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.     

Synthesis of free ATP from membrane-bound ATP in chromatophores of Rhodospirillum rubrum

Chromatophores of Rhodospirillum rubrum were preincubated with ³²P_i in the absence of added nucleotides. Particles and reaction mixture were then separated by sucrose density gradient centrifugation. The labeled chromatophores thus obtained esterify ³²P_i into acid-soluble ATP (ATP_as) on the addition of ADP in the dark. Additional firmly bound ATP (ATP_fb) can be liberated on sodium dodecylsulfate treatment. Coinciding with the formation of acid-soluble ATP there is a decrease in the amount of firmly bound ATP. The isotopic concentration experiments in which labeled chromatophores were incubated with carrier-free ³²P_i and ADP in the dark, show that ATP_as might arise from ATP_fb not by a direct γ-phosphate transfer but by an esterification of the added ADP and free phosphate with a concomitant hydrolysis of the ATP_fb. On this basis we have proposed a new working hypothesis for the last step of electron transport-linked phosphorylations. It includes the following reactions: + P*_i → P* (i.e., ATP_fb) P* + ADP + P**_i → ATP**_as + P*_i

The hypothesis is compatible with the concept of conformational energy conservation.     

Transient kinetics of the electron transfer between P-700, plastocyanin and cytochrome f in chloroplasts suspended in fluid media at sub-zero temperatures

The kinetics of redox changes of P-700, plastocyanin and cytochrome f in chloroplasts suspended in a fluid medium at sub-zero temperatures have been studied following excitation of the chloroplasts with either a single-turnover flash, a series of flashes or continuous light. The results show that: (1) The kinetics of reduction of P-700⁺ and those of oxidation of plastocyanin are consistent with a bimolecular reaction between these two components as previously suggested (Olsen, L.F., Cox, R.P. and Barber, J. (1980) FEBS Lett. 122, 13–16). (2) Cytochrome f shows heterogeneity with respect to its kinetics of oxidation by Photosystem I. (3) In contrast to the situation when plastoquinol is the electron donor, reduction of cytochrome f by electrons derived from diaminodurene occurs with sigmoidal kinetics that shows a good fit to an apparent equilibrium constant of 12 between the cytochrome and P-700. (4) The rate of electron transfer from plastoquinol to Photosystem I depends on the redox state of the plastoquinone pool. (5) In relation to current ideas about the lateral heterogeneity of Photosystem I and Photosystem II in the thylakoid membrane, the results are consistent with the function of plastocyanin as a mobile carrier of electrons in the intrathylakoid space.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

Laser-induced reactions of P700 and cytochrome f in a blue-green alga, Plectonema boryanum

Kinetics of the absorption change of P700 (blue band) and cytochrome f in whole cells of a blue-green alga, Plectonema boryanum, have been studied by Q-switched ruby-laser flash excitation (694 nm; approx. 20 nsec) to elucidate the sequential relationship of these two components in photosynthetic electron transport. “P700” was photooxidized within 2 μsec and recovered in two phases t_1/2 10 μsec and 200 μsec). Under the same conditions cytochrome f was oxidized with a half time of 15 μsec. The magnitude of the fast phase of “P700” recovery, however, diminished at lower laser intensity while the cytochrome f change remained unaffected. The result suggests that cytochrome f and P700 may not be on the same electron-transport chain.     

Modification of the apparent redox reaction between cytochrome f and the rieske iron-sulfur protein

We have investigated the role of cytochrome f and the Rieske FeS protein in spinach chloroplasts using the quinone analogue 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). UHDBT inhibits electron transport at two different sites in spinach chloroplasts. Fluorescence yield measurements monitoring the redox state of Q, the first stable primary acceptor of Photosystem II, and polarographic measurements of electron transport show that at low concentrations UHDBT inhibits near Q. At higher concentrations UHDBT inhibits at a second site. Electron transfer from durohydroquinone to methyl viologen is inhibited (50% inhibition at 21 μM) but not the reaction dichlorophenolindophenol to methyl viologen. Spectroscopic measurements of the kinetics of cytochrome f show that UHDBT inhibits the dark reduction rate of the cytochrome following a 100 ms flash (50% inhibition at 15 μM). By contrast, the oxidation kinetics of cytochrome f following a single-turnover flash are altered little by UHDBT; the initial rates are indistinguishable, and the half-time increases from 220 μs in the control to 285 μs in the presence of 15 μM UHDBT, largely because the extent of the cytochrome f oxidation is enhanced 1.4-fold in the presence of the inhibitor. In a single-turnover flash in the absence of UHDBT, we observe 38–48% of the total cytochrome f turning over, while in the presence of UHDBT we observe 60–69% of the cytochrome turning over. We interpret these results in terms of a linear rapid donor pool to Photosystem I, FeS → cytochrome f → plastocyanin → P-700, in which UHDBT inhibits by interacting with the Rieske FeS center. We conclude that the enhanced extent of cytochrome f oxidation in the presence of UHDBT is due to the removal of the Rieske FeS center from the rapid donor pool. As a consequence, removal of a single electron from the pool results in a greater cytochrome f oxidation. These results indicate that the Rieske FeS center and cytochrome f equilibrate in a time period comparable to the oxidation time of the cytochrome.     

