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Marcellus Ubbink - One of the best experts on this subject based on the ideXlab platform.
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loss oF electrostatic interactions causes increase oF dynamics within the plastocyanin Cytochrome F complex
Biochemistry, 2013Co-Authors: Sandra Scanu, Johannes M Foerster, Monika Timmer, Matthias G Ullmann, Marcellus UbbinkAbstract:: Recent studies on the electron transFer complex Formed by Cytochrome F and plastocyanin From Nostoc revealed that both hydrophobic and electrostatic interactions play a role in the process oF complex Formation. To study the balance between these two types oF interactions in the encounter and the Final state, the complex between plastocyanin From Phormidium laminosum and Cytochrome F From Nostoc sp. PCC 7119 was investigated using NMR spectroscopy and Monte Carlo docking. Cytochrome F has a highly negative charge. Phormidium plastocyanin is similar to that From Nostoc, but the net charge oF the protein is negative rather than positive. NMR titrations oF Zn-substituted Phormidium plastocyanin and Nostoc Cytochrome F indicated that a complex with an aFFinity intermediate between those oF the Nostoc and Phormidium complexes is Formed. Plastocyanin was Found in a head-on orientation, as determined using pseudocontact shiFts, similar to that in the Phormidium complex, in which the hydrophobic patch represents the main site oF interaction on plastocyanin. However, the interaction in the cross-complex is dependent on electrostatics, similar to that in the Nostoc complex. The negative charge oF plastocyanin decreases, but not abolishes, the attraction to Cytochrome F, resulting in the Formation oF a more diFFuse encounter complex than in the Nostoc case, as could be determined using paramagnetic relaxation spectroscopy. This work illustrates the subtle interplay oF electrostatic and hydrophobic interactions in the Formation oF transient protein complexes. The results are discussed in the context oF a model For association on the basis oF hydrophobic contacts in the encounter state.
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role oF hydrophobic interactions in the encounter complex Formation oF the plastocyanin and Cytochrome F complex revealed by paramagnetic nmr spectroscopy
Journal of the American Chemical Society, 2013Co-Authors: Sandra Scanu, Johannes M Foerster, Matthias G Ullmann, Marcellus UbbinkAbstract:Protein complex Formation is thought to be at least a two-step process, in which the active complex is preceded by the Formation oF an encounter complex. The interactions in the encounter complex are usually dominated by electrostatic Forces, whereas the active complex is also stabilized by noncovalent short-range Forces. Here, the complex oF Cytochrome F and plastocyanin, electron-transFer proteins involved in photosynthesis, was studied using paramagnetic relaxation NMR spectroscopy. Spin labels were attached to Cytochrome F, and the relaxation enhancements oF plastocyanin nuclei were measured, demonstrating that a large part oF the Cytochrome F surFace area is sampled by plastocyanin. In contrast, plastocyanin is always oriented with its hydrophobic patch toward Cytochrome F. The complex was visualized using ensemble docking, showing that the encounter complex is stabilized by hydrophobic as well as electrostatic interactions. The results suggest a model oF electrostatic preorientation beFore the prote...
