Reduction plastocyanin

These mobile electron carriers are relatively low-molecular-weight proteins and are both linked to the PS-1 reaction-center complex by electrostatic forces. Plastocyanin is located on the lumen side and is the electron donor to P700, while ferredoxin is located on the stroma side and receives electrons from the terminal, membrane-bound iron-sulfur proteins FeS-A/B. Water is the ultimate source of electrons for plastocyanin reduction. The electron from water is generated by its oxidation by photosystem 11, and transferred by way of cytochrome/in the cytochrome b(f complex. Reduction of NADP by ferredoxin requires mediation by the enzyme ferredoxin-NADP -reductase orFNR. Figure 1, right is a schematic representation of the relationship of the PS-1 reaction center to the peripheral electron carriers plastocyanin and ferredoxin as well as the protein catalyst ferredoxin-NADP -reductase. 

The reaction-center proteins for Photosystems I and II are labeled I and II, respectively. Key Z, the watersplitting enzyme which contains Mn P680 and Qu the primary donor and acceptor species in the reaction-center protein of Photosystem II Qi and Qt, probably plastoquinone molecules PQ, 6-8 plastoquinone molecules that mediate electron and proton transfer across the membrane from outside to inside Fe-S (an iron-sulfur protein), cytochrome f, and PC (plastocyanin), electron carrier proteins between Photosystems II and I P700 and Au the primary donor and acceptor species of the Photosystem I reaction-center protein At, Fe-S a and FeSB, membrane-bound secondary acceptors which are probably Fe-S centers Fd, soluble ferredoxin Fe-S protein and fp, is the flavoprotein that functions as the enzyme that carries out the reduction of NADP+ to NADPH. 

Results obtained for the blue Cu protein plastocyanin are considered here in detail as illustrative of different approaches yielding relevant information. The reduction of plastocyanin PCu(II) with cytochrome c(II) is also considered as an example of a protein-protein reaction. 

Summary of reduction potentials and rate constants for reactions with plastocyanin PCu(I) and PCu(II) at 25<>C, pH 7-8,... 

Figure 7. The variation of rate constants k (25 °C) with pH for the reduction of parsley plastocyanin PCu(II) with Ru(NH3)5py2+ (%) and cytochrome c(II) (A)...

Reaction with Cr(III) Modified Plastocyanin. From thermo-lysin proteolysis experiments Farver and Pecht (20) have concluded that reduction of PCu(II) with labelled Cr0 20)52+ (1 1 mole amounts) at pH 7 gives a product in which Cr(III) is attached to the peptide chain 40-49. Coordination of the Cr at one or two carboxylates in the 42-45 patch is favoured. It has now been demonstrated that rate constants (25oC) for the reaction of PCu(I).Cr(III) + Co(phen)33+ are decreased by 16%. 

PS1 The PS 1-prep, introduced in this communication is the first reported with a polyhistidine tag fused to the N-terminus of the PsaF subunit. This construct was possible due to the fact that cyanobacterial PsaF-deletion mutants show no impact on photoautotrophic growth - in contrast to Chlamydomonas reinhardtii, where inactivation of PsaF results in a severe reduction of electron transfer from plastocyanin to PS 1 [Hippier et al. 1997], Also, the N-terminus of the F-subunit which was decorated by the tag is located towards the lumen side which enables an attachment of the isolated PS1 with the lumen-exposed /donor-side to the electrode surface in our hydrogen-producing device. 

It is interesting to note that the plastocyanin from the alga Anabaena variabilis has a similar structure to that isolated from poplar, while having a different amino acid sequence.58 This results in an overall positive charge of +2. Thus, as shown in Figure 32a, it affords a wellshaped response for the Cu(II)/Cu(I) reduction E° = + 0.32 V vs. NHE) using a perpendicular pyrolytic graphite electrode, without the presence of additives. 

Negative values for redox couple entropy have also been obtained for the Cu(II)/Cu(I) reduction, in aqueous medium, of the blue copper proteins stellacyanin, plastocyanin and azurin.14 The decrease in molecular disorder has been attributed in this case to the fact that the charge neutralization of the redox site (from + 1 to 0) favours the formation of hydrogen bonds between the solvent (water) and the copper centre.17... 

Reduction potentials (Eg) for different plastocyanins, the PCu(II)/PCu(I) couple, have been determined by spectrophotometric titration against, e.g. [Fe(CN)e] . At pH 7.5 for higher plant and green algal plastocyanins values are close to 370 mV at 25 °C, 1=0.10 M(NaCl) [1]. Thus French bean gives a value 360 mV and S. obliquus 363 mV [50]. However, A. variabilis gives an... 

