Studies on photosystem I. I. Relationship of plastocyanin, cytochrome f and P700

DEAE-Sephadex column chromatography now has been used for the final step in purification of d-amino acid oxidase apoenzyme. A specific enzymatic activity of 35–37 units/mg has been obtained for the pure holoenzyme. The purity has been established by disc and SDS gel electrophoreses and by sedimentation equilibrium. The molecular weight per enzyme monomer has been found to be 38,000 ± 1000. Each enzyme monomer binds one FAD and one benzoate with dissociation constants at 23 °C and pH 8.5 of 5.35 × 10^?7m and 1.96 × 10^?6m, respectively. The holoenzyme is more negatively charged than the apoenzyme at alkaline pH. The amino acid composition and some other physicochemical properties of the oxidase are reported.     

Electron transfer reactions in Zn-substituted cytochrome P450cam

We have investigated photoinduced electron transfer (ET) reactions between zinc-substituted cytochrome P450cam (ZnP450) and several inorganic reagents by using the laser flash photolysis method, to reveal roles of the electrostatic interactions in the regulation of the ET reactions. The laser pulse irradiation to ZnP450 yielded a strong reductant, the triplet excited state of ZnP450, (3)ZnP450, which was able to transfer one electron to anionic redox partners, OsCl(6)(2-) and Fe(CN)(6)(3-), with formation of the porphyrin pi-cation radical, ZnP450(+). In contrast, the ET reactions from (3)ZnP450 to cationic redox partners, such as Ru(NH(3))(6)(3+) and Co(phen)(3)(3+), were not observed even in the presence of 100-fold excess of the oxidant. One of the possible interpretations for the preferential ET to the anionic redox partner is that the cationic patch on the P450cam surface, a putative interaction site for the anionic reagents, is located near the heme (less than 10 A from the heme edge), while the anionic surface is far from the heme moiety (more than 16 A from the heme edge), which would yield 8000-fold faster ET rates through the cationic patch. The ET rate through the anionic patch to the cationic partner would be substantially slower than that of the phosphorescence process in (3)ZnP450, resulting in no ET reactions to the cationic reagents. These results demonstrate that the asymmetrical charge distribution on the protein surface is critical for the ET reaction in P450cam.     

Role of charges on cytochrome f from the cyanobacterium Phormidium laminosum in its interaction with plastocyanin

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 was neutralized at five acidic residues individually and introduced at a residue close to the interface between the two proteins. The effects on the kinetics of the reaction were measured using stopped-flow spectrophotometry, and the midpoint potentials of the mutant proteins were determined. The dependence of the bimolecular rate constant of reaction, k(2), on ionic strength was determined for the reactions of the cytochrome f mutants with wild-type and mutant forms of plastocyanin. Double mutant cycle analysis was carried out to probe for the presence of specific electrostatic interactions. The effects of mutations on Cyt f were smaller than those seen previously for mutants of plastocyanin [Schlarb-Ridley, B. G. et al. (2002) Biochemistry 41, 3279-3285]. One specific short-range interaction between charged residues of wild-type plastocyanin (Arg93) and wild-type cytochrome f (Asp63) was identified. The kinetic evidence from this study and that of Schlarb-Ridley et al., 2002, appears to conflict with the NMR structure of the P. laminosum complex, which suggests the absence of electrostatic interactions in the final complex [Crowley, P. et al. (2001) J. Am. Chem. Soc. 123, 10444-10453]. The most likely explanation of the apparent paradox is that the overall rate is diffusion controlled and that electrostatics specifically influence the encounter complex and not the reaction complex.     

The surface-exposed tyrosine residue Tyr83 of pea plastocyanin is involved in both binding and electron transfer reactions with cytochrome f.

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.     

Photosynthetic electron transfer at the level of cytochrome f and plastocyanin]

Light-induced redox conversions of cytochrome f and plastocyanin in situ and electron transporst from H2O to NADP+ were studied by a dual-wave differential spectrophotometry under identical conditions and subsequently compared. The analysis in red and far red light, treatment by inhibitors, e. g. diurone and dibromothymoquinone, and the analysis of photoreactions during the greening of etiolated seedlings demonstrated that cytochrome f functions only in the non-cyclic chain of electron transport, whereas plastocyanin--both in the non-cyclic and in the cyclic electron transport chains. The jositions of cytochrome f and plastocyanin in various electron-transport chains are proposed.     