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Handbook oF Metalloproteins - Cytochrome F/Plastocyanin Complex
Handbook of Metalloproteins, 2006Co-Authors: Marcellus UbbinkAbstract:In the photosynthetic electron transFer chain in plants, Cytochrome F, a subunit oF the Cytochrome b6F complex, acts as electron donor For plastocyanin. Plastocyanin is a small soluble protein, which shuttles electrons to photosystem 1. The structure oF the short-lived and weak complex oF the soluble domain oF Cytochrome F and plastocyanin has been determined using nuclear magnetic resonance. It shows that the heme iron oF Cytochrome F and the copper ion oF plastocyanin are in close proximity, enabling Fast electron transFer. The copper ligand His87 is located close to the iron ligand Tyr1, suggesting that electron transFer occurs via the hydrophobic patch oF plastocyanin. Electrostatic interactions are also important in the complex. These Findings are discussed in the light oF studies oF the electron transFer kinetics. 3D Structure Keywords: electron transFer; Cytochrome F; plastocyanin; photosynthesis; paramagnetic; NMR
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the transient complex oF poplar plastocyanin with Cytochrome F eFFects oF ionic strength and ph
Biochimica et Biophysica Acta, 2005Co-Authors: Christian Lange, Irene Diazmoreno, Tobias Cornvik, Marcellus UbbinkAbstract:Abstract The orientation oF poplar plastocyanin in the complex with turnip Cytochrome F has been determined by rigid-body calculations using restraints From paramagnetic NMR measurements. The results show that poplar plastocyanin interacts with Cytochrome F with the hydrophobic patch oF plastocyanin close to the heme region on Cytochrome F and via electrostatic interactions between the charged patches on both proteins. Plastocyanin is tilted relative to the orientation reported For spinach plastocyanin, resulting in a longer distance between iron and copper (13.9 A). With increasing ionic strength, From 0.01 to 0.11 M, all observed chemical-shiFt changes decrease uniFormly, supporting the idea that electrostatic Forces contribute to complex Formation. There is no indication For a rearrangement oF the transient complex in this ionic strength range, contrary to what had been proposed earlier on the basis oF kinetic data. By decreasing the pH From pH 7.7 to pH 5.5, the complex is destabilized. This may be attributed to the protonation oF the conserved acidic patches or the copper ligand His87 in poplar plastocyanin, which are shown to have similar p K a values. The results are interpreted in a two-step model For complex Formation.
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Plastocyanin-Cytochrome F interactions: the inFluence oF hydrophobic patch mutations studied by NMR spectroscopy.
Biochemistry, 2002Co-Authors: Peter B Crowley, Nadejda Vintonenko, George S. Bullerjahn, Marcellus UbbinkAbstract:Transient complex Formation between plastocyanin From Prochlorothrix hollandica and Cytochrome F From Phormidium laminosumwas investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived From NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant oF 6 ((2) 10 3 M -1 For complex Formation, 1 order oF magnitude larger than that For the physiological plastocyanin-Cytochrome F complex From Ph. laminosum. Chemical- shiFt perturbation mapping indicates that the hydrophobic patch oF plastocyanin is involved in the complex interFace. When the unusual hydrophobic patch residues oF P. hollandica plastocyanin were reverted to the conserved residues Found in most other plastocyanins (Y12G/P14L), the binding constant For the interaction with Cytochrome F was unaFFected. However, the chemical shiFt perturbation map was considerably diFFerent, and the size oF the average perturbation decreased by 40%. The complexes oF both the wild-type and double mutant plastocyanin with Cytochrome F were sensitive to ionic strength, contrary to the physiological complex. The possible implications oF these Findings For the mechanism oF transient complex Formation are discussed.
A. B. Rubin - One of the best experts on this subject based on the ideXlab platform.
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comparative analysis oF plastocyanin Cytochrome F complex Formation in higher plants green algae and cyanobacteria
Physiologia Plantarum, 2019Co-Authors: V.a. Fedorov, I. B. Kovalenko, D. M. Ustinin, S. S. Khruschev, Taras Kornelievich Antal, G. Yu. Riznichenko, A. B. RubinAbstract:: Mechanisms oF the complex Formation between plastocyanin and Cytochrome F in higher plants (Spinacia oleracea and Brassica rapa), green microalgae Chlamydomonas reinhardtii and two species oF cyanobacteria (Phormidium laminosum and Nostoc sp.) were investigated using combined Brownian and molecular dynamics simulations and hierarchical cluster analysis. In higher plants and green algae, electrostatic interactions Force plastocyanin molecule close to the heme oF Cytochrome F. In the subsequent rotation oF plastocyanin molecule around the point oF electrostatic contact in the vicinity oF Cytochrome F, copper (Cu) atom approaches Cytochrome heme Forming a stable conFiguration where Cytochrome F molecule behaves as a rather rigid body without conFormational changes. In Nostoc plastocyanin molecule approaches Cytochrome F in a diFFerent orientation (head-on) where the stabilization oF the plastocyanin-Cytochrome F complex is accompanied by the conFormational changes oF the G188E189D190 loop that stabilizes the whole complex. In cyanobacterium P. laminosum, electrostatic preorientation oF the approaching molecules was not detected, thus indicating that random motions rather than long-range electrostatic interactions are responsible For the proper mutual orientation. We demonstrated that despite the structural similarity oF the investigated electron transport proteins in diFFerent photosynthetic organisms, the complexity oF molecular mechanisms oF the complex Formation increases in the Following sequence: non-heterocystous cyanobacteria - heterocystous cyanobacteria - green algae - Flowering plants.
