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 of cytochrome f with flavin semiquinones and with plastocyanin. Importance of protein-protein electrostatic interactions and of donor-acceptor coupling.

Reduction of turnip ferricytochrome f by flavin semiquinones and oxidation of this ferrocytochrome f by French bean cupriplastocyanin are studied by laser flash photolysis over a wide range of ionic strengths. Second-order rate constants (+/- 15%) at extreme values of ionic strength, all at pH 7.0 and 22 degrees C, are as follows: with FMN semiquinone at 1.00 and 0.0040 M, 5.0 x 10(7) and 3.9 x 10(8) M-1 s-1; with riboflavin semiquinone at 1.00 and 0.0040 m, 1.7 x 10(8) and 1.9 x 10(8) M-1 s-1; with lumiflavin semiquinone at 1.00 and 0.0045 M, 1.8 x 10(8) and 4.5 x 10(8) M-1 s-1; with cupriplastocyanin at 1.00 and 0.100 M, 1.4 x 10(6) and 2.0 x 10(8) M-1 s-1. These reactions of cytochrome f are governed by the local positive charge of the interaction domain (the exposed heme edge), not by the overall negative charge of the protein. Lumiflavin semiquinone behaves as if it carried a small negative charge, probably because partial localization of the odd electron gives this electroneutral molecule some polarity; local charge seems to be more important than overall charge even for relatively small redox agents. The dependence of the rate constants on ionic strength was fitted to the equation of Watkins; this model recognizes the importance of local charges of the domains through which redox partners interact. There is kinetic evidence that a noncovalent complex between cytochrome f and plastocyanin exists at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Preparation and characterization of a chemically modified plastocyanin.

The reaction of plastocyanin with tetranitromethane results in the nitration of only one of the three tyrosyl residues present in the protein. The modification does not affect the blue copper chromophore as both the characteristic visible spectrum of the chromophore and the redox potential of the protein are unchanged. Photochemical assays show that the modified plastocyanin is fully active in the reduction of photooxidized P700 and in the photooxidation of cytochrome f. The pK of the nitro-tyrosyl residue is about 7.3 indicating that the modified residue may be located in a negatively charged environment. Examination of the recently published X-ray structure of poplar plastocyanin suggests that Tyr-80 would be a likely candidate for the site of modification.     

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 transfers amongst cytochrome f, plastocyanin and photosystem I: kinetics and mechanisms

The review covers the theory and practice of the determination of kinetic constants for the electron transfer reactions in chloroplast thylakoid membranes between plastocyanin and cytochrome f in cytochrome bf complexes, and between plastocyanin and the reaction centre of photosystem I. Effects of ionic strength and pH are featured. The contribution of mutant studies is included. It is concluded that nearly all data from in vitro experiments can be interpreted with a reaction scheme in which an encounter complex between donor and acceptor is formed by long-range electrostatic attraction, followed by rearrangement during which metal centres become close enough for rapid intra-complex electron transfer. In vivo experiments so far cast doubt on this particular sequence, but their interpretation is not straightforward. Means of modelling the bimolecular complex between cytochrome f and plastocyanin are outlined, and two likely structures are illustrated. The complex formed by plastocyanin and photosystem I in higher plants involves the PsaF subunit, but its structure has not been fully determined.     

Electrostatic properties of cytochrome f: implications for docking with plastocyanin.

The electrostatic properties of cytochrome f (cyt f), a member of the cytochrome b6f complex and reaction partner with plastocyanin (PC) in photosynthetic electron transport, are qualitatively studied with the goal of determining the mechanism of electron transfer between cyt f and PC. A crystal structure for cyt f was analyzed with the software package GRASP, revealing a large region of positive potential generated by a patch of positively charged residues (including K58, K65, K66, K122, K185, K187, and R209) and reinforced by the iron center of the heme. This positive field attracts the negative charges of the two acidic patches on the mobile electron carrier PC. Three docked complexes are obtained for the two proteins, based on electrostatic or hydrophobic interactions or both and on steric fits by manual docking methods. The first of these three complexes shows strong electrostatic interactions between K187 on cyt f and D44 on PC and between E59 on PC and K58 on cyt f. Two other manually docked complexes are proposed, implicating H87 on PC as the electron-accepting site from the iron center of cyt f through Y1. The second complex maintains the D44/K187 cross-link (but not the E59/K58 link) while increasing hydrophobic interactions between PC and cyt f. Hydrophobic interactions are increased still further in the third complex, whereas the link between K187 on cyt f and D44 on PC is broken. The proposed reaction mechanism, therefore, involves an initial electrostatic docking complex that gives rise to a nonpolar attraction between the regions surrounding H87 on PC and Y1 on cyt f, providing for an electron-transfer active complex.     