Equilibration between cytochrome f and P700 in intact leaves

Electron transport between the two photosynthetic reaction centres of high plants is mediated by plastoquinone, a rieske iron-sulfur centre, cytochrome f and plastocyanin. Measurements of redox equilibration amongst these have produced confusing results, with apparent equilibrium constants being estimated that are inconsistent with in vitro measurements of redox midpoint potentials of the components concerned. We have critically reexamined methods for deconvoluting cytochrome f absorbance signals in intact leaves. We have determined the decay of cytochrome f+ following light to dark transitions from steady state and compared this with the decay of the oxidised photosystem I primary donor, P700+. Measurements across a wide range of different irradiances and CO2 concentrations were all consistent with cyt f and P700 existing in redox equilibrium, with a potential difference of around 117 mV. These results are discussed in relation to our understanding of the organisation of the photosynthetic electron transport. They also have implications for measurements of PSI electron flux--provided more than about 20% of P700+ is oxidised in the light, then the initial decay in the concentration of P700+ following a light to dark transition provides a good estimate of electron flux through PSI. Where P700 is largely reduced in the light, net reduction of cyt f+ might need to be corrected for.     

Electron transfer between plastocyanin and P700 in highly-purified photosystem I reaction center complex. Effects of pH, cations, and subunit peptide composition

Treatment of isolated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel chromatographies yielded a highly purified P700-chlorophyll a protein complex complex which consists of five polypeptides. The protein complex is virtually free of chlorophyll b (Ch1 alpha/Ch1 b greater than 10) with approximately 30 chlorophylls per P700, and contains iron-sulfur centers A, B, and X. At pH values higher than 6, divalent cations, but not monovalent or trivalent cations, efficiently accelerated the electron transfer from reduced spinach plastocyanin to the photooxidized P700 in the P700-chlorophyll alpha protein complex. At pH values lower than 6, the reaction rate drastically increased with decreasing pH with a maximum at about pH 4.3 without cations. Divalent salts as well as monovalent or trivalent salts decreased the P700 reduction rate at low pH, indicating the involvement of electrostatic interaction in those pH regions. The rate of electron transfer from plastocyanin to the photooxidized P700 in the reaction center protein, which consists of only the largest peptide subunit and no iron-sulfur centers, was reduced only 50% at pH 7.0 in the presence of MgCl2 as compared to the case of P700-chlorophyll alpha protein complex. Essentially similar effects of pH and metal ions on this electron transfer reaction were observed as in the case of P700-chlorophyll alpha protein complex. These results strongly suggest that plastocyanin donates electrons directly to the largest peptide of P700-chlorophyll alpha protein complex and the observed effects of pH and cations are mainly due to the interaction between the largest peptide of P700-chlorophyll alpha protein complex and plastocyanin. The four small subunits in the protein complex seemed to have only a minor role in the reaction with plastocyanin.     

Brownian dynamics study of cytochrome f interactions with cytochrome c6 and plastocyanin in Chlamydomonas reinhardtii plastocyanin, and cytochrome c6 mutants

Using Brownian dynamics simulations, all of the charged residues in Chlamydomonas reinhardtii cytochrome c(6) (cyt c(6)) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f (cyt f) were modeled. Systematic mutation of charged residues on both PC and cyt c(6) confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c(6) displayed similar inhibition classes. Our results indicate a difference between the two acidic clusters on PC. Mutations D44A and E43A of the lower cluster showed greater inhibition than do any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c(6) that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. In PC residues D42, E43, D44, D53, D59, D61, and E85, and in cyt c(6) residues D2, E54, K57, D65, R66, E70, E71, and the heme had significant electrostatic contacts with cyt f charged residues. PC and cyt c(6) showed different binding sites and orientations on cyt f. As there are no experimental cyt c(6) mutation data available for algae, our results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement, which supports the predictive power of Brownian dynamics simulations in mutagenesis studies.     

Effects of hydrostatic pressure on the kinetics of electron transfer in an isolated system of chloroplast cytochrome bf complex,plastocyanin and P700

