of the enzyme was suggested. A comparative study indicated no changes in the temperature dependance of ATPase activity when the enzyme was solubilized. However, possible conformation changes could have caused a decrease in the Km values for the (Ca-ATP)2− and (Mg-ATP)2− and in the Ki for free Mg2+ ions and ATP. The Ki for free Ca2+ ions increased on solubilization of the coupling factor. ATPase activity was inhibited by dicyclohexylcarbodiimide both in the soluble and in the membrane-bound coupling factor.

Membrane vesicles (heavy chromatophores) prepared from the photosynthetic bacteria Rhodopseudomonas capsulata catalyze photophosphorylation of exogenous ADP and also take up [3H]ADP from the external medium. The rate of uptake depends on the concentration of external ADP reaching half-maximal velocity at 2.7 m m . The rate increases also with the increase in the concentration of internal ADP. Vesicles, preloaded with [3H]ADP release the radioactive nucleotide when ADP is included in the external medium. Regular chromatophores, which are inside-out membrane vesicles also take up [3H]ADP from the external medium when preloaded with ADP. These results are interpreted to indicate the existence of nucleotide transport across the cytoplasmic membrane of these bacteria which is catalyzed by an ADP exchange carrier.

Chromatophores from Rhodopseudomonas capsulata cells grown semiaerobically in the dark oxidize NADH, succinate, and dichlorophenolindophenol. In the presence of N3− these activities are inhibited, but light induces oxidation of dichlorophenolindophenol with O2 as a terminal electron acceptor. Cyanide also inhibits electron transport but much higher concentrations are required to inhibit the photooxidation than the dark oxidation. The photooxidation was studied in a mutant strain of Rhodopseudomonas capsulata (YIV) which cannot grow anaerobically in the light, but similarly to the wild type, grows in the presence of oxygen. Chromatophores from YIV mutant catalyze photophosphorylation and dark oxidation activities with the same properties as those of the wild type. However, the rate of photooxidation in the mutant is only one-third that of the wild type. Based on the differential inhibitor sensitivity and on the mutation it is suggested that the photooxidase is different from the two respiratory oxidases and that this photooxidation activity might be essential for growth of the cells under anaerobic conditions in the light.

Cytochrome c2 was removed by washing from heavy chromatophores prepared from Rhodopseudomonas capsulata cells. The easy removal of the cytochrome could indicate that it was attached on the outside of the membrane. Therefore, the membrane was probably oriented inside out in relation to the membrane of regular chromatophores, from which cytochrome c2 could not be removed. Washing of the heavy chromatophores caused loss of photphosphorylation activity. The activity was restored to the resolved heavy chromatophores by the supernatant obtained during the washing or by the native cytochrome c2, which was found to be the active component in this supernatant. The activity could not be restored by other c-type cytochromes. Ascorbate, which enhanced photophosphorylation activity in the heavy chromatophores at the optimal concentration of 8 m m , restored this activity to the washed heavy chromatophores, but at an optimum concentration of 50 m m . Cytochrome c2 and dichlorophenol indophenol reduced the optimum of the ascorbate concentration to 7 m m . This might indicate that the effect of ascorbate is mediated through cytochrome c2. Washing the heavy chromatophores caused 70% loss of the light-induced electron transport from ascorbate and from ascorbate-reduced dichlorophenol indophenol to O2. However, this effect was only observed with the lower concentrations of ascorbate and the dye. The activity was restored either by the supernatant obtained from the washing or by various c-type cytochromes, reduced by ascorbate. Washing the heavy chromatophores did not affect succinate oxidation in the dark. It is suggested that cytochrome c2 is one of the cytochromes catalyzing the photosynthetic cyclic electron transport, as has been seen from its high specificity in the reconstitution experiments. Light can induce oxidation of various c-type cytochromes and other redox reagents. However, reduction was specific for cytochrome c2 from Rps. capuslata, since it was the only one which could be both reduced and oxidized as required from a component which is part of a cyclic electron transport chain. It is also suggested that cytochrome c2 was not part of the succinate oxidase system.

Proton uptake by chloroplasts was induced by light-triggered ATPase activity. A quotient of two was obtained when the initial rate of proton uptake was divided by the rate of P i released from ATP. Gramicidin accelerated the rate of ATPase activity and reduced the H + /P i ratio to 1.4. The results were found to be consistent with the chemiosmotic theory.

1. 1. The rate of decay of the light-triggered state of ATPase in chloroplasts was decreased in the presence of Pi and accelerated by ADP in a phosphateless medium. Phosphate and arsenate inhibited the effect of ADP. 2. 2. Compared with other dinucleotides, ADP was found to be highly specific for the acceleration of the decay, having an apparent Km of 1.1·10−6 M. A lower apparent Km for ADP was obtained in the presence of phosphate. The apparent Km for the inhibition of the effect of ADP by Pi was lowered with increased concentrations of ADP. 3. 3. These and other effects on ATPase and ATP-Pi exchange activities were discussed in terms of changes in the permeability of ADP and Pi across the membrane of the chloroplast.