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.     

Brownian dynamics study of the interaction between plastocyanin and cytochrome f.

The electrostatic interaction between plastocyanin (PC) and cytochrome f (cyt f), electron transfer partners in photosynthesis was studied using Brownian dynamics (BD) simulations. By using the software package MacroDox, which implements the BD algorithm of Northrup et al. (Northrup, S. H., J. O. Boles, and J. C. L. Reynolds. 1987. J. Phys. Chem. 91:5991-5998), we have modeled the interaction of the two proteins based on crystal structures of poplar PC and turnip cyt f at pH 7 and a variety of ionic strengths. We find that the electrostatic attraction between positively charged residues (K58, K65, K187, and R209, among others) on cyt f and negatively charged residues (E43, D44, E59, and E60, among others) on PC steers PC into a single dominant orientation with respect to cyt f, and furthermore, that the single dominant orientation that we observe is one that we had predicted in our previous work (Pearson, D. C., E. L. Gross, and E. S. David. 1996. Biophys. J. 71:64-76). This dominant orientation permits the formation of hydrophobic interactions, which are not implemented in the MacroDox algorithm. This proposed complex between PC and cyt f implicates H87, a copper ligand on PC, as the residue that accepts electrons from the heme on cyt f (and possibly through Y1 as we proposed previously). We argue for the existence of this single dominant complex on the basis of observations that the most favorable orientations of the interaction between PC and cyt f, as determined by grouping successful BD trajectories on the basis of closest contacts of charged residues, tend to overlap one another and have very close distances between the metal centers on the two proteins (copper on PC, iron on cyt f). We use this knowledge to develop a model for PC/cyt f interaction that places a reaction between the two proteins occurring when the copper-to-iron distance is between 16 and 17 A. This reaction distance gives a good estimate of the experimentally observed rate constant for PC-cyt f interaction. Analysis of BD results as a function of ionic strength predicts an interaction that happens less frequently and becomes less specific as ionic strength increases.     

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.     

The effect of ethylenediamine chemical modification of plastocyanin on the rate of cytochrome f oxidation and P-700+ reduction

Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6-8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42-45 and Nos. 59-61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42-45 and Nos. 59-61. Modification of plastocyanin at residues Nos. 42-45 showed no effect on the rate of P-700+ reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700+ reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59-61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 +/- 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42-45 or Nos. 59-61.     

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.     

The purification of cytochrome f and plastocyanin using affinity chromatography

Both plastocyanin and cytochrome f were purified using a combination of affinity chromatography together with established methods. Plastocyanin was partially purified using the method of Davis and San Pietro (Anal. Biochem. 95 (1979) 254-259), after which it was further purified using a column of cytochrome c covalently attached to Sepharose 4B. The affinity column was prepared using the method of Godinot and Gautheron (Methods Enzymol. 54 (1979) 112-114). The final purity index ratio (A278/A597) was less than 1.2, which is equal to that obtained using the more expensive FPLC procedure (Anderson, G.P., Sanderson, D.G., Lee, C.H., Durell, S., Anderson, L.B. and Gross, E.L. (1987) Biochim. Biophys. Acta 894, issue 3). Cytochrome f was partially purified using a modification of the method of Matazaki et al. (Plant Cell. Physiol. 16 (1975) 237-246) and bound to an affinity column of plastocyanin covalently attached to Sepharose 4B. Cytochrome f purified using this procedure had a purity index ratio (A554.5/A277) of 1.2. Both proteins are tyrosine proteins containing no tryptophan residues. After the affinity chromatography step, the fluorescence emission spectrum of either plastocyanin or cytochrome f was typical of a tyrosine protein free from tryptophan contamination.     

Electron transfer reactions of cytochrome c confined within erythrocyte ghosts

We have prepared and characterized resealed erythrocyte ghosts in which the only discernible pigment is cytochrome c. The resealed ghosts have the normal orientation and are free of 'leaky' species; they are stable and can be maintained at 4 degrees C for many days without lysis. The internal cytochrome c participates in redox reactions with both soluble and insolubilized cytochrome c present externally, and with external cytochrome b5. No reaction was observed with plastocyanin, cytochrome c oxidase or NADPH-cytochrome c reductase. A study has been made of the reaction of the internal cytochrome c with the low molecular weight reductants, ascorbate and glutathione. Complex kinetics are observed with both reagents: with ascorbate the results are best explained by assuming the existence, in the membrane, of a redox-active species able to undergo dedimerization. A protein bound disulfide bond would satisfy the requirement.