Biomedical subjects

E L Gross

Publications and source records attributed to E L Gross.

At least 19 recordsLinked to original sources

PubMed1998

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.

Algorithms↗

PubMed1997

Reconstitution and characterization of a divergent plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica, expressed in Escherichia coli.

Plastocyanin (PC) is a copper protein that serves as a mobile electron carrier between cytochrome f and Photosystem I in the light reactions of photosynthesis. Despite large variability in amino acid sequences and isoelectric points, PCs from cyanobacterial and chloroplast sources reveal considerable similarities with respect to their secondary and tertiary structures. In this paper, we have expressed in Escherichia coli a PC from the prokaryote Prochlorothrix hollandica, and efficiently reconstituted the protein with copper under conditions yielding the characteristics of a native holoPC, as judged by redox titration (Eo' = +376 mV), near and far UV circular dichroism, and electron paramagnetic resonance (EPR) spectroscopy. By comparison of amino acid sequences, P. hollandica PC is the most divergent homolog identified to date, and analysis of this reconstituted preparation may reveal new insights as to the structural requirements for electron transport between the PC copper center and neighboring reaction partners.

Circular Dichroism↗

PubMed1996

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.

Binding Sites↗

PubMed1992

Thermal denaturation of plastocyanin: the effect of oxidation state, reductants, and anaerobicity.

The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and EDTA. It was found that the copper center and its ligands play a crucial role in maintaining the stability of PC. Thermal denaturation was monitored by using far-uv circular dichroism (CD) spectra to monitor changes in secondary structure, the near-uv CD ellipticity at 280 nm to monitor changes in tertiary structure, and the absorbance at 597 nm and the 255-nm CD transition to monitor changes in the copper center. Reduced PC (Tm = 71 degrees C) was found to be more stable than the oxidized form (Tm = 61 degrees C). The Tm was increased by addition of reductants, removal of oxygen, or addition of EDTA. Two distinct denatured forms (designated D1 and D2) were separated by anion exchange fast protein liquid chromatography. Neither form contained a native copper center. Form D2 retained the characteristic 280-nm CD band but showed an altered far-uv CD spectrum. Its formation was inhibited by the addition of reductants or the removal of oxygen. It could be refolded to form native, Cu-PC upon incubation with copper plus a reductant such as dithionite. These results suggest that its formation involves the reversible oxidation of a group on the PC molecule, possibly a ligand to the copper such as Cys 84 or Met 92. Form D1 occurred in the presence of ferricyanide or at high temperatures in the presence of oxygen. EDTA inhibited its formation. Form D1 lost the 280-nm CD transition and its far-uv CD spectrum was altered. No renaturation was observed suggesting that Form D1 is the product of an irreversible oxidation step possibly involving a histidine ligand to the copper. Forms D1 and D2 are not interconvertible and represent the endpoints of two different denaturation pathways.

Anaerobiosis↗

PubMed1991

The interaction of nitrotyrosine-83 plastocyanin with cytochromes f and c: pH dependence and the effect of an additional negative charge on plastocyanin.

Spinach plastocyanin was selectively modified using tetranitromethane which incorporates a nitro group ortho to the hydroxyl group of tyrosine 83 (Anderson, G.P., Draheim, J.E. and Gross, E.L. (1985) Biochim. Biophys. Acta 810, 123-131). This tyrosine residue has been postulated to be part of the cytochrome f binding site on plastocyanin. Since the hydroxyl moiety of nitrotyrosine 83 is deprotonated above its pK of 8.3, it provides a useful modification for studying the effect of an extra negative charge on the interaction of plastocyanin with cytochrome f. No effect on cytochrome f oxidation was observed at pH 7 under conditions in which the hydroxyl moiety is protonated. However, the rate of cytochrome f oxidation increased at pH values greater than 8, reaching a maximum at pH 8.6 and decreasing at still higher pH values. The increase was half-maximal at pH 8.3 which is the pK for the hydroxyl moiety on nitrotyrosine 83. In contrast, the rate of cytochrome f oxidation for control plastocyanin was independent of pH from pH 7 to 8.6. These results show that increasing the negative charge on plastocyanin at Tyr-83 increases the ability to react with cytochrome f, supporting the hypothesis that cytochrome f interacts with plastocyanin at this location. In contrast, the reaction of Ntyr-83 plastocyanin with mammalian cytochrome c was independent of pH, suggesting that its mode of interaction with plastocyanin is different from that of cytochrome f. A comparison of the effects of Ntyr-83 modification of plastocyanin with the carboxyl- and amino-group modifications reported previously suggests that plastocyanin binds to cytochrome f in such a way that electrons could be donated to plastocyanin at either of its two binding sites.

