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.     

Role of electrostatics in the interaction between cytochrome f and plastocyanin of the cyanobacterium Phormidium laminosum

The role of charged residues on the surface of plastocyanin from the cyanobacterium Phormidium laminosum in the reaction with soluble cytochrome f in vitro was studied using site-directed mutagenesis. The charge on each of five residues on the eastern face of plastocyanin was neutralized and/or inverted, and the effect of the mutation on midpoint potentials was determined. The dependence of the overall rate constant of reaction, k(2), on ionic strength was investigated using stopped-flow spectrophotometry. Removing negative charges (D44A or D45A) accelerated the reaction and increased the dependence on ionic strength, whereas removing positive charges slowed it down. Two mutations (K46A, K53A) each almost completely abolished any influence of ionic strength on k(2), and three mutations (R93A, R93Q, R93E) each converted electrostatic attraction into repulsion. At low ionic strength, wild type and all mutants showed an inhibition which might be due to changes in the interaction radius as a consequence of ionic strength dependence of the Debye length or to effects on the rate constant of electron transfer, k(et). The study shows that the electrostatics of the interaction between plastocyanin and cytochrome f of P. laminosum in vitro are not optimized for k(2). Whereas electrostatics are the major contributor to k(2) in plants [Kannt, A., et al. (1996) Biochim. Biophys. Acta 1277, 115-126], this role is taken by nonpolar interactions in the cyanobacterium, leading to a remarkably high rate at infinite ionic strength (3.2 x 10(7) M(-1) s(-1)).     

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.     

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.     

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.     

Kinetic studies on a cross-linked complex between plastocyanin cytochrome f

A cross-linked complex between plastocyanin and cytochrome f was prepared by incubation in the presence of a water soluble carbodiimide and its kinetic properties were studied. The optical spectra, oxidation-reduction potentials and isoelectric pH of plastocyanin and cytochrome f did not change upon the formation of the cross-linked complex. Studies on the ionic strength effect on the electron transfer rate from cross-linked plastocyanin to ferricyanide indicated that the negative charge on the reaction site of plastocyanin was masked upon the cross-linking. It was also suggested that the sign of the net charge near the cytochrome f heme edge changed from positive to negative upon the cross-linking. On the other hand, electrostatic interactions between cross-linked plastocyanin and P700 seemed to be essentially the same as those in the case of native plastocyanin, although the rate of electron transfer from cross-linked plastocyanin to P700 was severely reduced. We also measured the intra-complex electron transfer from cytochrome f to plastocyanin. This suggested that the covalently cross-linked complex is a valid model of the electron transfer encounter complex. Based on these results, the reaction sites of plastocyanin with P700 and cytochrome f were discussed.     

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.     

Computer simulation of plastocyanin interaction with cytochrome f and photosystem I in cyanobacterium Phormidium laminosum

The paper is devoted to computer simulation of complex formation of protein plastocyanin with transmembrane pigment-protein complex photosystem I and subunit f of cytochrome b ₆ f complex in the cyanobacterium Phormidium laminosum. The computer algorithm considers diffusion and electrostatic interactions of protein molecules. The computer models have shown that electrostatic interactions in the cyanobacterium play a less important role than in higher plants because of different electrostatic potentials created by charged amino acid residues on the protein surfaces.     

A Brownian dynamics computational study of the interaction of spinach plastocyanin with turnip cytochrome f: the importance of plastocyanin conformational changes

Brownian Dynamics (BD) computer simulations were used to study electrostatic interactions between turnip cytochrome f (cyt f) and spinach plastocyanin (PC). Three different spinach PC structures were studied: The X-ray crystal structure of Xue and coworkers [(1998) Protein Sci 7:2099–2105] and the NMR structure of Musiani et al. [(2005) J Biol Chem 280:18833–18841] and Ubbink and co-workers [(1998) Structure 6:323–335]. Significant differences exist in the backbone conformation between the PC taken from Ubbink and coworkers and the other two PC structures particularly the regions surrounding G10, E59–E60, and D51. Complexes formed in BD simulations using the PC of Ubbink and colleagues had a smaller Cu–Fe distance than the other two. These results suggest that different PC conformations may exist in solution with different capabilities of forming electron-transfer-active docks. All three types of complexes show electrostatic contacts between D42, E43, and D44 on PC and K187 on cyt f as well as between E59 on PC and K58 on cyt f. However, the PC of Ubbink and coworkers reveals additional contacts between D51 and cyt f as a result of the difference in backbone configuration. A second minor complex component was observed for the PC of Ubbink and co-workers and Xue and co-workers which had contacts between K187 on cyt f and E59 and E60 on PC rather than between K187 on cyt f and D42-D44 on PC as observed for the major components. This second type of complex may represent an earlier complex which rearranges to form a final complex capable of electron transfer. Professor Elizabeth L. Gross, Professor Emeritus of Biochemistry, The Ohio State University, passed away on June 27, 2007.     

