Search

The development of novel phytoremediation strategies to mitigate mercury contamination, an increasingly important worldwide threat, could be enhanced by identifying Hg-tolerant legume cultivars. A fast method was thus developed to screen a germplasm collection of the model legume Medicago truncatula for Hg tolerance, testing 258 accessions at the seedling stage in a miniaturized hydroponic system and using root growth as an indicator of tolerance. To validate the results of the screening, the Hg-tolerant cultivars identified were analyzed in a referenced hydroponic growth assay. The different growth parameters measured in this assay were then subjected to a factorial analysis (principal component analysis method) in order to establish indices of tolerance, which further supported the rapid method of screening. Accordingly, four Hg-tolerant M. truncatula cultivars were selected that might serve as source material for genetic improvement, as model cultivars to study Hg tolerance in legumes and/or to develop soil phytoremediation approaches.

Aim: To determine whether expression of a cyanobacterial flavodoxin in soil bacteria of agronomic interest confers protection against the widely used herbicides paraquat and atrazine. Methods and Results: The model bacterium Escherichia coli, the symbiotic nitrogen-fixing bacterium Ensifer meliloti and the plant growth-promoting rhizobacterium Pseudomonas fluorescens Aur6 were transformed with expression vectors containing the flavodoxin gene of Anabaena variabilis. Expression of the cyanobacterial protein was confirmed by Western blot. Bacterial tolerance to oxidative stress was tested in solid medium supplemented with hydrogen peroxide, paraquat or atrazine. In all three bacterial strains, flavodoxin expression enhanced tolerance to the oxidative stress provoked by hydrogen peroxide and by the reactive oxygen species-inducing herbicides, witnessed by the enhanced survival of the transformed bacteria in the presence of these oxidizing agents. Conclusions: Flavodoxin overexpression in beneficial soil bacteria confers tolerance to oxidative stress and improves their survival in the presence of the herbicides paraquat and atrazine. Flavodoxin could be considered as a general antioxidant resource to face oxidative challenges in different micro-organisms. Significance and Impact of the study: The use of plant growth-promoting rhizobacteria or nitrogen-fixing bacteria with enhanced tolerance to oxidative stress in contaminated soils is of significant agronomic interest. The enhanced tolerance of flavodoxin-expressing bacteria to atrazine and paraquat points to potential applications in herbicide-treated soils.

Nitrogen fixation by legumes is very sensitive to salinity stress, which can severely reduce the productivity of legume crops and their soil-enriching capacity. Salinity is known to cause oxidative stress in the nodule by generating reactive oxygen species (ROS). Flavodoxins are involved in the response to oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by flavodoxin overexpression in bacteroids might lead to a protective effect on nodule functioning under salinity stress. Tolerance to salinity stress was evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that overexpressed a cyanobacterial flavodoxin compared with nodules produced by the wildtype bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme activities were determined. The decline in nitrogenase activity associated to salinity stress was significantly less in flavodoxin-expressing than in wild-type nodules.We detected small but significant changes in nodule antioxidant metabolism involving the ascorbate–glutathione cycle enzymes and metabolites, as well as differences in activity of the carbon metabolism enzyme sucrose synthase, and an atypical starch accumulation pattern in flavodoxincontaining nodules. Salt-induced structural and ultrastructural alterations were examined in detail in alfalfa wild-type nodules by light and electron microscopy and compared to flavodoxin-containing nodules. Flavodoxin reduced saltinduced structural damage, which primarily affected young infected tissues and not fully differentiated bacteroids. The results indicate that overexpression of flavodoxin in bacteroids has a protective effect on the function and structure of alfalfa nodules subjected to salinity stress conditions. Putative protection mechanisms are discussed

