Negatively charged residues in the H loop of PsaB subunit in Photosystem I from Synechocystis sp. PCC 6803 appear to be responsible for electrostatic repulsions with plastocyanin*.

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125-10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF-PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been.

Site-directed mutagenesis of cytochrome c6 from Synechocystis sp. PCC 6803. The heme protein possesses a negatively charged area that may be isofunctional with the acidic patch of plastocyanin.

This paper reports the first site-directed mutagenesis analysis of any cytochrome c6, a heme protein that performs the same function as the copper-protein plastocyanin in the electron transport chain of photosynthetic organisms. Photosystem I reduction by the mutants of cytochrome c6 from the cyanobacterium Synechocystis sp. PCC 6803 has been studied by laser flash absorption spectroscopy. Their kinetic efficiency and thermodynamic properties have been compared with those of plastocyanin mutants from the same organism. Such a comparative study reveals that aspartates at positions 70 and 72 in cytochrome c6 are located in an acidic patch that may be isofunctional with the well known "south-east" patch of plastocyanin. Calculations of surface electrostatic potential distribution in the mutants of cytochrome c6 and plastocyanin indicate that the changes in protein reactivity depend on the surface electrostatic potential pattern rather than on the net charge modification induced by mutagenesis. Phe-64, which is close to the heme group and may be the counterpart of Tyr-83 in plastocyanin, does not appear to be involved in the electron transfer to photosystem I. In contrast, Arg-67, which is at the edge of the cytochrome c6 acidic area, seems to be crucial for the interaction with the reaction center.

The 2.15 A crystal structure of a triple mutant plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803.

The crystal structure of the triple mutant A42D/D47P/A63L plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 has been determined by Patterson search methods using the known structure of the poplar protein. Crystals of the triple mutant A42D/D47P/A63L, which are stable for days in its oxidized form, were grown from ammonium sulfate, with the cell constants a = b = 34.3 A and c = 111.8 A belonging to space group P3(2)21. The structure was refined using restrained crystallographic refinement to an R-factor of 16.7% for 4070 independent reflections between 8.0 and 2.15 A with intensities greater than 2 sigma (I), with root mean square deviations of 0.013 A and 1.63 degrees from ideal bond lengths and bond angles, respectively. The final model comprises 727 non-hydrogen protein atoms within 98 residues, 75 water molecules and a single copper ion. The overall tertiary fold of Synechocystis plastocyanin consists of a compact ellipsoidal beta-sandwich structure made up of two beta-sheets embracing a hydrophobic core. Each sheet contains parallel and antiparallel beta-strands. In addition to the beta-sheets, the structure contains an alpha-helix from Pro47 to Lys54 that follows beta-strand 4. The three-dimensional structure of Synechocystis plastocyanin is thus similar to those reported for the copper protein isolated from eukaryotic organisms and, in particular, from the cyanobacterium Anabaena variabilis, the only cyanobacterial plastocyanin structure available so far. The molecule holds an hydrophobic region surrounding His87, as do other plastocyanins, but the lack of negatively charged residues at the putative distant remote site surrounding Tyr83 could explain why the Synechocystis protein exhibits a collisional reaction mechanism for electron transfer to photosystem I (PSI), which involves no formation of the transient plastocyanin-PSI complex kinetically observed in green algae and higher plants.

Changes in the reaction mechanism of electron transfer from plastocyanin to photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 as induced by site-directed mutagenesis of the copper protein.

The kinetic mechanism of plastocyanin oxidation by photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 is drastically changed by modifying the metalloprotein by site-directed mutagenesis. The mutations herein considered concern four specific residues, two in the east face and the other two in the hydrophobic patch of plastocyanin. The first set of mutants include D44A, D44K, D47A, and D47R, as well as the double mutants D44A/D47A and D44R/D47R; the second set consists of L12A and K33E. The kinetic efficiency of all these mutant plastocyanins has been analyzed by laser-flash absorption spectroscopy. The plastocyanin concentration dependence of the observed electron transfer rate constant (kobs) is linear with most mutant plastocyanins, as with wild-type plastocyanin, but exhibits a saturation plateau at high protein concentration with the double mutant D44R/D47R, which suggests the formation of a plastocyanin-PSI transient complex. The effect of ionic strength on kobs varies from the wild-type plastocyanin to some of the mutants, for instance D44K, for which the salt concentration dependence of kobs is just the reverse as compared to the wild-type protein. The ionic strength dependence of kobs with D44R/D47R exhibits a bell-shaped profile, which is similar to that of green algae and higher plants. These findings indicate that the double mutant D44R/D47R follows a reaction mechanism involving not only complex formation with PSI but also further reorientation to properly accommodate the redox centers prior to electron transfer, as is the case in most evolved species, whereas the wild-type copper protein reacts with PSI by following a simple collisional kinetic model.

Purification and physicochemical properties of the low-potential cytochrome C549 from the cyanobacterium Synechocystis sp. PCC 6803.

A soluble low-potential cytochrome c549 has been purified in milligram quantities from the cyanobacterium Synechocystis sp. PCC 6803. The protein exhibits an acid isoelectric point of 3.9, a molecular mass of 15.8 kDa, and a midpoint redox potential value of -250 mV at pH 7.0 EPR and 1H NMR studies suggest a low-spin heme iron with bis-histidine coordination at the fifth and sixth positions. EDTA-photoreduced 5-deazariboflavin has been used as the electron-donating system to study, by laser flash absorption spectroscopy, the electron transfer reactions between Synechocystis cytochrome c549 and redox proteins involved in the cyclic electron flow around photosystem I. The second-order rate constants (k2) obtained for ferredoxin (or flavodoxin) oxidation by Synechocystis cytochrome c549 are rather low (ca. 10(5) M-1 s-1), thus suggesting that this low-potential heme-protein does not operate as the primary electron carrier for either transferring electrons to the cytochrome b6f complex in cyclic photophosphorylation or to hydrogenase during anaerobic metabolism. The k2 values for plastocyanin reduction by cytochrome c549 are about 100 times higher (ca. 10(7) M-1 s-1), but it remains to be determined whether or not this reaction actually reflects a physiological process.