Iron Deficiency Promotes the Lack of Photosynthetic Cytochrome c550 and Affects the Binding of the Luminal Extrinsic Subunits to Photosystem II in the Diatom Phaeodactylum tricornutum.

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced QB and the S2/S3 states of the water-splitting machinery. However, electron transfer from QA to QB, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c550 subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c550 under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c550, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.

New Insights into the Evolution of the Electron Transfer from Cytochrome f to Photosystem I in the Green and Red Branches of Photosynthetic Eukaryotes.

In cyanobacteria and most green algae of the eukaryotic green lineage, the copper-protein plastocyanin (Pc) alternatively replaces the heme-protein cytochrome c6 (Cc6) as the soluble electron carrier from cytochrome f (Cf) to photosystem I (PSI). The functional and structural equivalence of 'green' Pc and Cc6 has been well established, representing an example of convergent evolution of two unrelated proteins. However, plants only produce Pc, despite having evolved from green algae. On the other hand, Cc6 is the only soluble donor available in most species of the red lineage of photosynthetic organisms, which includes, among others, red algae and diatoms. Interestingly, Pc genes have been identified in oceanic diatoms, probably acquired by horizontal gene transfer from green algae. However, the mechanisms that regulate the expression of a functional Pc in diatoms are still unclear. In the green eukaryotic lineage, the transfer of electrons from Cf to PSI has been characterized in depth. The conclusion is that in the green lineage, this process involves strong electrostatic interactions between partners, which ensure a high affinity and an efficient electron transfer (ET) at the cost of limiting the turnover of the process. In the red lineage, recent kinetic and structural modeling data suggest a different strategy, based on weaker electrostatic interactions between partners, with lower affinity and less efficient ET, but favoring instead the protein exchange and the turnover of the process. Finally, in diatoms the interaction of the acquired green-type Pc with both Cf and PSI may not yet be optimized.

The heterologous expression of a plastocyanin in the diatom Phaeodactylum tricornutum improves cell growth under iron-deficient conditions.

We have investigated if the heterologous expression of a functional green alga plastocyanin in the diatom Phaeodactylum tricornutum can improve photosynthetic activity and cell growth. Previous in vitro assays showed that a single-mutant of the plastocyanin from the green algae Chlamydomonas reinhardtii is effective in reducing P. tricornutum photosystem I. In this study, in vivo assays with P. tricornutum strains expressing this plastocyanin indicate that even the relatively low intracellular concentrations of holo-plastocyanin detected (≈4 µM) are enough to promote an increased growth (up to 60%) under iron-deficient conditions as compared with the WT strain, measured as higher cell densities, content in pigments and active photosystem I, global photosynthetic rates per cell, and even cell volume. In addition, the presence of plastocyanin as an additional photosynthetic electron carrier seems to decrease the over-reduction of the plastoquinone pool. Consequently, it promotes an improvement in the maximum quantum yield of both photosystem II and I, together with a decrease in the acceptor side photoinhibition of photosystem II-also associated to a reduced oxidative stress-a decrease in the peroxidation of membrane lipids in the choroplast, and a lower degree of limitation on the donor side of photosystem I. Thus the heterologous plastocyanin appears to act as a functional electron carrier, alternative to the native cytochrome c6 , under iron-limiting conditions.

The singular properties of photosynthetic cytochrome c550 from the diatom Phaeodactylum tricornutum suggest new alternative functions.

Cytochrome c550 is an extrinsic component in the luminal side of photosystem II (PSII) in cyanobacteria, as well as in eukaryotic algae from the red photosynthetic lineage including, among others, diatoms. We have established that cytochrome c550 from the diatom Phaeodactylum tricornutum can be obtained as a complete protein from the membrane fraction of the alga, although a C-terminal truncated form is purified from the soluble fractions of this diatom as well as from other eukaryotic algae. Eukaryotic cytochromes c550 show distinctive electrostatic features as compared with cyanobacterial cytochrome c550 . In addition, co-immunoseparation and mass spectrometry experiments, as well as immunoelectron microscopy analyses, indicate that although cytochrome c550 from P. tricornutum is mainly located in the thylakoid domain of the chloroplast - where it interacts with PSII - , it can also be found in the chloroplast pyrenoid, related with proteins linked to the CO2 concentrating mechanism and assimilation. These results thus suggest new alternative functions of this heme protein in eukaryotes.

The photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum.

The photosynthetic cytochrome c 550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c 550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c 550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c 550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c 550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c 550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c 550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c 550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre.