Mercury contamination of soils constitutes a serious environmental problem worldwide. Rhizobium–legume symbioses represent an interesting potential tool in soil bioremediation. Besides their nitrogen-fixing capacity, they have the advantage of integrating microorganisms that may influence metal bioavailability and legume plants that may act as phytoextractors or phytostabilizers. Herbaceous forage legumes present additional advantages in that they grow rapidly, producing a high biomass, and they are easy to harvest. Analysing legume endosymbiont tolerance is essential to successfully manage these symbiotic associations in metal-polluted areas, although little information is as yet available regarding Hg-tolerant rhizobia. The aim of this study was to isolate and characterize rhizobia from nodules of Medicago spp. and Trifolium spp. plants that grow in Hg-contaminated soils. Fifty-nine bacterial strains were genetically characterized and identified as belonging to the Ensifer medicae and Rhizobium leguminosarum bv. trifolii species. Rhizobial strains highly tolerant to Hg and strains displaying multiple metal tolerances were identified. The bacteria showed important phenotypic diversity in terms of salt and pH tolerance, phosphate solubilization and tolerance to different heavy metals. Significant correlations were found between metal tolerance responses. The influences of the site of origin, the host plant and bacterial species on metal tolerance were analysed.

Photoprotection is a crucial ability that allows plants to live under stressful environments. Plants develop structural and chemical mechanisms of photoprotection to preserve their photosystems from light excess. We studied the photo-protective strategy and its physiological consequences in tara (Caesalpinia spinosa), a legume tree endemic of South America, showing the adaptative trait of closing leaflets under stressful conditions. Tara has a significant ecological and economic value thriving in many different ecosystems from semiarid shrub-lands to agricultural areas and dry or fog forests.

Mineral nitrogen deficiency is a frequent characteristic of arid and semi-arid soils....

We investigated the diversity and structure of the ectomycorrhizal (EM) fungal community associated with post-fire regenerated Pinus pinaster Ait., and the influence of fire severity and site slope on EM assemblage patterns. Seedlings were sampled in the first autumn and in both spring and autumn of the second growing season after fire, in a total of three samplings.EMpercentages per seedling were assessed, morphotypes described, and tentative identification of EMtypes performed by restriction fragment length polymorphism (RFLP) and sequencing of nrDNA internal transcribed spacer (ITS) region. Seedlings were highly mycorrhizal in all samplings, independently of the factors studied. A total of 45 EM types were identified, and richness and diversity significantly increased from the first to the second autumn after fire. Neither fire severity nor slope had a significant effect on fungal richness and diversity. Overall EM community composition was similar in all samplings, although fire severity, site slope and elapsed time after fire caused evident shifts in presence or in relative frequencies of anumberofEMtypes.No significant effect of fire severity or slope on EM assemblage patterns was detected in the first two samplings after fire. However, a significant effect of fire severity was observed at the end of the second growing season. The harvest of burned wood did not significantly affectEMfungal assemblages although the slope did.We conclude that there was a high potential of active EM inoculum in soil immediately after fire colonizing post-fire natural regenerated P. pinaster seedlings with high EM percentages, and that factors defining burn intensity, such as fire severity and topography, directly influenced the species composition and assemblage patterns of EM fungal communities, triggering replacements and succession of EM fungal species.

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxinexpressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbateglutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogenfixing activity.

Flavodoxins are electron carrier flavoproteins that are involved in the response to oxidative stress in bacteria and cyanobacteria. Recently, we obtained Sinorhizobium meliloti bacteria that overexpressed a flavodoxin from the cyanobacterium Anabaena variabilis [Redondo et al. (2009) Plant Physiology 149:1166–1178]. In the present work, tolerance to cadmium was evaluated in free-living transformed S. meliloti and in alfalfa plants nodulated by the flavodoxin-overexpressing rhizobia, in comparison with plants nodulated by wild-type bacteria. Overexpression of flavodoxin protected free-living S. meliloti from cadmium toxicity and had a positive effect on nitrogen fixation of alfalfa plants subjected to cadmium stress. Flavodoxin notably reduced cadmiuminduced structural and ultrastructural alterations in alfalfa nodules. Putative protection mechanisms in flavodoxin-overexpressing nodules are discussed. Flavodoxin could have applications as a biotechnological tool to improve the symbiotic performance of alfalfa and other legumes in cadmium polluted soils.