The effects of pressure on the kinetics of redox reactions in and around the chloroplast cytochrome bf complex were studied using a reconstituted system consisting of Photosystem I (PS I) particles, cytochrome bf complex and plastocyanin (PC), all derived from pea chloroplasts. There were no significant permanent effects of pressure in the range 0.1–191 MPa on the reaction kinetics, or on the shape of the absorption spectra of components studied. Discernable effects on rate-coefficients of increasing pressure were observed on the reduction of P700⁺ by PC^I, on the reduction of PC^II by ascorbate, and on the oxidation of decyl plastoquinol by the bf complex. The volumes of activation ΔV^# were determined from the dependence of the rate-coefficient on pressure using: $$(\partial lnk/\partial P)_T = - \Delta V^\# /RT.$$ The volume of activation is the difference in partial molar volume between the activated state and the reactants for the redox reaction. Such data was sought to help define in detail those redox reactions and the corresponding activated states. For the reduction of P700⁺ by PC^I and the oxidation of decyl plastoquinol by the bf complex, the rate coefficient decreased with increase in pressure, whilst for the reduction of PC^II by ascorbate it increased. The corresponding volumes of activation were 9.6±0.6×10^-6 m³ mol^-1, 18±2×10^-6 m³ mol^-1 and -14±1×10^-6 m³ mol^-1, respectively. Much of the pressure-dependence of PC^II reduction by ascorbate was ascribed to an increase in ascorbate ionisation with increase in pressure. There was little effect of pressure on the kinetics of oxidation of ferrocytochrome f by PC^II, or on the equilibrium constant of the redox pair ferrocytochrome f/ferricytochrome f: PC^II/PC^I. Possible physical bases for these activation volumes are discussed, and they are compared with literature values.     

Direct simulation of plastocyanin and cytochrome f interactions in solution

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.     

Brownian dynamics simulations of the interaction of Chlamydomonas cytochrome f with plastocyanin and cytochrome c6

The interaction of Chlamydomonas cytochrome f (cyt f) with either Chlamydomonas plastocyanin (PC) or Chlamydomonas cytochrome c(6) (cyt c(6)) was studied using Brownian dynamics simulations. The two electron acceptors (PC and cyt c(6)) were found to be essentially interchangeable despite a lack of sequence homology and different secondary structures (beta-sheet for PC and alpha-helix for cyt c(6)). Simulations using PC and cyt c(6) interacting with cyt f showed approximately equal numbers of successful complexes and calculated rates of electron transfer. Cyt f-PC and cyt f-cyt c(6) showed the same types of interactions. Hydrophobic residues surrounding the Y1 ligand to the heme on cyt f interacted with hydrophobic residues on PC (surrounding the H87 ligand to the Cu) or cyt c(6) (surrounding the heme). Both types of complexes were stabilized by electrostatic interactions between K65, K188, and K189 on cyt f and conserved anionic residues on PC (E43, D44, D53, and E85) or cyt c(6) (E2, E70, and E71). Mutations on cyt f had identical effects on its interaction with either PC or cyt c(6). K65A, K188A, and K189A showed the largest effects whereas residues such as K217A, R88A, and K110A, which are located far from the positive patch on cyt f, showed very little inhibition. The effect of mutations observed in Brownian dynamics simulations paralleled those observed in experiments.     

Electron transfer and stability of the cytochrome b6f complex in a small domain deletion mutant of cytochrome f

The lumen segment of cytochrome f consists of a small and a large domain. The role of the small domain in the biogenesis and stability of the cytochrome b(6)f complex and electron transfer through the cytochrome b(6)f complex was studied with a small domain deletion mutant in Chlamydomonas reinhardtii. The mutant is able to grow photoautotrophically but with a slower rate than the wild type strain. The heme group is covalently attached to the polypeptide, and the visible absorption spectrum of the mutant protein is identical to that of the native protein. The kinetics of electron transfer in the mutant were measured by flash kinetic spectroscopy. Our results show that the rate for the oxidation of cytochrome f was unchanged (t(12) = approximately 100 micros), but the half-time for the reduction of cytochrome f is increased (t(12) = 32 ms; for wild type, t(12) = 2.1 ms). Cytochrome b(6) reduction was slower than that of the wild type by a factor of approximately 2 (t(12) = 8.6 ms; for wild type, t(12) = 4.7 ms); the slow phase of the electrochromic band shift also displayed a slower kinetics (t(12) = 5.5 ms; for wild type, t(12) = 2.7 ms). The stability of the cytochrome b(6)f complex in the mutant was examined by following the kinetics of the degradation of the individual subunits after inhibiting protein synthesis in the chloroplast. The results indicate that the cytochrome b(6)f complex in the small domain deletion mutant is less stable than in the wild type. We conclude that the small domain is not essential for the biogenesis of cytochrome f and the cytochrome b(6)f complex. However, it does have a role in electron transfer through the cytochrome b(6)f complex and contributes to the stability of the complex.     

The properties of cytochrome f and P700 in chloroplasts suspended in fluid media at sub-zero temperatures.