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The identiFication oF intermediate states oF the electron-transFer proteins plastocyanin and Cytochrome F diFFusional encounters
Biophysics, 2015Co-Authors: S. S. Khruschev, I. B. Kovalenko, A. M. Abaturova, G. Yu. Riznichenko, V.a. Fedorov, A. B. RubinAbstract:The Brownian dynamics method was used For qualitative analysis oF the events that lead to the Formation oF a Functionally active plastocyanin–Cytochrome F complex. The intermediate states oF this process were identiFied by density-based hierarchical clustering. DiFFusive entrapment oF plastocyanin by Cytochrome F is the key point oF the suggested putative scenario oF protein–protein encounter. The motion oF plastocyanin was characterized For diFFerent values oF protein–protein electrostatic interaction energy.
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multiparticle computer simulation oF plastocyanin diFFusion and interaction with Cytochrome F in the electrostatic Field oF the thylakoid membrane
Biophysics, 2010Co-Authors: O S Knyazeva, I. B. Kovalenko, A. M. Abaturova, E. A. Grachev, Yu G Riznichenko, A. B. RubinAbstract:A multiparticle computer model oF plastocyanin-Cytochrome F complex Formation in the thylakoid lumen has been designed, which takes into account the electrostatic interactions oF proteins and membrane. The Poisson-Boltzmann Formalism was used to determine the electrostatic potentials oF the electron carrier proteins and the thylakoid membrane at diFFerent ionic strengths. The membrane electrostatic Field was shown to inFluence plastocyanin diFFusion and interaction with Cytochrome F. The rate constants For plastocyanin-Cytochrome F complex Formation were calculated as a Function oF ionic strength and membrane surFace charge.
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Direct simulation oF plastocyanin and Cytochrome F interactions in solution
Physical Biology, 2006Co-Authors: I. B. Kovalenko, A. M. Abaturova, P. A. Gromov, D. M. Ustinin, E. A. Grachev, G. Yu. Riznichenko, A. B. RubinAbstract:Most biological Functions, including photosynthetic activity, are mediated by protein interactions. The proteins plastocyanin and Cytochrome F are reaction partners in a photosynthetic electron transport chain. We designed a 3D computer simulation model oF diFFusion and interaction oF spinach plastocyanin and turnip Cytochrome F in solution. It is the First step in simulating the electron transFer From Cytochrome F to photosystem 1 in the lumen oF thylakoid. The model is multiparticle and it can describe the interaction oF several hundreds oF proteins. In our model the interacting proteins are represented as rigid bodies with spatial Fixed charges. Translational and rotational motion oF proteins is the result oF the eFFect oF stochastic Brownian Force and electrostatic Force. The Poisson-Boltzmann Formalism is used to determine the electrostatic potential Field generated around the proteins. Using this model we studied the kinetic characteristics oF plastocyanin-Cytochrome F complex Formation For plastocyanin mutants at pH 7 and a variety oF ionic strength values.
Derek S Bendall - One of the best experts on this subject based on the ideXlab platform.