Cytokinin receptors (CRs) are hybrid-type histidine kinases, membrane proteins with a cytokinin-binding extracellular domain. CRs initiate and propagate cytokinin signaling by means of phosphorylation and phosphotransfer to downstream proteins. In legumes, some members of the CR multigenic family are essential for nodulation. In two recent works, we investigated the involvement of two new CRs, MsHK1 from Medicago sativa, and LaHK1 from Lupinus albus, in nodule morphogenesis, senescence and stress response. LaHK1 expression increased during the first stages of lupin nodule development, while MsHK1 expression was localized in the meristem and the invasion zone of alfalfa nodules pointing to a role for CRs in nodule cell proliferation and differentiation. Both CRs were also induced during nodule senescence. MsHK1 expression increased under osmotic stress and both genes were induced following dark stress, indicating that CRs are also likely to play a significant role in the response to stress. We propose multiple roles for CRs which, when analyzed jointly with recent results from other authors, suggest coordinated cross-talk of different signaling systems.

The molecular characterization of 62 rhizobial isolates obtained from root-nodules of Arachis hypogaea growing in north-western Morocco was performed. Bacteria were firstly characterized by restriction of the 16S-rDNA region, and phylogeny was inferred from 16S gene sequences. Phylogenetically, isolates were grouped with species belonging to the Bradyrhizobium and Rhizobium genera. A high degree of variability was detected among isolates in terms of their nitrogen-fixing ability. This is, to our knowledge, the first study on genetic diversity and symbiotic effectiveness of rhizobia isolated from peanut nodules grown in Morocco. This characterization provides a basis for the selection of peanut-nodulating rhizobia which may have applications in the formulation of appropriate inocula to improve peanut crop yield on Moroccan soils.

Here we report the isolation of a new cytokinin receptor homologue, LaHK1, from lupin (Lupinus albus) root nodules. LaHK1 transcript accumulation was detected in different plant organs, and expression was analyzed throughout nodule development. We observed notably higher expression in nodule primordia and young nodules compared to the root or to mature nodules. We also detected elevated transcript accumulation in naturally senescent nodules and in senescent nodules subjected to foliar dark stress. The results could be an indication of a putative role of this cytokinin receptor homologue in nodule development, from morphogenesis through senescence.

A new cytokinin receptor homologue, MsHK1, was isolated from Medicago sativa root nodules. MsHK1 expression was induced in alfalfa seedlings by exogenous application of the cytokinin trans-zeatin. Transcript accumulation was detected in diVerent plant organs. MsHK1 expression was induced by salt stress in alfalfa roots, leaves and nodules, and transcript accumulation in the vascular bundles pointed to a putative role in osmosensing for MsHK1 and/or other close cytokinin receptor homologues. Expression in the meristem and the invasion zone of the nodule suggest a role for cytokinin receptors in cytokinin sensing during nodule cell division and diferentation

The aimof this work is to describe the diversity and phylogeny of rhizobial bacteria associated to nodules of Ononis tridentata L. in different geographical regions of Spain. Twenty-two bacterial isolates were characterized using several molecular techniques (16S amplified ribosomal deoxyribonucleic acid restriction analysis, fingerprinting, and sequencing) and phylogenies were inferred from their 16S and nodC gene sequences. Phylogenetically, the isolates grouped with the genera Rhizobium, Mesorhizobium, Phylobacterium, and Bosea. The nodC gene, essential for nodulation, was detected for the first time in isolates close to the genera Bosea and Phyllobacterium. The bacteria isolated showed a high diversity at the genus, species, and strain level regardless of the geographical origin of the host plant. This is the first report describing bacteria associated to nodules of O. tridentata. This shrub legume is highly prized for the revegetation of gypsum soils in semiarid Mediterranean areas. Our molecular description of bacteria associated to this legume improves the current understanding of the ecology of this plant species. Our findings have implications