Cytochrome c Group↗

PubMed1991

The use of monoclonal antibodies to study the structure and function of cytochrome f.

Monoclonal antibodies (MAbs) were prepared against native cytochrome f (cyt f) isolated from turnip leaves. The two MAbs obtained, designated MAb-JB2 and MAb-ED4, were Western blot positive to purified turnip cytochrome f and also reacted with inside-out (ISO) but not right-side-out (RSO) spinach thylakoid membranes. MAb-ED4 reacted with a covalent adduct formed by crosslinking cyt f and plastocyanin (PC), whereas MAb-JB2 did not. In contrast, MAb-JB2 reacted with the isolated cyt b6/f complex but MAb-ED4 did not. These results indicate that MAb-JB2 binds to cyt f at or near the PC binding site on f, whereas MAb-ED4 binds to a portion of cyt f which is not exposed in the cyt b6/f complex. The location of the epitopes in the primary sequence of cyt f was determined by trypsin hydrolysis, HPLC separation of tryptic peptides, and ELISA identification of the purified peptides. The molecular weights of the purified peptides, determined by gel exclusion chromatography, were found to be 5040 and 3130 Da for MAb-JB2 and MAb-ED4, respectively. Amino acid sequencing showed that the first eight amino acids of the MAb-ED4 positive peptide were L-D-Q-P-L-T-S-N. These results suggest that the 3130-Da peptide has 28 amino acids extending from Leu 223 to Arg 250. This peptide is located on the N-terminal (lumen) side of the postulated membrane-spanning sequence. The first eight amino acids of the MAb-JB2-positive peptide were N-I-L-V-I-G-P-V. This sequence and the peptide molecular weight indicate that the epitope for MAb-JB2 is located within a 44-amino acid peptide extending from Asn 111 to Arg 154.

Amino Acid Sequence↗

PubMed1990

Chemical modification of spinach plastocyanin using 4-chloro-3,5-dinitrobenzoic acid: characterization of four singly-modified forms.

Chemical modification of plastocyanin was carried out using 4-chloro-3,5-dinitrobenzoic acid, which has the effect of replacing positive charges on amino groups with negatively charged carboxyl groups. Four singly-modified forms were obtained which were separated using anion exchange FPLC. The four forms were modified at the N-terminal valine and at lysines 54, 71 and 77. The rates of reaction with mammalian cytochrome c were increased for all four modified plastocyanins. In contrast, the rates of reaction with cytochrome f were inhibited for the forms modified at residues 1, 54 and 77, whereas no effect was observed for the form modified at residue 71. Modification had no effect on either the midpoint redox potential or the reaction with K3Fe(CN)6. These results are consistent with a model in which charged residues on plastocyanin located at or near the binding site for cytochrome f recognize the positively-charged binding site on cytochrome f. In contrast, charged residues located at points on plastocyanin distant from the cytochrome f binding site recognize the net negative charge on the cytochrome f molecule. Based on these considerations, Glu-68 may be within the interaction sphere of cytochrome f, suggesting that cytochrome f may donate electrons to plastocyanin at either Tyr-83 or His-87.

Amino Acid Sequence↗

PubMed1990

Factor analysis of the near-ultraviolet absorption spectrum of plastocyanin using bilinear, trilinear, and quadrilinear models.