Iron-containing proteins in Chromatium I. Solubilization of membrane-bound cytochrome

Variations in the nature of the iron-containing proteins in Chromatium sp. strain D, grown under varying conditions of substrate and as a function of growth phase, are investigated. The results obtained show no significant changes induced by manipulation of these parameters. At least 80% of the heme protein present is membrane-bound and can be released only by detergent treatment. All but a small fraction is of c type, a minor protoheme component (cytochrome b′) being present. The membrane-bound cytochrome cannot be identified with those which are freely soluble.     

Transient inhibition by ribose 5-phosphate of photosynthetic O2 evolution in a reconstituted chloroplast system

Photosynthetic oxygen evolution by a reconstituted chloroplast system utilising sn-phospho-3-glycerol (3-phosphoglycerate) ceases upon the addition of ribose 5-phosphate even though the presence of this metabolite permits a rapid and immediate CO₂ fixation. The period of cessation is appreciable at 0.1 mM ribose 5-phosphate. It is lengthened as the amount of added ribose 5-phosphate is increased and by the addition of dithiothreitol, a known activator of ribulose-5-phosphate kinase. Ribulose 1,5-bisphosphate is without effect. A similar interruption of O₂ evolution may also be brought about by the addition of ADP or by ADP-generating systems such as glucose plus hexokinase. Spectrophotometric experiments indicate that the reoxidation of NADPH in the presence of sn-phospho-3-glycerol is similarly affected.The transient inhibition by ribose 5-phosphate is not observed in the presence of an active ATP-generating system or in the presence of sufficient dl-glyceraldehyde to inhibit ribulose-5-phosphate kinase activity.It is concluded that ribose 5-phosphate inhibits photosynthetic O₂ evolution by adversely affecting the steady-state ATP/ADP ratio and consequently the reduction of sn-phospho-3-glycerol to glyceraldehyde 3-phosphate. The results are discussed in their relation to ADP regulation of photosynthetic carbon assimilation and metabolite transport.     

Studies on the uptake of radioactive copper by sheep erythrocytes in vitro

From previous studies different mechanisms for uptake of Cu by mammalian tissues from buffer and media containing proteins have been proposed. The interpretation of some of these investigations may have been complicated by the binding of Cu to proteins in the media.The uptake of ⁶⁴Cu from both buffer and plasma has therefore been studied using sheep erythrocytes in order to determine the mechanism and the effects of protein. Cu uptake was proportional to concentration of added Cu and the kinetics were those of a first order reaction for both media. There was no evidence for the participation of a membrane carrier and studies with inhibitors indicated that active transport was not involveed. For a given concentration of added Cu the rate of uptake was much slower in plasma and the effective concentration was calculated to be about 13% of that added. Efflux from labelled cells was much faster into plasma than into buffer. The addition of histidine to the medium increased uptake from dialysed plasma but not from buffer.It is concluded that Cu is taken up by the erythrocyte by simple diffusion, and the reduced rate in plasma is due to the binding of Cu to plasma protein thus reducing the effective concentration. The effect of histidine is attributed to the formation of a Cu-histidine complex which exists in equilibrium with Cu bound to albumin and facilitates removal of Cu from the latter.     

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.     

Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids

The lateral distribution of plastocyanin in the thylakoid lumen of spinach and pea chloroplasts was studied by combining immunocytochemical localization and kinetic measurements of P700+ reduction at high time resolution. In dark-adapted chloroplasts, the concentration of plastocyanin in the photosystem I containing stroma membranes exceeds that in photosystem II containing grana membranes by a factor of about two. Under these conditions, the reduction of P700+ with a halftime of 12 microseconds after a laser flash of saturating intensity indicates that to greater than 95% of total photosystem I a plastocyanin molecule is bound. An analysis of the labeling densities, the length of the different lumenal regions, and the total amounts of plastocyanin and P700 shows that most of the remaining presumable mobile plastocyanin is found in the granal lumen. This distribution of plastocyanin is consistent with a more negative surface charge density in the stromal than in the granal lumen. During illumination the concentration of plastocyanin in grana increases at the expense of that in stroma lamellae, indicating a light-driven diffusion from stroma to grana regions. Our observations provide evidence that a high concentration of plastocyanin in grana in the light favors the lateral electron transport from cytochrome b6/f complexes in appressed grana across the long distance to photosystem I in nonappressed stroma membranes.     

Interaction of Pseudomonas cytochrome cd1 with the cytoplasmic membrane

Labelling with ferritin-conjugated antibody shows that Pseudomonas cytochrome cd₁ is associated with the inner surface of the cytoplasmic membrane. Cytochrome cd₁ is, however, enriched to the soluble fraction obtained after destruction of Pseudomonas spheroplasts. Comparison of the respiratory nitrite reductase activities, due to this cytochrome, between different cellular fractions and the purified enzyme shows that while the kinetic pattern and the temperature dependence of the activity remain almost the same the molecular activity is enhanced when the enzyme is released from cells.A new assay of respiratory nitrite reductase was developed in this study. The method is based on determination of the stoichiometrical proton consumption accompanying nitrite reduction.     

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.