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the interaction between Cytochrome F and plastocyanin or Cytochrome c 6
2016Co-Authors: Derek S Bendall, Christopher J. HoweAbstract:Cytochrome F is bound to the Cytochrome b6F complex embedded in the thylakoid membrane, but plastocyanin is a small protein that diFFuses between Cytochrome F and photosystem I in the thylakoid lumen. The essential Feature oF electron transFer From Cytochrome F to plastocyanin is that a weak, transient complex is Formed so as to allow both rapid Formation oF the reaction complex, and rapid dissociation oF products. The reaction has been studied with the proteins in solution, using preparations oF the large, heme-containing luminal domain oF Cytochrome F, by kinetic methods, NMR and computer simulation. The reaction sites have been identiFied as small areas oF hydrophobic residues on the surFace immediately above the heme or Cu atom and including ligand residues (Tyr1 For Cytochrome F and His87 For plant plastocyanin). A model oF the reaction has been developed in which an intermediate encounter complex is Formed covering a relatively large area oF the surFace and serving the purpose oF steering the proteins towards a reactive conFiguration. With plant and algal proteins the main attractive Force Forming the encounter complex is electrostatic, but the reactive complex depends on desolvation energy to which electrostatics can make little contribution. With cyanobacterial proteins there is considerable species variation. Hydrophobic attraction plays a signiFicant role in Formation oF the encounter complex as well as the reaction complex and sometimes may dominate. NMR relaxation studies and Brownian dynamics simulations suggest that rather than a single reaction complex, several (encounter) conFigurations contribute to ket. There are additional Features governing the reaction in vivo. Plastocyanin diFFuses within the tight luminal space which Forms a sheet no more than about 10 nm thick, obstructed by the large luminal domains oF photosystem II and other complexes. Solution studies with added viscogens suggest that other soluble luminal proteins would cause little inhibition. The lumen is now thought to expand in the light, contrary to previous evidence, raising the intriguing possibility that expansion and contraction oF the thylakoid lumen is a signiFicant method oF regulation oF electron transport. The role oF electrostatic attraction in vivo has been tested with Chlamydomonas mutants. The surprising result was that charge neutralisation had very little eFFect on either cell growth or the rate oF oxidation oF Cytochrome F, suggesting that the dominant attraction Forming the encounter complex in vivo is probably hydrophobic in all oxygenic organisms.
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The UnFinished Story oF Cytochrome F.
Photosynthesis research, 2004Co-Authors: Derek S BendallAbstract:Cytochrome F is a unique, integral membrane protein. The background to its discovery by Robert Hill (1899-1991) and Ronald Scarisbrick over 60 years ago and the inFluence oF David Keilin (1887-1963) and Frederick Gowland Hopkins (1861-1947) are discussed. The development oF methods For isolating Cytochrome F is outlined, emphasizing the remarkable achievement oF Hill and Scarisbrick at a time when Few iF any membrane proteins had been isolated, and the importance oF the discovery oF a natural proteolysis in Brassica spp., stimulated by organic solvents, by Eijiro Yakushiji and coworkers and by Masa-aki Takahashi and Kozi Asada in 1975. The signiFicance oF diFFerent types oF instrumentation in the study oF Cytochrome F is discussed, drawing attention to the importance oF the microspectroscope ocular For its discovery, to types oF spectrophotometer developed especially by Britton Chance For spectrophotometric measurements on turbid suspensions oF cells and plastids, and to the history oF stopped-Flow spectrophotometry. The stopped-Flow instrument originated in the bucket-scale Flow methods oF Hartridge and Roughton (1923), and was later developed on the microscale by Chance. Finally, the problems that remain For understanding the behavior oF Cytochrome F in the thylakoid lumen are contrasted with the signiFicance oF in vitro studies that provide a paradigm For transient protein-protein interactions in the wider Field oF biology as a whole.
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relation between interFace properties and kinetics oF electron transFer in the interaction oF Cytochrome F and plastocyanin From plants and the cyanobacterium phormidium laminosum
Biochemistry, 2003Co-Authors: Beatrix G Schlarbridley, Derek S Bendall, Christopher J. HoweAbstract:Cytochrome F and plastocyanin From the cyanobacterium Phormidium laminosum react an order oF magnitude Faster than their counterparts From chloroplasts when long-range electrostatic interactions have been screened out by high salt concentration [Schlarb-Ridley, B. G., et al. (2002) Biochemistry 41, 3279-3285]. To investigate the relative contributions oF the reaction partners to these diFFerences, the reactions oF turnip Cytochrome F with P. laminosum plastocyanin and P. laminosum Cytochrome F with pea plastocyanin were examined. Exchanging one oF the plant reaction partners with the corresponding cyanobacterial protein nearly abolished electron transFer at low ionic strength but increased the rate at high ionic strength. This increase was larger For P. laminosum Cytochrome F than For P. laminosum plastocyanin. To identiFy molecular Features oF P. laminosum Cytochrome F that contribute to the increase, the eFFect oF mutations in the N-terminal heme-shielding peptide on the reaction with P. laminosum plastocyanin was determined. Phenylalanine-3 was converted to valine and tryptophan-4 to phenylalanine or leucine. The mutations lowered the rate constant at 0.1 M ionic strength by Factors oF 0.71 For F4V, 0.42 For W4F, and 0.63 For W4L while introducing little change in the shape oF the ionic strength dependence curve. When the N-terminal tetrapeptide (sequence YPFW) was converted into that Found in the chloroplast oF Chlamydomonas reinhardtii (YPVF), the reaction was slowed Further (Factor oF 0.26). The N-terminal heme-shielding peptide was Found to be responsible For 75% oF the kinetic diFFerences between Cytochrome F From chloroplasts and the cyanobacterium when electrostatic interactions were eliminated.