Cyt c6-3: A New Isoform of Photosynthetic Cyt c6 Exclusive to Heterocyst-Forming Cyanobacteria.

All known cyanobacteria contain Cyt c6, a small soluble electron carrier protein whose main function is to transfer electrons from the Cyt b6f complex to PSI, although it is also involved in respiration. We have previously described a second isoform of this protein, the Cyt c6-like, whose function remains unknown. Here we describe a third isoform of Cyt c6 (here called Cytc6-3), which is only found in heterocyst-forming filamentous cyanobacteria. Cyt c6-3 is expressed in vegetative cells but is specifically repressed in heterocysts cells under diazotrophic growth conditions. Although there is a close structural similarity between Cyt c6-3 and Cyt c6 related to the general protein folding, Cyt c6-3 presents differential electrostatic surface features as compared with Cyt c6, its expression is not copper dependent and has a low reactivity towards PSI. According to the different expression pattern, functional reactivity and structural properties, Cyt c6-3 has to play an as yet to be defined regulatory role related to heterocyst differentiation.

Iron Deficiency Induces a Partial Inhibition of the Photosynthetic Electron Transport and a High Sensitivity to Light in the Diatom Phaeodactylum tricornutum.

Iron limitation is the major factor controlling phytoplankton growth in vast regions of the contemporary oceans. In this study, a combination of thermoluminescence (TL), chlorophyll fluorescence, and P700 absorbance measurements have been used to elucidate the effects of iron deficiency in the photosynthetic electron transport of the marine diatom P. tricornutum. TL was used to determine the effects of iron deficiency on photosystem II (PSII) activity. Excitation of iron-replete P. tricornutum cells with single turn-over flashes induced the appearance of TL glow curves with two components with different peaks of temperature and contributions to the total signal intensity: the B band (23°C, 63%), and the AG band (40°C, 37%). Iron limitation did not significantly alter these bands, but induced a decrease of the total TL signal. Far red excitation did not increase the amount of the AG band in iron-limited cells, as observed for iron-replete cells. The effect of iron deficiency on the photosystem I (PSI) activity was also examined by measuring the changes in P700 redox state during illumination. The electron donation to PSI was substantially reduced in iron-deficient cells. This could be related with the important decline on cytochrome c 6 content observed in these cells. Iron deficiency also induced a marked increase in light sensitivity in P. tricornutum cells. A drastic increase in the level of peroxidation of chloroplast lipids was detected in iron-deficient cells even when grown under standard conditions at low light intensity. Illumination with a light intensity of 300 µE m(-2) s(-1) during different time periods caused a dramatic disappearance in TL signal in cells grown under low iron concentration, this treatment not affecting to the signal in iron-replete cells. The results of this work suggest that iron deficiency induces partial blocking of the electron transfer between PSII and PSI, due to a lower concentration of the electron donor cytochrome c 6. This decreased electron transfer may induce the over-reduction of the plastoquinone pool and consequently the appearance of acceptor side photoinhibition in PSII even at low light intensities. The functionality of chlororespiratory electron transfer pathway under iron restricted conditions is also discussed.

Interaction of photosystem I from Phaeodactylum tricornutum with plastocyanins as compared with its native cytochrome c6: Reunion with a lost donor.

In the Phaeodactylum tricornutum alga, as in most diatoms, cytochrome c6 is the only electron donor to photosystem I, and thus they lack plastocyanin as an alternative electron carrier. We have investigated, by using laser-flash absorption spectroscopy, the electron transfer to Phaeodactylum photosystem I from plastocyanins from cyanobacteria, green algae and plants, as compared with its own cytochrome c6. Diatom photosystem I is able to effectively react with eukaryotic acidic plastocyanins, although with less efficiency than with Phaeodactylum cytochrome c6. This efficiency, however, increases in some green alga plastocyanin mutants mimicking the electrostatics of the interaction site on the diatom cytochrome. In addition, the structure of the transient electron transfer complex between cytochrome c6 and photosystem I from Phaeodactylum has been analyzed by computational docking and compared to that of green lineage and mixed systems. Taking together, the results explain why the Phaeodactylum system shows a lower efficiency than the green systems, both in the formation of the properly arranged [cytochrome c6-photosystem I] complex and in the electron transfer itself.

Molecular recognition in the interaction of chloroplast 2-Cys peroxiredoxin with NADPH-thioredoxin reductase C (NTRC) and thioredoxin x.