1. 1. Mg 2+ -ATPase (light-triggered Mg 2+ -dependent ATPase) activity in chloroplasts was stimulated by atebrin, NH 4 Cl and gramicidin when the uncouplers were added after light triggering. The stimulation was followed in time by inhibition when the reaction took place in the dark. 2. 2. This inhibition of Mg 2+ -ATPase activity was overcome when the reaction was carried out under continuous illumination. 3. 3. The energy transfer inhibitors of photophosphorylation, phlorizin and Dio-9, inhibited Mg 2+ -ATPase activity and the extent of inhibition increased with time. The inhibition by Dio-9 was partially reversed by light while that of phlorizin was not. 4. 4. Light-triggered ATP-P i exchange activity in chloroplasts was inhibited by both atebrin and phlorizin. The extent of the inhibition increased with time. 5. 5. The activity of a soluble Ca 2+ -ATPase was inhibited by Dio-9, phlorizin, NH 4 Cl and atebrin. The kinetics of activity was linear with time, except in the presence of phlorizin. 6. 6. These results are interpreted as indicating a requirement of a high-energy state for triggering and maintenance of Mg 2+ -ATPase and ATP-P i exchange reactions. The relation of ATPase activity to the coupling mechanism in chloroplasts is discussed.

The requirement for ADP and Pi in exchange reactions in chloroplasts was assumed to indicate that the reactions leading to ATP synthesis do not involve a covalently bound enzyme intermediate [l] . It is known, however, that the chloroplast membrane is rather impermeble to various anions and cations [2]. Therefore, the reactants concentration at the catalytic site is not necessarily reflected by their concentration in the outside medium. In this work a disparity between the internal and external concentrations of ADP and Pi was indicated by the effect which the addition and the removal of these reagents had on light-triggered ATPase and on ATP-Pi exchange reactions in chloroplasts. The requirements for ADP and Pi in light-triggered ATP-Pi exchange reaction indicated that the exchange was a result of a dynamic reversal of photophosphorylation.

The cell envelope of Gram-negative bacteria such as Rhodopseudomonas capsulata contains an inner cytosplasmic membrane and an outer membrane [ 11. The space between the two membranes is the periplasma, which contains the periplasmic proteins and enzymes [2]. Several of the reactions catalyzed by the periplasmic enzymes, such as cell wall and capsule synthesis, require energy. The possible function of specific transport systems for UDP-glucose was suggested [3] . However, a general mechanism for energy transfer from the cytoplasm to the periplasm was not found. In photosynthetic bacteria both oxidative and photosynthetic phosphorylation systems are located in the cytoplasmic membranes [4] and the reversible ATPase is attached on the cytoplasmic side of this membrane [ 51. A system which could translocate nucleotides across the cytoplasmic membrane would provide energy for the periplasmic enzymes. The outer membrane was found to exclude macromolecules [l] and even oligosaccharides having molecular dimensions larger than 600 daltons [6]. Except for the case of Bdellovibrio bacteriovants [7] nucleotides were found to be excluded from bacteria [8] . It is possible, therefore, that the outer membrane could serve as a barrier to prevent the diffusion of nucleotides from the periplasmic space to the outer medium. The possible existence of nucleotide transport across the cytoplasmic membrane was indicated from the finding of photophosphorylation of exogenous ADP to ATP by heavy chromatophores [9]. These vesicles, prepared from Rhodopseudomonas capsulata, retain the membrane polarity of the intact cell as

Coupling factor 1 (CFI) from chloroplasts is directly involved in the terminal steps of photophosphorylation. The enzyme can also catalyze ATPase activity which is probably a result of the reversal of ATP synthesis. However, ATPase activity is latent both in the membrane bound and in the isolated soluble pro

Purple membranes from Halobacterium halobium contain bacteriorhodopsin which is a light-driven electrogenic proton pump [ 11. After light excitation the chromophore which is retinal-bound through a Schiff base to a lysyl residue in the opsin, forms several photointermediates (designated K, L, M, 0) returning to the ground state at the end of the cycle. The major photochemical and biochemical events involve isomerization of the retinal from all-pans to 13-cis [2] and dissociation of a proton from the retinal Schiff base [3]. The electrogenic transfer of protons should also involve charge separation within and across the protein. Measurements of photoelectric transient response in oriented purple membranes were assumed to reflect these changes. The experimental systems included either purple membranes or bacteriorhodopsin liposomes attached to lipid-impregnated filters, lipid bilayer, teflon films or multilayers of purple membranes [4-71. We have used molecular probes to monitor surface potential changes in purple membranes [8,9]. Spin probes were used to measure the photostationary and pulse-induced surface potential charges. However, because of the relatively low sensitivity and the slow response of EPR, the measurements were performed only under experimental conditions which induced a slowdown in the photocycle. Here, we have developed a highly sensitive optical method having a fast time resolution. The method involves measurements of absorption changes in a pH indicator adsorbed to the purple membranes suspended in a highly buffered solution. It was shown [lo] that fixed charges alter the apparent pK, of adsorbed indicators. Measurements of the kinetics of

washed in 0.1 M tricine-NaOH, pH 7.8 and stored under nitrogen at -2O’C. The cells were ground with alumina in 0.1 M tricine-NaOH, pH 7.8, then centrifuged at 12 000 X g for 15 min to remove debris. The chromatophores were sedimented by cen- trifugation at 144 000 X g for 1 h, resuspended in a small volume of a solution containing 0.1 M tricine

In most ATPases the substrate is found in the form of a complex of ATP, with a metal ion, such as Ca++ or Mg++. We have therefore attempted to replace these labile metal ions by Co3+ which, by virtue of the inertness of its complexes, could remain attached to its ligands.