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role oF charges on Cytochrome F From the cyanobacterium phormidium laminosum in its interaction with plastocyanin
Biochemistry, 2003Co-Authors: Sarah E Hart, Beatrix G Schlarbridley, Derek S Bendall, Christine Delon, Christopher J. HoweAbstract:The role oF charge on the surFace oF Cytochrome F From the cyanobacterium Phormidium laminosum in the reaction with plastocyanin was investigated in vitro using site-directed mutagenesis. Charge wa...
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Tryptophan-heme π-electrostatic interactions in Cytochrome F oF oxygenic photosynthesis
Biochemistry, 2000Co-Authors: Mikhail V. Ponamarev, Derek S Bendall, Christopher J. Carrell, Beatrix G. Schlarb, Christopher J. Howe, Janet L. Smith, William A. CramerAbstract:Cytochrome F oF oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures oF the chloroplast and cyanobacterial Cytochromes F were compared to explain spectral and redox diFFerences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis oF Cytochrome F From the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 → Trp substitution in the N-terminal domain was unique in causing a red shiFt oF 1 and 2 nm in the Cytochrome Soret (γ) and Q (α) visible absorption bands, respectively. The resulting α band peak at 556 nm is characteristic oF the cyanobacterial Cytochrome. Conversely, a Trp4 → Phe mutation in the expressed Cytochrome From the cyanobacterium Phormidium laminosum caused a blue ...
Janet L. Smith - One of the best experts on this subject based on the ideXlab platform.
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Interruption oF the internal water chain oF Cytochrome F impairs photosynthetic Function.
Biochemistry, 2000Co-Authors: G. Sainz, William A. Cramer, Christopher J. Carrell, M. V. Ponamarev, G. M. Soriano, Janet L. SmithAbstract:The structure oF Cytochrome F includes an internal chain oF Five water molecules and six hydrogen-bonding side chains, which are conserved throughout the phylogenetic range oF photosynthetic organisms From higher plants, algae, and cyanobacteria. The in vivo electron transFer capability oF Chlamydomonas reinhardtii Cytochrome F was impaired in site-directed mutants oF the conserved Asn and Gln residues that Form hydrogen bonds with water molecules oF the internal chain [Ponamarev, M. V., and Cramer, W. A. (1998) Biochemistry 37, 17199-17208]. The 251-residue extrinsic Functional domain oF C. reinhardtii Cytochrome F was expressed in Escherichia coli without the 35 C-terminal residues oF the intact Cytochrome that contain the membrane anchor. Crystal structures were determined For the wild type and three "water chain" mutants (N168F, Q158L, and N153Q) having impaired photosynthetic and electron transFer Function. The mutant Cytochromes were produced, Folded, and assembled heme at levels identical to that oF the wild type in the E. coli expression system. N168F, which had a non-photosynthetic phenotype and was thus most aFFected by mutational substitution, also had the greatest structural perturbation with two water molecules (W4 and W5) displaced From the internal chain. Q158L, the photosynthetic mutant with the largest impairment oF in vivo electron transFer, had a more weakly bound water at one position (W1). N153Q, a less impaired photosynthetic mutant, had an internal water chain with positions and hydrogen bonds identical to those oF the wild type. The structure data imply that the waters oF the internal chain, in addition to the surrounding protein, have a signiFicant role in Cytochrome F Function.
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Tryptophan-heme π-electrostatic interactions in Cytochrome F oF oxygenic photosynthesis
Biochemistry, 2000Co-Authors: Mikhail V. Ponamarev, Derek S Bendall, Christopher J. Carrell, Beatrix G. Schlarb, Christopher J. Howe, Janet L. Smith, William A. CramerAbstract:Cytochrome F oF oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures oF the chloroplast and cyanobacterial Cytochromes F were compared to explain spectral and redox diFFerences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis oF Cytochrome F From the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 → Trp substitution in the N-terminal domain was unique in causing a red shiFt oF 1 and 2 nm in the Cytochrome Soret (γ) and Q (α) visible absorption bands, respectively. The resulting α band peak at 556 nm is characteristic oF the cyanobacterial Cytochrome. Conversely, a Trp4 → Phe mutation in the expressed Cytochrome From the cyanobacterium Phormidium laminosum caused a blue ...