In addition to the standard NADPH thioredoxin reductases (NTRs), plants hold a plastidic NTR (NTRC), with a thioredoxin module fused at the C-terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs). The interaction of NTRC and chloroplastic thioredoxin x with 2-Cys Prxs has been confirmed in vivo, by bimolecular fluorescence complementation (BiFC) assays, and in vitro, by isothermal titration calorimetry (ITC) experiments. In comparison with thioredoxin x, NTRC interacts with 2-Cys Prx with higher affinity, both the thioredoxin and NTR domains of NTRC contributing significantly to this interaction, as demonstrated by using the NTR and thioredoxin modules of the enzyme expressed separately. The presence of the thioredoxin domain seems to prevent the interaction of NTRC with thioredoxin x.

Structural and functional analysis of novel human cytochrome C targets in apoptosis.

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals.

External loops at the ferredoxin-NADP(+) reductase protein-partner binding cavity contribute to substrates allocation.

Ferredoxin-NADP(+) reductase (FNR) is the structural prototype of a family of FAD-containing reductases that catalyze electron transfer between low potential proteins and NAD(P)(+)/H, and that display a two-domain arrangement with an open cavity at their interface. The inner part of this cavity accommodates the reacting atoms during catalysis. Loops at its edge are highly conserved among plastidic FNRs, suggesting that they might contribute to both flavin stabilization and competent disposition of substrates. Here we pay attention to two of these loops in Anabaena FNR. The first is a sheet-loop-sheet motif, loop102-114, that allocates the FAD adenosine. It was thought to determine the extended FAD conformation, and, indirectly, to modulate isoalloxazine electronic properties, partners binding, catalytic efficiency and even coenzyme specificity. The second, loop261-269, contains key residues for the allocation of partners and coenzyme, including two glutamates, Glu267 and Glu268, proposed as candidates to facilitate the key displacement of the C-terminal tyrosine (Tyr303) from its stacking against the isoalloxazine ring during the catalytic cycle. Our data indicate that the main function of loop102-114 is to provide the inter-domain cavity with flexibility to accommodate protein partners and to guide the coenzyme to the catalytic site, while the extended conformation of FAD must be induced by other protein determinants. Glu267 and Glu268 appear to assist the conformational changes that occur in the loop261-269 during productive coenzyme binding, but their contribution to Tyr303 displacement is minor than expected. Additionally, loop261-269 appears a determinant to ensure reversibility in photosynthetic FNRs.

A hydrogen bond network in the active site of Anabaena ferredoxin-NADP(+) reductase modulates its catalytic efficiency.

Ferredoxin-nicotinamide-adenine dinucleotide phosphate (NADP(+)) reductase (FNR) catalyses the production of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) in photosynthetic organisms, where its flavin adenine dinucleotide (FAD) cofactor takes two electrons from two reduced ferredoxin (Fd) molecules in two sequential steps, and transfers them to NADP(+) in a single hydride transfer (HT) step. Despite the good knowledge of this catalytic machinery, additional roles can still be envisaged for already reported key residues, and new features are added to residues not previously identified as having a particular role in the mechanism. Here, we analyse for the first time the role of Ser59 in Anabaena FNR, a residue suggested by recent theoretical simulations as putatively involved in competent binding of the coenzyme in the active site by cooperating with Ser80. We show that Ser59 indirectly modulates the geometry of the active site, the interaction with substrates and the electronic properties of the isoalloxazine ring, and in consequence the electron transfer (ET) and HT processes. Additionally, we revise the role of Tyr79 and Ser80, previously investigated in homologous enzymes from plants. Our results probe that the active site of FNR is tuned by a H-bond network that involves the side-chains of these residues and that results to critical optimal substrate binding, exchange of electrons and, particularly, competent disposition of the C4n (hydride acceptor/donor) of the nicotinamide moiety of the coenzyme during the reversible HT event.

Photosystem I reduction in diatoms: as complex as the green lineage systems but less efficient.

Diatoms occupy a key branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. Here, the electron transfer reaction mechanism from cytochrome c6 to photosystem I from the diatom Phaeodactylum tricornutum has been analyzed by laser-flash absorption spectroscopy. Kinetic traces of photosystem I reduction fit to biphasic curves, the analysis of the observed rate constants indicating that electron transfer occurs in a cytochrome c6/photosystem I transient complex, which undergoes a reorganization process from the initial encounter complex to the optimized final configuration. The mild ionic strength dependence of the rate constants makes evident the relatively weak electrostatically attractive nature of the interaction. Taken together, these results indicate that the "red" Phaeodactylum system is less efficient than "green" systems, both in the formation of the properly arranged (cytochrome c6/photosystem I) complex and in the electron transfer itself. The results obtained from cross-reactions with cytochrome c6 and photosystem I from cyanobacteria, green algae, and plants shed light on the different evolutionary pathway of the electron transfer to photosystem I in diatoms with regard to the way that it evolved in higher plants.