Factor analysis was used to resolve the spectral components in the near-uv absorption spectrum of plastocyanin. The data set was absorption as a function of four variables: wavelength, species of plastocyanin, oxidation state of the copper center, and environmental pH. The data were fit with the traditional bilinear model, as well as with trilinear and quadrilinear models. Trilinear and quadrilinear models have the advantage that they uniquely define the components, avoiding the indeterminacy of bilinear models. Bilinear analysis using the absorption spectra of tyrosine and copper metallothionein as targets resulted in a two-component solution which was nearly identical to that obtained using trilinear and quadrilinear models, for which no targets are required. The two-component models separate the absorption into tyrosine and copper center components. The absorption of tyrosine is found to be pH dependent in reduced plastocyanin, and the absorption magnitude of the reduced copper center is the same in the four different plastocyanin species. Further resolution is provided by a three-component quadrilinear model. The results indicate that there are at least two different electronic transitions which cause the absorption of the reduced copper center and that one of them couples to a tyrosine residue. It is the absorption of this coupled tyrosine residue which is pH dependent. Correlation of the results with previous studies indicates that it is Tyr 83 which is the perturbed residue. The separation of the absorption of the copper center and Tyr 83 provides spectroscopic probes for the conformations of the north pole and east face reaction sites on the plastocyanin protein.

Binding Sites↗

PubMed1990

Modeling of the electrostatic potential field of plastocyanin.

The DelPhi computer program is used to calculate the electrostatic potential field of the photosynthetic electron transport protein plastocyanin. Knowledge of the potential field is important for understanding the mechanisms by which plastocyanin interacts with other charged reagents. The program uses a macroscopic, continuum approach in which the protein and solvent are assigned different dielectric constants, the crystal structure of the protein defines the dielectric boundary, and the ionic strength of the solvent is taken into account. The potential field is determined by numerically solving the Poisson-Boltzmann equation. The field surrounding plastocyanin is characterized by a region of positive potential over the copper center active site, and a region of negative potential over the adjacent association site containing tyrosine 83. The shape and magnitude of the potential field shows a strong dependence on the ionic strength and pH of the solvent. The program is able to accurately predict the effect of the copper center oxidation state on the pKa of a tetranitromethane derivative of tyrosine 83 using an intrinsic protein dielectric constant of 2 to 4. Evidence is also presented that the glutamate 68 side chain is exposed to the solvent to a greater extent in the solution structure of plastocyanin than in the crystal structure.

Amino Acid Sequence↗

PubMed1988

Analysis of the near-ultraviolet absorption and circular dichroic spectra of parsley plastocyanin for the effects of pH and copper center conformation changes.

The absorption and circular dichroic (CD) spectra of parsley plastocyanin (PC) were measured in order to determine the effects of changes in primary amino acid sequence on both the copper center and protein components of the PC molecule. The near-ultraviolet (uv) absorption and CD spectra of parsley PC were found to be qualitatively similar to those of spinach, poplar, and lettuce PC, except for the near-uv CD spectrum of the reduced form at low pH (ca. pH 5.0). The CD spectrum of reduced parsley PC in the 250-265 nm wavelength region changes from positive to negative ellipticity upon reduction of pH, and is characterized by a pKa value of 5.7. This pKa value is the same as that for the protonation of the histidine 87 copper ligand, observed by NMR, and the change in conformation of the copper center. Similar processes are believed to occur in the other PC species at lower pH values. Thus, the pH-dependent perturbations of the near-uv CD spectra of reduced PC are interpreted as due to transitions in the reduced copper center. The increase in the near-uv absorption spectrum of reduced PC can be divided into pH-independent and pH-dependent portions. The pH-independent portion resembles the absorption spectrum of tetrahedral Cu(I) metallothionein, suggesting the presence of Cu(I)-Cys 84 and/or Cu(I)-Met 92 charge transfer transitions in the near-uv absorption spectra of reduced PC. The pH dependence of the absorption spectrum changes and the pH difference absorption spectrum indicate that tyrosine residues may contribute to at least a part of the pH-dependent portion of the absorption increase of reduced PC.

Chlorophyll↗

PubMed1987

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.

Amino Acid Sequence↗

PubMed1987

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.