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Structure oF the soluble domain oF Cytochrome F From the cyanobacterium Phormidium laminosum.
Biochemistry, 1999Co-Authors: Christopher J. Carrell, Derek S Bendall, William A. Cramer, Beatrix G. Schlarb, Christopher J. Howe, Janet L. SmithAbstract:Cytochrome F From the photosynthetic Cytochrome b6F complex is unique among c-type Cytochromes in its Fold and heme ligation. The 1.9-A crystal structure oF the Functional, extrinsic portion oF Cytochrome F From the thermophilic cyanobacterium Phormidium laminosumdemonstrates that an unusual buried chain oF Five water molecules is remarkably conserved throughout the biological range oF Cytochrome F From cyanobacteria to plants (Martinez et al. (1994) Structure 2 ,9 5-105). Structure and sequence conservation oF the Cytochrome F extrinsic portion is concentrated at the heme, in the buried water chain, and in the vicinity oF the transmembrane helix anchor. The electrostatic surFace potential is variable, so that the surFace oF P. laminosum Cytochrome F is much more acidic than that From turnip. Cytochrome F is unrelated to Cytochrome c1, its Functional analogue in the mitochondrial respiratory Cytochrome bc1 complex, although other components oF the b6F and bc1 complexes are homologous. Identical Function oF the two complexes is inFerred For events taking place at sites oF strong sequence conservation. Conserved sites throughout the entire Cytochrome b6F/bc1 Family include the cluster-binding domain oF the Rieske protein and the heme b and quinone-binding sites on the electrochemically positive side oF the membrane within the b Cytochrome, but not the putative quinone-binding site on the electrochemically negative side.
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the heme redox center oF chloroplast Cytochrome F is linked to a buried Five water chain
Protein Science, 1996Co-Authors: Sergio E Martinez, William A. Cramer, D Huang, M Ponomarev, Janet L. SmithAbstract:The crystal structure oF the 252-residue lumen-side domain oF reduced Cytochrome F, a subunit oF the proton-pumping integral Cytochrome b6F complex oF oxygenic photosynthetic membranes, was determined to a resolution oF 1.96 A From crystals cooled to -35 degrees. The model was reFined to an R-Factor oF 15.8% with a 0.013-A RMS deviation oF bond lengths From ideality. Compared to the structure oF Cytochrome F at 20 degrees, the structure at -35 degrees has a small change in relative orientation oF the two Folding domains and signiFicantly lower isotropic temperature Factors For protein atoms. The structure revealed an L-shaped array oF Five buried water molecules that extend in two directions From the N delta 1 oF the heme ligand His 25. The longer branch extends 11 A within the large domain, toward Lys 66 in the prominent basic patch at the top oF the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature Factors are comparable to those oF protein atoms. Virtually all residues that Form hydrogen bonds with the water chain are invariant among 13 known Cytochrome F sequences. The water chain has many Features that optimize it as a proton wire, including insulation From the protein medium. It is suggested that this chain may Function as the lumen-side exit port For proton translocation by the Cytochrome b6F complex.
John C. Gray - One of the best experts on this subject based on the ideXlab platform.
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Assembly oF Cytochrome F into the Cytochrome bF complex in isolated pea chloroplasts
European journal of biochemistry, 2001Co-Authors: Ruth M. Mould, Aliki Kapazoglou, John C. GrayAbstract:Structural Features oF Cytochrome F necessary For assembly into the Cytochrome bF complex were examined in isolated pea chloroplasts Following import oF (35)S-labelled chimeric precursor proteins, consisting oF the presequence oF the small subunit oF Rubisco Fused to the turnip Cytochrome F precursor. Assembly was detected by nondenaturing gel electrophoresis oF dodecyl maltoside-solubilized thylakoid membranes. A Cytochrome F polypeptide unable to bind haem because oF mutagenesis oF Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to Cytochrome F is not necessary For assembly oF the protein into the Cytochrome bF complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type Cytochrome F but was not assembled into the complex. This indicates that the C-terminal region oF Cytochrome F is important For assembly into the complex. A mutant Cytochrome F unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence oF the C-terminal membrane anchor, haem-less Cytochrome F is recognized by a thylakoid proteolytic system.