New Arabidopsis thaliana cytochrome c partners: a look into the elusive role of cytochrome c in programmed cell death in plants.

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.

Communication between (L)-galactono-1,4-lactone dehydrogenase and cytochrome c.

UNLABELLED: l-galactono-1,4-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plant mitochondria. Here we investigated the communication between Arabidopsis thaliana GALDH and its natural electron acceptor cytochrome c (Cc). Using laser-generated radicals we observed the formation and stabilization of the GALDH semiquinone anionic species (GALDHSQ ). GALDHSQ oxidation by Cc exhibited a nonlinear dependence on Cc concentration consistent with a kinetic mechanism involving protein-partner association to form a transient bimolecular complex prior to the electron transfer step. Oxidation of GALDHSQ by Cc was significantly impaired at high ionic strength, revealing the existence of attractive charge-charge interactions between the two reactants. Isothermal titration calorimetry showed that GALDH weakly interacts with both oxidized and reduced Cc. Chemical shift perturbations for (1) H and (15) N nuclei of Cc, arising from the interactions with unlabeled GALDH, were used to map the interacting surface of Cc. For Arabidopsis Cc and yeast Cc, similar residues are involved in the interaction with GALDH. These residues are confined to a single surface surrounding the heme edge. The range of chemical shift perturbations for the physiological Arabidopsis Cc-GALDH complex is larger than that of the non-physiological yeast Cc-GALDH complex, indicating that the former complex is more specific. In summary, the results point to a relatively low affinity GALDH-Cc interaction, similar for all partner redox states, involving protein-protein dynamic motions. Evidence is also provided that Cc utilizes a conserved surface surrounding the heme edge for the interaction with GALDH and other redox partners. DATABASE: NMR assignment of the backbone amide resonances of Arabidopsis CcRED has been deposited in BMRB database (BMRB accession number 18828). L-galactono-1,4-lactone dehydrogenase (L-galactono-1,4-lactone: ferricytochrome c oxidoreductase, EC 1.3.2.3).

Electron transfer pathways and dynamics of chloroplast NADPH-dependent thioredoxin reductase C (NTRC).

NADPH-dependent thioredoxin reductases (NTRs) contain a flavin cofactor and a disulfide as redox-active groups. The catalytic mechanism of standard NTR involves a large conformational change between two configurations. Oxygenic photosynthetic organisms possess a plastid-localized NTR, called NTRC, with a thioredoxin module fused at the C terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs) and thus is involved in the protection against oxidative stress, among other functions. Although the mechanism of electron transfer of canonical NTRs is well established, it is not yet known in NTRC. By employing stopped-flow spectroscopy, we have carried out a comparative kinetic study of the electron transfer reactions involving NTRC, the truncated NTR module of NTRC, and NTRB, a canonical plant NTR. Whereas the three NTRs maintain the conformational change associated with the reductive cycle of catalysis, NTRC intramolecular electron transfer to the thioredoxin module presents two kinetic components (k(ET) of ~2 and 0.1 s(-1)), indicating the occurrence of additional dynamic motions. Moreover, the dynamic features associated with the electron transfer to the thioredoxin module are altered in the presence of 2-Cys Prx. NTRC shows structural constraints that may locate the thioredoxin module in positions with different efficiencies for electron transfer, the presence of 2-Cys Prx shifting the conformational equilibrium of the thioredoxin module to a specific position, which is not the most efficient.

ArsH from the cyanobacterium Synechocystis sp. PCC 6803 is an efficient NADPH-dependent quinone reductase.