A Brownian dynamics study of the interaction of Phormidium laminosum plastocyanin with Phormidium laminosum cytochrome f

The interaction of Phormidium laminosum plastocyanin (PC) with P. laminosum cytochrome f (cyt f) was studied using Brownian dynamics (BD) simulations. Few complexes and a low rate of electron transfer were observed for wild-type PC. Increasing the positive electrostatic field on PC by the addition of a Zn(2+) ion in the neighborhood of D44 and D45 on PC (as found in crystal structure of plastocyanin) increased the number of complexes formed and the calculated rates of electron transfer as did PC mutations D44A, D45A, E54A, and E57A. Mutations of charged residues on Phormidium PC and Phormidium cyt f were used to map binding sites on both proteins. In both the presence and absence of the Zn(2+) ion, the following residues on PC interact with cyt f: D44, D45, K6, D79, R93, and K100 that lie in a patch just below H92 and Y88 and D10, E17, and E70 located on the upper portion of the PC molecule. In the absence of the Zn(2+) ion, K6 and K35 on the top of the PC molecule also interact with cyt f. Cyt f residues involved in binding PC, in the absence of the Zn(2+) ion, include E165, D187, and D188 that are located on the small domain of cyt f. The orientation of PC in the complexes was quite random in accordance with NMR results. In the presence of the Zn(2+) ion, K53 and E54 in the lower patch of the PC molecule also interact with cyt f and PC interacts with E86, E95, and E123 on the large domain of cyt f. Also, the orientation of PC in the complexes was much more uniform than in the absence of the Zn(2+) ion. The difference may be due to both the larger electrostatic field and the greater asymmetry of the charge distribution on PC observed in the presence of the Zn(2+) ion. Hydrophobic interactions were also observed suggesting a model of cyt f-PC interactions in which electrostatic forces bring the two molecules together but hydrophobic interactions participate in stabilizing the final electron-transfer-active dock.     

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.     

Dynamic interaction of plastocyanin with the cytochrome bf complex

The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of plastocyanin, which has been proposed to be the site of electron transfer from cytochrome f. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of plastocyanin to the intact cytochrome bf complex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.     

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.     

Relation between interface properties and kinetics of electron transfer in the interaction of cytochrome f and plastocyanin from plants and the cyanobacterium Phormidium laminosum

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. laminosumplastocyanin. 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.     

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.     

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.     

Plastocyanin cytochrome f interaction

Spinach plastocyanin and turnip cytochrome f have been covalently linked by using a water-soluble carbodiimide to yield an adduct of the two proteins. The redox potential of cytochrome f in the adduct was shifted by -20 mV relative to that of free cytochrome f, while the redox potential of plastocyanin in the adduct was the same as that of free plastocyanin. Solvent perturbation studies showed the degree of heme exposure in the adduct to be less than in free cytochrome f, indicating that plastocyanin was linked in such a way as to bury the exposed heme edge. Small changes were also observed when the resonance Raman spectrum of the adduct was compared to that of free cytochrome f. The adduct was incapable of interacting with or donating electrons to photosystem I. Peptide mapping and sequencing studies revealed two sites of linkage between the two proteins. In one site of linkage, Asp-44 of plastocyanin is covalently linked to Lys-187 of cytochrome f. This represents the first identification of a group on cytochrome f that is involved in the interaction with plastocyanin. The other site of linkage involves Glu-59 and/or Glu-60 of plastocyanin to as yet unidentified amino groups on cytochrome f. Euglena cytochrome c-552 could also be covalently linked to turnip cytochrome f, although with a lower efficiency than spinach plastocyanin. In contrast, a variety of cyanobacterial cytochrome c-553's and a cyanobacterial plastocyanin could not be covalently linked to turnip cytochrome f.     

Interaction between immobilized plastocyanin and cytochrome f and the photosystem I reaction center in a reconstituted system

The effects of redox conversions of plastocyanin copper chromophore on the formation of plastocyanin complexes with cytochrome f and the reaction center of photosystem I from pea chloroplasts were studied. In order to investigate the complex formation plastocyanin and cytochrome f were immobilized on Sephadex G-200. The cytochrome f and reaction center assembly takes place on the immobilized plastocyanin, which is necessary for cytochrome f photooxidation. It was found that in a reconstituted system the reduced plastocyanin forms more stable complexes with the proteins than the oxidized one, which is due to its lower pI value.