Papers by Chanoch Carmeli
PubMed, Dec 1, 2000
Isolated chloroplast ATP synthase (CF0F1) was used for determination of the structure-function re... more Isolated chloroplast ATP synthase (CF0F1) was used for determination of the structure-function relation by measuring the effect of divalent metal ions on the properties of ATPase. Mg2+ ions were more efficient catalysts than Ca2+ ions as indicated by Kcat/Km of 55.2 and 5.4, respectively. Other activity parameters related to binding, such as the Km of MATP and Ki of MADP, indicated a stronger binding in the presence of Mg2+ as seen from a Mg2+/Ca2+ ratio of 2.8 and 3.8, respectively. Strong binding of Ca2+ ions with a Kd of 0.03 +/- 00.6 microM-1 was detected only in the presence of ADP probably because of the positive interactive effect of CaADP as indicated in the inhibition properties. Mg2+ ions were more efficient catalysts also in other forms of the enzyme such as in the thylakoid membrane, in isolated CF0F1 and in CF1. The Mg2+/Ca2+ ratio of Kcat/Km was 5.3, 10.2 and 1.5 for the thylakoid membrane enzyme, the isolated CF0F1 and the soluble CF1 respectively. This indicated that Ca2+ ions became less efficient catalysts in the more intact and integrated enzyme while Mg2+ ions were as efficient in all forms of the enzyme. Unlike Mg2+, Ca2+ ions also did not support proton-coupled ATP synthesis and ATP driven proton pumping. It is suggested that the differences in the ligand structure of these two ions might be the reason for the differential function. An average 0.3 A shorter bond length of octahedral first coordination in Ca2+ ions caused a weaker binding of CaATP than that of MgATP. The effect of differential binding is discussed in relation to the binding of the transition state intermediate and to the rate of product release.
Biochimica Et Biophysica Acta - Bioenergetics, Feb 1, 2009
Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, ... more 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 Å and 2.69 Å, 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 Å. 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.
Journal of Biological Chemistry, 1993
The activity of CF1-ATPase was inhibited by vanadate in an allosteric manner with respect to CaAT... more 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 Caa+ ions and of free divalent metal ions during assay of the activity. The strongest cooperative interaction with a Hill coefficient of 5.3 f 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 K , of 12.4 f 1.2 I.~M for vanadate inhibition was determined under these conditions. Direct measurements with the aid of 'lV NMR indicated that vanadate binds to CFl in the presence of Ca2+ and ADP in a positive cooperative manner with a Hill coefficient of 2.3 f 0.2 and an average & of 0.3 f 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 CFl. The proton-translocating reversible ATPase contains two major sections: hydrophobic proteins (CFo), situated in the membrane and serving as a proton channel, to which coupling factor 1 (CF,)' is attached (1). The catalytic sector (CF,) of the chloroplast H+-ATPase is composed of five types of subunits designated 3a, 3p, y, 6, and € (2). Several lines of evidence indicating the /3 subunits as the possible location of the active site included a high sequence homology of the , f 3 subunits from various organisms, a conserved region in the vicinity of the nucleotide binding sequence (3), and affinity labeling studies with ADP and ATP analogues (4). CF, was suggested to have a total of six nucleotide binding sites (4,5). Three of the sites were assigned to the p subunits and three to the a at the interface of the /3 subunits. Based on specificity, capacity to synthesize (6) or to hydrolyze (7) ATP, and affinity labeling studies (8), the catalytic sites were suggested to be on the /3 subunits while noncatalytic sites were assigned to the a subunits. The mechanism of the ATP synthase is probably described best by the "alternating site" hypothesis (9). ATP is formed from tightly bound ADP and Pi or hydrolyzed from bound ATP, alternately, at the three catalytic sites on the F,. The release of the product is induced by a confor-*This work was supported by Grant 706/92 from The Israel Academy of Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: CFI, coupling factor 1 of chloroplast H*-ATPase; Tricine, N-tris(hydroxymethy1)methylglycine; MOPS, 3-(N-morpholino)propanesulfonic acid.