The cyanobacterium Synechocystis sp. PCC 6803 possesses an arsenic resistance operon that encodes, among others, an ArsH protein. ArsH is a flavin mononucleotide (FMN)-containing protein of unknown function and a member of the family of NADPH-dependent FMN reductases. The nature of its final electron acceptor and the role of ArsH in the resistance to arsenic remained to be clarified. Here we have expressed and purified Synechocystis ArsH and conducted an intensive biochemical study. We present kinetic evidence supporting a quinone reductase activity for ArsH, with a preference for quinones with hydrophobic substituents. By using steady-state activity measurements, as well as stopped-flow and laser-flash photolysis kinetic analyses, it has been possible to establish the mechanism of the process and estimate the values of the kinetic constants. Although the enzyme is able to stabilize the anionic semiquinone form of the FMN, reduction of quinones involves the hydroquinone form of the flavin cofactor, and the enzymatic reaction occurs through a ping-pong-type mechanism. ArsH is able to catalyze one-electron reactions (oxygen and cytocrome c reduction), involving the FMN semiquinone form, but with lower efficiency. In addition, arsH mutants are sensitive to the oxidizing agent menadione, suggesting that ArsH plays a role in the response to oxidative stress caused by arsenite.

Specific nitration of tyrosines 46 and 48 makes cytochrome c assemble a non-functional apoptosome.

Under nitroxidative stress, a minor fraction of cytochrome c can be modified by tyrosine nitration. Here we analyze the specific effect of nitration of tyrosines 46 and 48 on the dual role of cytochrome c in cell survival and cell death. Our findings reveal that nitration of these two solvent-exposed residues has a negligible effect on the rate of electron transfer from cytochrome c to cytochrome c oxidase, but impairs the ability of the heme protein to activate caspase-9 by assembling a non-functional apoptosome. It seems that cytochrome c nitration under cellular stress counteracts apoptosis in light of the small amount of modified protein. We conclude that other changes such as increased peroxidase activity prevail and allow the execution of apoptosis.

Probing the reactivity of different forms of azurin by flavin photoreduction.

The reactivity of a variant of the blue copper protein, azurin from Pseudomonas aeruginosa, was investigated with laser flash photolysis and compared with the reactivity of the wild-type (WT) protein. The variant was obtained by changing the Cu ligating His117 for a glycine. The mutation creates a gap in the ligand shell of the Cu that can be filled with external ligands or water molecules. The crystal structure of the H117G variant is reported. It shows that the immediate surrounding of the Cu site in the variant exhibits less rigidity than in the WT protein and that the loop containing the Cu ligands Cys112, His117 and Met121 in the WT protein has gained flexibility in the H117G variant. Flash photolysis experiments were performed with 5-deazariboflavin and 8α-imidazolyl-(N-propylyl)-amino riboflavin as electron donors to probe the reactivity of WT and H117G azurin, and of H117G azurin for which the gap in the Cu co-ordination shell was filled with imidazole. 8α-Imidazolyl-(N-propylyl)-amino riboflavin appears one to two orders less efficient as a photo-flash reductant than 5-deazariboflavin. The reactivity of the H117G variant in the absence of external ligands appears to be 2.5-fold lower than the WT reactivity (second-order rate constants of 51 ± 2 × 10(7) m(-1) ·s(-1) versus 21 ± 1 × 10(7) m(-1) ·s(-1) ), whereas the addition of imidazole restores reactivity to above the WT level (71 ± 4 × 10(7) m(-1) ·s(-1) ). The differences are discussed in terms of structural modifications and changes in reorganizational energy and electronic coupling. Database Structural data are available in the Protein Data Bank under the accession number 3N2J.

Effect of crowding on the electron transfer process from plastocyanin and cytochrome c6 to photosystem I: a comparative study from cyanobacteria to green algae.

Plastocyanin and cytochrome c(6), the alternate donor proteins to photosystem I, can be acidic, neutral or basic; the role of electrostatics in their interaction with photosystem I vary accordingly for cyanobacteria, algae and plants. The effect of different crowding agents on the kinetics of the reaction between plastocyanin or cytochrome c(6) and photosystem I from three different cyanobacteria, Synechocystis PCC 6803, Nostoc PCC 7119 and Arthrospira maxima, and a green alga, Monoraphidium braunii, has been investigated by laser flash photolysis, in order to elucidate how molecular crowding affects the interaction between the two donor proteins and photosystem I. The negative effect of viscosity on the interaction of the two donors with photosystem I for the three cyanobacterial systems is very similar, as studied by increasing sucrose concentration. Bovine serum albumin seems to alter the different systems in a specific way, probably by means of electrostatic interactions with the donor proteins. Ficoll and dextran behave in a parallel manner, favouring the interaction by an average factor of 2, although this effect is somewhat less pronounced in Nostoc. With regards to the eukaryotic system, a strong negative effect of viscosity is able to overcome the favourable effect of any crowding agent, maybe due to stronger donor/photosystem I electrostatic interactions or the structural nature of the eukaryotic photosystem I-enriched membrane particles.