1. The properties of P700 and cytochrome f have been studied at sub-zero temperatures in chloroplasts suspended in a medium containing 50% (v/v) ethylene glycol. The dark reduction of these components after a period of illumination provided information about the rate-limiting step of photosynthetic electron transport under these conditions. 2. The oxidation of P700 on illumination in the presence of methyl viologen and its subsequent dark reduction can be observed at -35 degrees C. This cycle of reactions could be repeated many times. The rate of reduction was increased by NH4Cl and reduction was inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. 3. The oxidation and reduction of cytochrome f could also be observed under similar conditions. The activation energies for the reduction of cytochrome f and P700 are similar (about 75 kJ mol-1) and the reduction of cytochrome f is also inhibited by dichlorophenyldimethylurea and stimulated by NH4Cl. 4. The reduction of both cytochrome f and P700 seemed to follow first-order kinetics, but the t1/2 for the redcution of the cytochrome was at least three times that for the reduction of P700 at the same temperature. It was concluded that the results were only compatible with a model in which the main pathway of electrons from plastoquinone to P700 involved cytochrome f if the equilibrium constant between the cytochrome and P700 was very much less than that expected from their redox potentials.     

Preparation of a covalently linked adduct between plastocyanin and cytochrome f

Plastocyanin can be covalently cross-linked to the monomeric cytochrome f from turnip by incubation in the presence of a water-soluble carbodiimide. The adduct between the two proteins has a molecular weight of approximately 43,000 suggesting a 1:1 stoichiometry between the two proteins of the adduct. This stoichiometry has been verified by spectral characterization of the adduct. The efficiency of the cross-linking reaction is pH dependent with a higher degree of cross-linking being observed at pH 6.5 than at pH 7.0.     

Electrostatic analysis and Brownian dynamics simulation of the association of plastocyanin and cytochrome f.

The oxidation of cytochrome f by the soluble cupredoxin plastocyanin is a central reaction in the photosynthetic electron transfer chain of all oxygenic organisms. Here, two different computational approaches are used to gain new insights into the role of molecular recognition and protein-protein association processes in this redox reaction. First, a comparative analysis of the computed molecular electrostatic potentials of seven single and multiple point mutants of spinach plastocyanin (D42N, E43K, E43N, E43Q/D44N, E59K/E60Q, E59K/E60Q/E43N, Q88E) and the wt protein was carried out. The experimentally determined relative rates (k(2)) for the set of plastocyanin mutants are found to correlate well (r(2) = 0.90 - 0.97) with the computed measure of the similarity of the plastocyanin electrostatic potentials. Second, the effects on the plastocyanin/cytochrome f association rate of these mutations in the plastocyanin "eastern site" were evaluated by simulating the association of the wild type and mutant plastocyanins with cytochrome f by Brownian dynamics. Good agreement between the computed and experimental relative rates (k(2)) (r(2) = 0.89 - 0.92) was achieved for the plastocyanin mutants. The results obtained by applying both computational techniques provide support for the fundamental role of the acidic residues at the plastocyanin eastern site in the association with cytochrome f and in the overall electron-transfer process.     

Structure of the intermolecular complex between plastocyanin and cytochrome f from spinach

In oxygenic photosynthesis, plastocyanin shuttles electrons between the membrane-bound complexes cytochrome b6f and photosystem I. The homologous complex between cytochrome f and plastocyanin, both from spinach, is the object of this study. The solution structure of the reduced spinach plastocyanin was determined using high field NMR spectroscopy, whereas the model structure of oxidized cytochrome f was obtained by homology modeling calculations and molecular dynamics. The model structure of the intermolecular complex was calculated using the program AUTODOCK, taking into account biological information obtained from mutagenesis experiments. The best electron transfer pathway from the heme group of cytochrome f to the copper ion of plastocyanin was calculated using the program HARLEM, obtaining a coupling decay value of 1.8 x 10(-4). Possible mechanisms of interaction and electron transfer between plastocyanin and cytochrome f were discussed considering the possible formation of a supercomplex that associates one cytochrome b6f, one photosystem I, and one plastocyanin.     

Different modes of interaction in cyanobacterial complexes of plastocyanin and cytochrome f

The highly efficient electron-transfer chain in photosynthesis demonstrates a remarkable variation among organisms in the type of interactions between the soluble electron-transfer protein plastocyanin and it partner cytochrome f. The complex from the cyanobacterium Nostoc sp. PCC 7119 was studied using nuclear magnetic resonance spectroscopy and compared to that of the cyanobacterium Phormidium laminosum. In both systems, the main site of interaction on plastocyanin is the hydrophobic patch. However, the interaction in the Nostoc complex is highly dependent on electrostatics, contrary to that of Phormidium, resulting in a binding constant that is an order of magnitude larger at low ionic strength for the Nostoc complex. Studies of the mixed complexes show that these differences in interactions are mainly attributable to the surface properties of the plastocyanins.