Journal of Synchrotron Radiation, 1999
The structure of vanadate, a phosphate analogue which functions in the presence of tightly bound ... more The structure of vanadate, a phosphate analogue which functions in the presence of tightly bound ADP and divalent cations as a transition state inhibitor of CFI-ATPase, was investigated by X-ray absorption spectroscopy. There was a decrease in the intensity of the pre-edge transition and a change in the shape and energy of the K-edge upon binding of ADP-vanadate-Mg(II) to CF 1. The changes were due to alterations in the structure of vanadium from tetrahedral to five-coordinated trigonal bipyramidal geometry. Simulation of the edge shape and energies and EXAFS analysis confirmed the presence of pentacoordinated vanadium bound to the enzyme. This structure was analogous to the proposed transition state of the phosphate during the synthesis and the hydrolysis of ATP by CF 1 .
FEBS Letters, 1970
Proton uptake by chloroplasts was induced by light-triggered ATPase activity. A quotient of two w... more 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 Pi released from ATP. Gramicidin accelerated the rate of ATPase activity and reduced the H+/Pi ratio to 1.4. The results were found to be consistent with the chemiosmotic theory.
FEBS Letters, 1978
Successful attempts have been made to separate coupling factor from chromatophores of several pho... more Successful attempts have been made to separate coupling factor from chromatophores of several photosynthetic bacteria [l-.5]. Highly-purified coupling factor which contained five subunits was obtained only from Rh. rubrum [6]. We have reported [7] on the properties of a partially-purified coupling factor from Chromatium chromatophores. It is the purpose of this work to present a procedure for the preparation of a highly purified ATPase from Chromatium and to describe its properties. 2. Materials and methods 2.1. Isolation of chromatophores Chromatium strain D bacteria were grown anaerobically in the light on the medium [8] supplemented with 0.2% malate for 4 days at 32°C. The harvested bacteria were 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 centrifugation at 144 000 X g for 1 h, resuspended in a small volume of a solution containing 0.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, ... more 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 Å and 2.69 Å, 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 Å. 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.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2002
Intra-subunit interactions in the environment of the iron-sulfur cluster F X in photosystem I (PS... more 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, a conserved aspartate (D566) adjacent to one of the cysteine ligands of F X C565 was replaced by glutamate. The resulting D566E mutation in subunit PsaB of the heterodimer core prevented functional assembly of PS I in the thylakoids of the cyanobacterium. A stable functional PS I containing all three iron-sulfur centers, F X and F A/B, was assembled in the second site-suppressor mutant D566E/L416P. A modified yet stable PS I was also formed in a second site-directed mutant C565S/D566E. The double mutants C556S/D566E, C556H/D566E and C565H/D566E did not assemble PS I. Cells with the mutations C565S/D566E and D566E/L416P grew autotrophically in light. This is first report of selections of a second site-suppressor mutation and a second site-directed mutation within the subunit that rectified deficiencies caused by mutations in PsaB. The results may suggest that a stable F X was required for assembly of PS I. It is suggested that the stability of F X is supported by a salt bridge formed between D566, which is adjacent to one of the cysteine ligands of the iron-sulfur cluster, and R703. The positively charged residue is stabilized through short-and long-range interactions of the inter-helical loops between helices VIII-IX (h-i), X-XI (j-k) and VI-VII (f-g), respectively.
Langmuir, 2020
In biological systems, membrane proteins play major roles in energy conversion, transport, sensin... more 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.
This report was done with support from the Department of Energy. Any conclusions or opinions expr... more This report was done with support from the Department of Energy. Any conclusions or opinions expressed in this report represent solely those of the author(s) and not necessarily those of The Regents of the University of California. the Lawrence Berkeley Laboratory or the Department of Energy. Reference to a company or product name does not imply approval or recommendation of the product by the University of California or the U.S. Department of Energy to the exclusion of others that may be suitable.
AIP Conference Proceedings, 2010
We present a chemical route to covalently couple the photosystem I (PS I) to carbon nanotubes (CN... more We present a chemical route to covalently couple the photosystem I (PS I) to carbon nanotubes (CNTs). Small linker molecules are used to connect the PS I to the CNTs. Hybrid systems, consisting of CNTs and the PS I, promise new photo-induced transport phenomena due to the outstanding electro-optical properties of the robust cyanobacteria membrane protein PS I.
The Journal of Physical Chemistry C, 2008
The electronic coupling between the photoactive proteins and semiconductors can be used for fabri... more 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.
Proceedings of the National Academy of Sciences, 1980
The surface potential of purple membrane fragments, determined from the distribution of the aqueo... more 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...
Nano Letters, 2009
An extremely fast electron transfer through an electronically coupled junction between covalently... more 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.
Journal of the American Chemical Society, 2007
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Papers by Chanoch Carmeli