Chromatography, Affinity↗

PubMed1986

Determination of the redox potential and diffusion coefficient of the protein plastocyanin using optically transparent filar electrodes.

Direct and mediated electrolysis of the protein plastocyanin at a gold filar electrode is described. The filar electrode used is of a unique design that allows potentiometric measurements, steady-state voltammetry and absorption spectrophotometry to be performed on a few microliters of solution containing 0.1-1.0 mM protein. As a result, we have determined the formal potential and diffusion coefficient of the blue copper protein, plastocyanin, to be 372 +/- 5 mV vs. normal hydrogen electrode and 8.9 X 10(-7) cm2 X s-1, respectively. The same value of the formal potential is obtained from a steady-state current experiment, an equilibrium spectrophotometric experiment, and a twin-electrode steady-state spectrophotometric experiment. The fact that the diffusion coefficient is measured under conditions of steady-state current, results in significant improvement in signal to background over techniques that monitor a transient current, while the potential is changing.

Electrochemistry↗

PubMed1986

Plastocyanin conformation. An analysis of its near ultraviolet absorption and circular dichroic spectra.

The near-ultraviolet absorption and circular dichroic spectra of plastocyanin are dependent upon the redox state, solution pH, and ammonium sulfate concentration. This dependency was observed in plastocyanin isolated from spinach, poplar, and lettuce. Removal of the copper atom also perturbed the near-ultraviolet spectra. Upon reduction there are increases in both extinction and ellipticity at 252 nm. Further increases at 252 nm were observed upon formation of apo plastocyanin eliminating charge transfer transitions as the cause. The spectral changes in the near-ultraviolet imply a flexible tertiary conformation for plastocyanin. There are at least two charge transfer transitions at approximately 295-340 nm. One of these transitions is sensitive to low pH's and is attributed to the His 87 copper ligand. The redox state dependent changes observed in the near-ultraviolet spectra of plastocyanin are attenuated either by decreasing the pH to 5 or by increasing the ammonium sulfate concentration to 2.7 M. This attenuation cannot be easily explained by simple charge screening. Hydrophobic interactions probably play an important role in this phenomenon. The pH and redox state dependent conformational changes may play an important role in regulating electron transport.

Circular Dichroism↗

PubMed1985

Plastocyanin conformation: the effect of the oxidation state on the pKa of nitrotyrosine-83.

Plastocyanin treated with tetranitromethane was nitrated at a single location, Tyr-83. Tyr-83 and its neighboring negative charges have been implicated as a binding site for positively charged redox agents (Chapman, S.K., Watson, A.D. and Sykes, A.G. (1983) J. Chem. Soc. Dalton Trans. 1983, 2543-2548). No effect was observed on either the plastocyanin midpoint redox potential or its reaction kinetics with P-700+ and cytochrome f. This makes nitration an ideal spectroscopic probe for monitoring changes in the environment of Tyr-83. The pKa of the nitrotyrosine was 8.6 and 8.3 for reduced and oxidized plastocyanin, respectively, indicating that the charge on the copper atom is 'felt' at Tyr-83. The high pKa value for both forms indicates that Tyr-83 is in a negatively charged environment, near residues Nos. 42-45 and Nos. 59-61. The extinction of the nitrotyrosine chromophore at 360 nm was not affected by a change in redox state. However, the ellipticity of this transition was greater for the oxidized form, indicating that environment of Tyr-83 is dependent upon the charge on the copper atom. This suggests an electrostatically driven conformational change at Tyr-83. A conformational change at Tyr-83 could regulate the binding of plastocyanin with its reaction partners in order to promote smooth electron transport.

Chlorophyll↗

PubMed1985

Plastocyanin conformation. The effect of nitrotyrosine modification and pH.