Coupling factor, isolated from lettuce chloroplasts, contained several binding sites for Mn2+ ions. Three of these sites showed strong cooperative interactions having a Hill coefficient of 2.9 +/- 0.20 and a Kd of 14.7 +/- 0.44 microM. Three additional non-interacting Mn2+-binding sites were found with a Kd of 46.7 +/- 2.3 microM. Chemical modification with naphthylglyoxal of 1 arginyl residue/chloroplast coupling factor 1, which inhibited ATPase activity, inhibited the cooperativity among the sites but did not prevent Mn2+ binding to the enzyme. It is suggested that the cooperative interaction among the Mn2+-binding sites is an expression of the interaction among the active sites of the enzyme which is required for catalysis.

Two fractions of membrane preparations, a heavy and a light one were isolated from mildly broken Rhodopseudomonas capsulata cells. The light fraction which contained vesicles similar to the regular chromatophores obtained by sonication and a heavy fraction which appeared in electron micrographs to consist of cell fragments which were designated as heavy chromatophores and were composed of broken cell envelopes containing closely packed vesicles enclosed within the cytoplasmic membrane. Both types of chromatophores catalyzed photophosphorylation. However, cytochrome c2 could be washed out only from the heavy chromatophores. Photophosphorylation activity which was lost by the removal of the cytochrome could be restored by addition of either cytochrome c2 or phenazine methosulphate. Light induced proton efflux in heavy chromatophores in contrast to proton influx in regular chromatophores. The washed heavy chromatophores did not lose the light induced proton movement. Light induced quenching of 9-aminoacridine and atebrin fluorescence in chromatophores, while the fluorescence was enhanced in the heavy chromatophores. The washing did not affect the fluorescence changes of the heavy chromatophores but caused a reduction of the steady state of the carotenoid absorbance shift. It is suggested that the membrane in the heavy chromatophores is oriented inside out with respect to the membrane in regular chromatophores. Cytochrome c2 which is attached to that side of the membrane facing the outside medium could be removed from the heavy chromatophores and reconstituted to them. The role of cytochrome c2 in photophosphorylation is discussed.

The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O 2 . Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c , K 4 Fe(CN) 6 , 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c 2 from the heavy chromatophores caused a decrease in the light-induced O 2 uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c 2 from Rps. capsulata . It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c 2 level which faces the outside medium. However, cytochrome c 2 is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c 2 in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O 2 whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O 2 consumed is 4, which means that the end product of O 2 reduction is H 2 O.

1 Chromatophores obtained from the photosynthetic bacteria Chromatium strain D catalyzed Mg2+ and Ca2+-dependent ATP hydrolysis. The rate of ATPase activity was lower than the rate of photophosphorylation. 2 Tryptic digestion of the Chromatophores activated both Mg2+- and Ca2+-dependent ATPase activities while inhibiting only 15% of the phosphorylation. 3 Incubation of the Chromatophores in a low salt medium (5 mM tricine-NaOH), followed by centrifugation, yielded resolved Chromatophores which completely lost their capacity for photophosphorylation and most of their Mg2+- and Ca2+-dependent ATPase activities. Incubation of the resolved particles with the supernatant fluid obtained after centrifugation, in the presence of Mg2+, restored photophosphorylation and ATPase activity. 4 The soluble fraction (the crude coupling factor) had a low Mg2+- and Ca2+-dependent ATPase activities. Both ATPase activities in the crude coupling factor could be activated by trypsin and inhibited by N,N′-dicyclohexylcarbodiimide. 5 There was a distinct difference in the requirements for ATP and cations between the membrane-bound and the soluble ATPase The effect of the changes in the environment on the mode of binding of the substrate to the enzyme is discussed.

1. Proton uptake was induced by ATP in the dark following light triggering of ATPase activity in chloroplasts. The accumulated protons were released when ATPase activity was inhibited by the energy transfer inhibitor DIO-9. 2. Approximately two protons were taken up for each ATP hydrolyzed at pH 8. A drop in H+/ATP ratio was caused by uncouplers such as NH4Cl and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These uncouplers caused an increase in the rate of ATP hydrolysis without a corresponding increase in proton uptake. 3. The energy transfer inhibitor dicyclocarbodiimide inhibited both ATPase activity and the rate of proton uptake without changing the H+/ATP ratio. 4. The antibiotic valinomycin caused an increase in the rate of both proton uptake and ATP hydrolysis without altering the ratio of H+/ATP. The H+/ATP ratio varied with changes in the external pH. The results were discussed in view of the chemiosmotic theory of oxidative and photosynthetic phosphorylation.