Earlier suggestions that the two uncoordinated and invariant residues His35 (inaccessible to solvent and covered by polypeptide) and His83 (remote and 13 A from Cu) are, from effects of [H ] on rate constants (and related pKg values), sites for electron transfer may require some re-examination. Thus, it has been demonstrated in plastocyanin studies [50] that a surface protonation can influence the reduction potential at the active site, in which case its effect is transmitted to all reaction sites. In other words, an effect of protonation on rate constants need not necessarily imply that the reaction occurs at the site of protonation. His35 is thought to be involved in pH-dependent transitions between active and inactive forms of reduced azurin [53]. The proximity of... 

The kinetics of the reduction of spinach plastocyanin PCu(II) by the optically active complexes 2,6-bis[3-(S)- or 3-(/ )-carboxyl-2-azabutyl] pyridine, here abbreviated to (S,S)- or (f ,/ )-ALAMP have been studied [107]. The latter enantiomer (A-configuration) reacts 1.6-2.0 times faster at different values of pH and temperature than the S,S form. Activation parameters have shown that the observed stereoselectivity is a consequence of the difference in activation... 

Whatever the explanation, the sensitivity of the remote pK to oxidation state of the Cu is of potential importance in relation to the functional role of plastocyanin. Plastocyanin and its physiological electron transport partner cytochrome f are believed to have complementary surfaces which lead to efficient interaction prior to electron transfer. As will be seen below there is substantial evidence for cytochrome f(II) (as reductant) reacting at the remote site of PCu(II). One problem which may be anticipated here is how dissociation of the product... 

Reduction potentials of the S. obliqms His59 Ru(NH3)5-modified protein have been determined by cyclic voltammetry using as electrode the oxidized surface obtained by polishing the edge plane of pyrolytic graphite [137]. The modified protein responds well at the electrode, whereas the native protein requires multi-eharged cations, e.g. Mg or [Cr(NH3)g] as mediators to give satisfactory reversibility. Separate reduction potentials at 1=0.10 M(NaCl) for native S. obliquus plastocyanin (389 mV) and [Ru(NH3)5 (imidazole)]... 

The kinetics of the reactions of horse cytochrome c(II), M, 12,400, (charge 8+) reduction potential 260 mV, with parsley and French bean plastocyanins PCu(II) (charges — 7 and — 8 respectively), have been studied. As in the case of HIPIP, cytochrome c is not a physiologically relevant protein. It is nevertheless important in assessing different approaches prior to investigating the reactions of physiological redox partners. In the case of the reaction of parsley PCu(II) with cytochrome c(II), the rate constant (25 °C) is 1.5 X 10 s at pH7.6, 1 = 0.10 M(NaCl) [141]. There is no evidence... 

Table 11. Rate eonstants/M s (10°C) for the reduction of different plastocyanins, PCu(II), by cabbage (Brassica oleracea) cytochrome f(Il) at two different pH s, 1=0.20 M(NaCl) ...

Four products were obtained containing between 2 and 6 moles of amine per plastocyanin. More recently the reaction has been carried out to yield single and double modified products. No effect of modification at 42-45 on the rate of the PC u(I) reduction of P700 is reported, suggesting that these residues are not involved in the reaction. In a further elaboration the reaction of plastocyanin, cross-linked (by carbodiimide treatment) to cytochrome f at the remote site, with P700 has been studied and the effects of Mg " and Na" explored [158]. 

The reduction of the ruthenated plastocyanin protein PCu"Ru by CO 5 results in 72% PCu Ru and 28% PCu Ru . This very fast stage is followed by a slower one in which PCu Ru" is converted by a first order process into PCu Ru For this conversion... 

Blue copper proteins, 36 323, 377-378, see also Azurin Plastocyanin active site protonations, 36 396-398 charge, 36 398-401 classification, 36 378-379 comparison with rubredoxin, 36 404 coordinated amino acid spacing, 36 399 cucumber basic protein, 36 390 electron transfer routes, 36 403-404 electron transport, 36 378 EXAFS studies, 36 390-391 functional role, 36 382-383 occurrence, 36 379-382 properties, 36 380 pseudoazurin, 36 389-390 reduction potentials, 36 393-396 self-exchange rate constants, 36 401-403 UV-VIS spectra, 36 391-393 Blue species... 

Fig. 7 Geometry of a typical type I copper site in plastocyanin. Bond lengths and bond angles are relatively constant upon reduction or oxidation of the copper site (J. M. Guss, P. R. Harrowell, M. Murata et al.,J. Mol. Biol. 1986, 792, 361-387).

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