Plastocyanin isolated from several species including spinach, poplar, and lettuce showed conformational changes both upon reduction and upon lowering the pH as determined by near-ultraviolet absorption and fluorescence measurements. The fluorescence excitation maximum was at 278 nm for all species of plastocyanin measured. In the case of spinach, the emission maximum was at 310-312 nm, similar to a tyrosine residue in solution. The fluorescence intensity increased 22% upon reduction of plastocyanin at pH 7.0. In poplar plastocyanin, the emission maximum was shifted to 335 nm and increased only 10% upon reduction. The 335 nm emission peak observed in poplar plastocyanin is attributed to Tyr 80 which is hydrogen bonded to a carbonyl group on the protein backbone. Tyr 83 was also shown to undergo fluorescence changes upon reduction since the redox state-dependent fluorescence changes decreased for a nitrotyrosine (nitrotyrosine-plastocyanin) derivative of this residue. These results show that the east face of the molecule, which contains both Tyr 80 and 83 as well as a possible binding site, undergoes conformational changes upon reduction. These conformational changes may be involved in promoting smooth electron transport between plastocyanin and its reaction partners. Both the absorption and fluorescence were found to be pH dependent. The quantum yield for fluorescence increased sharply below pH 6 for both oxidized and reduced spinach plastocyanin. This may be related to the appearance of a redox-inactive form of reduced plastocyanin. The conformational changes observed at low pH may provide a mechanism for control of electron transport by the proton gradient. Low concentrations of CaCl2 (10 mM) had no effect on plastocyanin fluorescence. However, addition of 2.7 M (NH4)2SO4 eliminated the redox-dependent fluorescence changes.

Chloroplasts↗

PubMed1985

Conformational changes in plastocyanin.

The visible and near-uv absorption and circular dichroic spectra were determined for spinach and poplar plastocyanin under a variety of conditions. The visible spectra showed that the copper center was invariant to changes in species, chemical modification with ethylenediamine, and addition of high concentrations of salt [2.7 M (NH4)2SO4]. In contrast, the near-uv spectra were sensitive to these conditions. Reduction of plastocyanin also altered its near-uv absorption and circular dichroic spectra. It is unlikely that these spectral changes were due to charge transfer bands since the near-uv CD spectrum of apo-plastocyanin was almost identical to that of reduced plastocyanin. There were no corresponding changes in the far-uv spectra which monitor protein secondary structure. The most likely explanation is that the protein has a flexible tertiary conformation. Conformational changes may be important in regulating electron transport. If plastocyanin is a mobile electron carrier, differential binding of the oxidized and reduced forms of plastocyanin to its reaction partners cytochrome f and P700 could facilitate electron transport.

Apoproteins↗

PubMed1984

Variable fluorescence of photosystem I particles and its application to the study of the structure and function of photosystem I.

Chlorophyll a fluorescence in Photosystem I (PSI) particles isolated according to the method of Bengis and Nelson [J. Biol. Chem. 252, 4564-4569 (1977)] was found to be dependent on the redox state of both P700 and X (an acceptor on the reducing side of PSI). Addition of dithionite plus neutral red to PSI caused an increase in fluorescence intensity and a shift of the main fluorescence peak from 689 to 674 nm. Addition of electron acceptors such as ferredoxin and methyl viologen decreased the fluorescence yield when added to PSI incubated under anaerobic conditions in the presence of excess dichlorophenol indophenol (DCIPH2). The Km for ferredoxin agreed with that determined from direct measurements of ferredoxin reduction, showing that X is a quencher of fluorescence. P700 was also found to be a quencher of fluorescence, since electron donors such as DCIPH2, TMPD, and plastocyanin decreased fluorescence with Km's nearly identical to those observed for P700+ reduction. Chemical modification of PSI (with ethylene diamine + a water-soluble carbodiimide) to make it positively charged increased the fluorescence yield and shifted the 689-nm peak to 674 nm. The Km's for DCIPH2 and ferredoxin were decreased. In contrast, modification of PSI with succinic anhydride, which increased the net negative charge, increased the Km for ferredoxin. Salts affected the interaction of methyl viologen with PSI. Both anion and cation selectivity were observed. Limited proteolysis increased the Km for both methyl viologen and ferredoxin, indicating that their binding site on PSI was altered. These results suggest that the binding site for ferredoxin is on either the 70- or the 20-kDa subunit of PSI.

Chlorophyll↗