Photosynthetic electron transfer through the cytochrome b6f complex can bypass cytochrome f

The cytochrome b(6)f complex is an obligatory electron transfer and proton-translocating enzyme in all oxygenic photosynthesis. Its operation has been described by the "Q-cycle." This model proposes that electrons are transferred from plastoquinol to plastocyanin (the reductant of P700 in Photosystem I) through, obligatorily in series, the iron-sulfur and the cytochrome f redox centers in the cytochrome b(6)f complex. However, here we demonstrate that (a) the iron-sulfur center-dependent reductions of plastocyanin and P700 are much faster than cytochrome f reduction, both in Chlamydomonas reinhardtii cytochrome f mutants and in the wild type, and (b) the steady-state photosynthetic electron transport does not correlate with strongly inhibited cytochrome f reduction kinetics in the mutants. Thus, cytochrome f is not an obligatory intermediate for electrons flowing through the cytochrome b(6)f complex. The oxidation equivalents from Photosystem I are delivered to the high potential chain of the cytochrome b(6)f complex both at the cytochrome f level and, independently, at another site connected to the quinol-oxidizing site, possibly the iron-sulfur center.     

On the interaction between cytochrome f and plastocyanin

The interaction between cytochrome f and its electron acceptor plastocyanin (PC) was studied. To address the question of which specific regions and which of the positively charged residues of cytochrome f are important for the interaction with the negatively charged residues of PC we have used two different experimental approaches. Cytochrome f was proteolytically cleaved and fragments that could bind to a PC-affinity column were isolated. The smallest of these fragments was analysed to give information on the minimum structural requirement for binding to PC. By this procedure, we identified a peptide of approx. 11 kDa, containing the heme binding site, and having an N-terminal sequence identical to that of the mature cytochrome f. This finding suggests that the first 90 amino acids of cytochrome f contain at least some of the residues interacting with PC. The second approach involved modification of Arg residues of cytochrome f with the specific chemical modifier, hydroxyphenylglyoxal (HPG). Cytochrome f modification was performed in the absence of PC to enable identification of residues that are protected from modification when PC is bound to cytochrome f. Two peptides containing Arg residues which are modified in the absence of PC, but are not modified when PC is present, were isolated. Sequence analysis of these two peptides revealed that Arg residues no. 88 and 154 of cytochrome f are the residues that are protected from modification when cytochrome f is bound to PC, suggesting a role for these residues in the binding of cytochrome f to PC.     

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.     

Photoinduced electron transfer within complexes between plastocyanin and ruthenium bisbipyridine dicarboxybipyridine cytochrome c derivatives

A new technique has been developed to measure intracomplex electron transfer between cytochrome c and its redox partners. Cytochrome c derivatives labeled at single lysine amino groups with ruthenium bisbipyridine dicarboxybipyridine were prepared as previously described [Pan, L.P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Excitation of RuII with a short light pulse resulted in the formation of the excited-state RuII*, which rapidly transferred an electron to the ferric heme group to form FeII and RuIII. Aniline was included in the buffer to reduce RuIII to RuII, leaving the heme group in the ferrous state. This process was complete within the lifetime of the light pulse. When plastocyanin was present in the solution, electron transfer from the ferrous heme of cytochrome c to CuII in plastocyanin was observed. All of the ruthenium cytochrome c derivatives formed electrostatic complexes with plastocyanin at low ionic strength, allowing intracomplex electron-transfer rate constants to be measured. The rate constants for derivatives modified at the indicated lysines were as follows: Lys 13, 1920 s-1; Lys 8, 1480 s-1; Lys 7, 1340 s-1; Lys 86, 1020 s-1; Lys 25, 820 s-1; Lys 72, 800 s-1; Lys 27, 530 s-1. It is interesting that the derivative modified at lysine 13 at the top of the heme crevice had the largest rate constant, while lysine 27 at the right side of the heme crevice had the smallest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of electron transfer between plastocyanin and the soluble CuA domain of cyanobacterial cytochrome c oxidase

It has been shown that efficient functioning of photosynthesis and respiration in the cyanobacterium Synechocystis PCC 6803 requires the presence of either cytochrome c6 or plastocyanin. In order to check whether the blue copper protein plastocyanin can act as electron donor to cytochrome c oxidase, we investigated the intermolecular electron transfer kinetics between plastocyanin and the soluble CuA domain (i.e. the donor binding and electron entry site) of subunit II of the aa3-type cytochrome c oxidase from Synechocystis. Both copper proteins were expressed heterologously in Escherichia coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (5.1+/-0.2) x 10(4) M(-1) s(-1) and (8.5+/-0.4) x 10(5) M(-1) s(-1), respectively (20 mM phosphate buffer, pH 7). This corresponds to an apparent equilibrium constant of 0.06 in the physiological direction (reduction of CuA), which is similar to Keq values calculated for the reaction between c-type cytochromes and the soluble fragments of other CuA domains. The potential physiological role of plastocyanin in cyanobacterial respiration is discussed.     

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.     

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.     

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.     

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.     

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.     

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.     

Electron transfer reactions of chemically modified plastocyanin with P700 and cytochrome f. Importance of local charges

Chemically modified spinach plastocyanin, in which negatively charged carboxyl residues are replaced with positively charged amino residues, has been prepared. Four distinct species of chemically modified plastocyanin, having 1 to 4 mol of modified carboxyl residue per mol of plastocyanin, could be separated by ion-exchange chromatography on DEAE-Sephacel. The rate of electron transfer from reduced cytochrome f to oxidized singly substituted plastocyanin was 30% of that of the native unmodified plastocyanin, and the reaction rate decreased further with increasing number of modified carboxyl residues. These results indicate the importance of electrostatic interactions between the negative charges on plastocyanin and the positive charges on cytochrome f in this reaction. Since the overall net charge of cytochrome f is negative at neutral pH, the positive charges on cytochrome f involved in the reaction should be localized ones. On the other hand, the rates of electron transfer from reduced singly and doubly substituted plastocyanin to photooxidized P700 in the P700-chlorophyll alpha protein complex were similar to that of native plastocyanin, which suggests that these carboxyl residues have only a minor role in the electron transfer to P700. Although divalent cation is essential for the electron transfer from native plastocyanin to P700 at neutral pH, the triply substituted plastocyanin could donate electrons to P700 even without MgCl2, and the rate of this reaction reached the maximum at a low concentration of MgCl2 (less than 2.5 mM). The modification of four carboxyl residues per plastocyanin molecule activated this reaction to the maximum level without MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reverse electron transfer from the cytochrome c level in cyanide-resistant mitochondria of the yeast, Candida lipolytica]

The ability of cyanide-resistant mitochondria of yeast Candida lipolytica to perform reverse electron transfer from cytochrome c to alternative oxidase was studied. It was shown that the energy for such a transfer can be provided by high energy intermediates or membrane potential but not by ATP. Reverse electron transfer from cytochrome c is impossible due to energy of NADH and alpha-glycerophosphate oxidation via alternative pathway in the presence of cyanide. These results prove once again that electron transfer via alternative pathway is not connected with the energy accumulation.     

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.     

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.