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The role oF individual lysine residues in the basic patch on turnip Cytochrome F For electrostatic interactions with plastocyanin in vitro.
European journal of biochemistry, 2000Co-Authors: Xiaoâsong Gong, Derek S Bendall, Christopher J. Howe, Jiang Qi Wen, Nicholas E. Fisher, Simon Young, John C. GrayAbstract:The role oF electrostatic interactions in determining the rate oF electron transFer between Cytochrome F and plastocyanin has been examined in vitro with mutants oF turnip Cytochrome F and mutants oF pea and spinach plastocyanins. Mutation oF lysine residues Lys58, Lys65 and Lys187 oF Cytochrome F to neutral or acidic residues resulted in decreased binding constants and decreased rates oF electron transFer to wild-type pea plastocyanin. Interaction oF the Cytochrome F mutant K187E with the pea plastocyanin mutant D51K gave a Further decrease in electron transFer rate, indicating that a complementary charge pair at these positions could not compensate For the decreased overall charge on the proteins. Similar results were obtained with the interaction oF the Cytochrome F mutant K187E with single, double and triple mutants oF residues in the acidic patches oF spinach plastocyanin. These results suggest that the lysine residues oF the basic patch on Cytochrome F are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis oF the data using thermodynamic cycles provided evidence For the interaction oF Lys187 oF Cytochrome F with Asp51, Asp42 and Glu43 oF plastocyanin in the complex, in agreement with a structural model oF a Cytochrome F–plastocyanin complex determined by NMR.
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Cytochrome F: Structure, Function and biosynthesis.
Photosynthesis research, 1992Co-Authors: John C. GrayAbstract:Cytochrome F is an intrinsic membrane component oF the Cytochrome bF complex, transFerring electrons From the Rieske FeS protein to plastocyanin in the thylakoid lumen. The protein is held in the thylakoid membrane by a single transmembrane span located near its C-terminus with a globular hydrophilic domain extending into the lumen. The globular domain oF the turnip protein has recently been crystallised, oFFering the prospect oF a detailed three-dimensional structure. Reaction with plastocyanin involves localised positive charges on Cytochrome F interacting with the acidic patch on plastocyanin and electron transFer via the surFace-exposed tyrosine residue (Tyr83) oF plastocyanin. ApoCytochrome F is encoded in the chloroplast genome and is synthesised with an N-terminal presequence which targets the protein to the thylakoid membrane. The synthesis oF Cytochrome F is coordinated with the synthesis oF the other subunits oF the Cytochrome bF complex.
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the surFace exposed tyrosine residue tyr83 oF pea plastocyanin is involved in both binding and electron transFer reactions with Cytochrome F
The EMBO Journal, 1991Co-Authors: Shiping He, Derek S Bendall, Sandeep Modi, John C. GrayAbstract:Abstract Site-directed mutants oF the pea plastocyanin gene in which the codon For the surFace-exposed Tyr83 has been changed to codons For Phe83 and Leu83 have been expressed in transgenic tobacco plants. The mutant proteins have been puriFied to homogeneity and their conFormations shown not to diFFer signiFicantly From the wild-type plastocyanin by 1H-NMR and CD. Overall rate constants For electron transFer (k2) From Cytochrome F to plastocyanin have been measured by stopped-Flow spectrophotometry and rate constants For binding (ka) and association constants (KA) have been measured From the enhanced Soret absorption oF Cytochrome F on binding plastocyanin. These measurements allow the calculation oF the intrinsic rate oF electron transFer in the binary complex. An 8-Fold decrease in the overall rate oF electron transFer to the Phe83 mutant is due entirely to a decreased association constant For Cytochrome F, whereas the 40-Fold decrease in the overall rate oF electron transFer to the Leu83 mutant is due to weaker binding and a lower intrinsic rate oF electron transFer. This indicates that Tyr83 is involved in binding to Cytochrome F and Forms part oF the main route oF electron transFer.
