Elizabeth L. Gross

Correlation between monovalent cation-induced decreases in chlorophyll a fluorescence and chloroplast structural changes

Abstract Low concentrations (~ 3 m m ) of salts of monovalent cations such as Na + , K + , and tetraethylammonium were found to decrease the turbidity of chloroplast suspensions. The turbidity changes (Δ 540 ) had the same kinetics, salt concentration dependence, and pH dependence as the monovalent cation-induced decreases in chlorophyll a fluorescence (9), suggesting that structural changes are the cause of the associated increases in spillover. Electron microscopy revealed that the grana are stacked when spillover is inhibited (in the absence of salts or the presence of divalent cations) and that monovalent cations cause the grana to unstack, thereby promoting spillover.

Monovalent cation-induced inhibition of chlorophyll a fluorescence: Antagonism by divalent cations

Abstract Salts of monovalent cations at concentrations less than 10 m m and buffers such as tricine were found to increase spillover from Photosystem II to Photosystem I in green plant photosynthesis as measured by a decrease in chlorophyll a fluorescence at room temperature. At 77 °K, they increased the fluorescence emission at 735 nm relative to the bands at 685 and 693 nm indicating that Photosystem I was receiving a greater part of the excitation energy. Divalent cations and monovalent cations at concentrations greater than 10 m m reversed the fluorescence changes.

Plastocyanin: Structure and function

The aim of this review is to analyze the current state of knowledge concerning the blue copper protein plastocyanin (PC) focusing on its interactions with its reaction partners cytochromef and P700. Plastocyanin is a 10 kD blue copper protein which is located in the lumen of the thylakoid where it functions as a mobileelectron carrier shuttling electrons from cytochromef to P700 in Photosystem I. PC is a typical β-barrel protein containing a single copper center which is ligated to two histidines, a methionine and a cysteine in a distorted tetrahedral geometry. PC has two potential binding sites for reaction partners. Site 1 consists of the H87 ligand to the copper and Site 2 consists of Y83 which is surrounded by two clusters of negative charges which are highly conserved in higher plant PCs.The interaction of PC with cytochromef has been studied extensively. It is electrostatic in nature with negative charges on PC interacting with positive charges on cytochromef. Evidence from cross-linking, chemical modification, kinetics and site-directed mutagenesis studies implicate Site 2 as the binding site for Cytf. The interaction is thought to occur in two stages: an initial diffusional approach guided by electrostatic interactions, followed by more precise docking to form a final electron transfer complex.Due to the multisubunit nature of the Photosystem I complex, the evidence is not as clear for the binding site for P700. However, a small positively-charged subunit (Subunit III) of Photosystem I has been implicated in PC binding. Also, both chemical modification and site-directed mutagenesis experiments have suggested that PC interacts with P700 via Site 1.

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.

Differentiation of chloroplast lamellae. Onset of cation regulation of excitation energy distribution.

Abstract A parallel pattern of appearance of (i) divalent cation regulation of excitation energy distribution, (ii) low affinity divalent cation binding sites, and (iii) synthesis of a light-harvesting chlorophyll a b protein were observed in greening chloroplast membranes. It is suggested that the pigment-protein complex possesses binding sites for cations. Cation binding to these sites may be involved in the primary process regulating excitation energy distribution between photosystems I and II.

Correlation between calcium ion binding to chloroplast membranes and divalent cation-induced structural changes and changes in chlorophyll a fluorescence

Abstract 1. 1. Two distinct sites were obseved for Ca2+ binding to chloroplast membranes. Site I has a number of sites equal to 0.65 μmole/mg chlorophyll and has a dissociation constant of 8 ± 3 μ M . This site is the same as that previously observed for Mn2+ (Gross, E. L. (1972) Arch. Biochem. Biophys. 150, 324–329) and is responsible for reversal of quaternary ammonium salt uncoupling (Gross, E. L. (1971) Arch. Biochem. Biophys. 147, 77–84). 2. 2. Site II has a number of sites equal to 0.5 ± 0.2 μ mole/mg chlorophyll and a dissociation constant of 51 ± 8 μ M . Other divalent cations such as Mn2+ and Mg2+ also bind to this site. 3. 3. Divalent cation binding to Site II was correlated with divalent cation-induced structural changes. 4. 4. Monovalent cations such as K+ and tetraethylammonium also bind to this site but do not cause structural changes. Another explanation for monovalent cation-induced structural changes must be found. 5. 5. Divalent cation binding was also correlated with divalent cation-induced changes in chlorophyll a fluorescence. 6. 6. The divalent cations were found to bind to previously unoccupied sites on the chloroplast membrane rather than to exchange for other cations already present.

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.

Protein-Protein interactions of light-harvesting pigment protein from spinach chloroplasts. I. Ca2+ binding and its relation to protein association

The role of divalent cations in the regulation of the distribution of excitation energy between the two photosystems involved in green plants photosynthesis has led us to search for a better understanding of how such phenomena might occur at the molecular level. Since small changes in orientation of and distance between pigment molecules could greatly affect the distribution of excitation energy, we have decided to study the effects of ions on the light-harvesting pigment protein from spinach chloroplasts. The light-harvesting pigment protein is shown to have two types of binding sites for Ca-2+. Binding studies and analytical ultracentrifugation indicate that site I (K-d equals 2.5 mu-M, n equals 1.5-4.0 mu-mol Ca-2+ bound/mg chlorophyll) is lost as the protein associates. Site II (K-d equals 32 mu-M, n equals 9,5 mu-mol Ca-2+/mg chlorophyll) is not affected by the association of the protein. This site is responsible, however, for a further divalent cation-dependent association of the protein. The possible role of this protein in grana stacking and control of spillover is discussed.

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.

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.