Lupin nodule cells maintain their ability to divide for several cycles after being infected by endosymbiotic rhizobia. The conformation of the cytoskeletal elements of nodule cells was studied by fluorescence labelling, immunocytochemistry, and laser confocal and transmission electron microscopy. The dividing infected cells showed the normal microtubule and actin patterns of dividing plant cells. The clustered symbiosomes were tethered to the spindle-pole regions and moved to the cell poles during spindle elongation. In metaphase, anaphase, and early telophase, the symbiosomes were found at opposite cell poles where they did not interfere with the spindle filaments or phragmoplast. This symbiosome positioning was comparable with that of the organelles (which ensures organelle inheritance during plant cell mitosis). Tubulin microtubules and actin microfilaments appeared to be in contact with the symbiosomes. The possible presence of actin molecular motor myosin in nodules was analysed using a monoclonal antibody against the myosin light chain. The antigen was detected in protein extracts of nodule and root cytosol as bands of approximately 20 kDa (the size expected). In the nodules, an additional polypeptide of 65 kDa was found. Immunogold techniques revealed the antigen to be localized over thin microfilaments linked to the cell wall, as well as over the thicker microfilament bundles and surrounding the symbiosomes. The pattern of cytoskeleton rearrangement in dividing infected cells, along with the presence of myosin antigen, suggests that the positioning of symbiosomes in lupin nodule cells might depend on the same mechanisms used to partition genuine plant cell organelles during mitosis

Colonisation of Pinus halepensis roots by GFP-tagged Pseudomonas fluorescens Aur6 was monitored by epifluorescence microscopy and dilution plating. Aur6-GFP was able to colonise and proliferate on P. halepensis roots. Co-inoculation with the ectomycorrhizal fungus Suillus granulatus did not affect the bacterial colonisation pattern whereas it had an effect on bacterial density. Bacterial counts increased during the first 20 days of seedling growth, irrespective of seedlings being mycorrhizal or not. After 40 days, bacterial density significantly decreased and bacteria concentrated on the upper two-thirds of the pine root. The presence of S. granulatus significantly stimulated survival of bacteria in the root elongation zone where fungal colonisation was higher. The number of mycorrhizas formed by S. granulatus was not affected by co-inoculation with Aur6-GFP. Neither Aur6-GFP nor S. granulatus stimulated P. halepensis development when inoculated alone, but a synergistic effect was observed on seedling growth when bacteria and fungus were co-inoculated.

The reactions of excess apoflavodoxin from Desulfovibrio vulgaris, Anabaena variabilis and Azotobacter vinelandii with ribo- flavin 5«-phosphate (FMN), riboflavin 3«,5«-bisphosphate and riboflavin are pseudo-first-order. The rates increase with decreasing pH in the range pH 5-8, and, in general, they increase with increasing ionic strength to approach a maximum at an ionic strength greater than 0.4 M. The rate of FMN binding in phosphate at high pH increases to a maximum at an ionic strength of about 0.1 M, and then decreases as the phosphate concentration is increased further. The dissociation constants for the complexes withFMNand riboflavin decrease with an increase of ionic strength. Inorganic phosphate stabilizes the complex with riboflavin. The effects of phosphate on riboflavin binding suggest that phosphate interacts with the apoprotein at the site normally occupied by the phosphate of FMN. Redox potentials determined for the oxidized}semiquinone and semiquinone} hydroquinone couples of the riboflavin and FMN complexes were used with Kd values for the complexes with the oxidized flavins to calculate values for Kd for the semiquinone and hydroquinone complexes. The hydroquinone complexes are all less stable than the complexes with the two other redox forms of the flavin. Destabilization of the hydroquinone is less marked in the complexes with riboflavin, supporting a proposal that the terminal phosphate group of FMN plays a role in decreasing the stability of the hydroquinone complex and in decreasing the redox potential of the semiquinone}hydroquinone couple.

Isolated D1 -D2-cytochrome b559 photosystem I1 reaction center preparations with pigment stoichiometry higher than 4 chlorophylls per 2 pheophytins can be contaminated with CP47 proximal antenna complex. Reaction centers prepared by a modification of the Nanba-Satoh procedure and containing about 6 chlorophylls per 2 pheophytins showed immuno-cross-reactivity when probed with a monoclonal antibody raised against the CP47 polypeptide. Furthermore, they could be fractionated successfully by Superose- 12 sieve chromatography into two different populations. The first few fractions off the column contained a more definitive 435 nm shoulder corresponding to increased chlorophyll content, and showed strong immuno-cross-reactivity with the CP47 antibody. The peak fractions off the column displayed a less prominent 435 nm shoulder, and did not cross-react with the antibody. Moreover, when a 6-chlorophyll preparation was mixed with Sepharose beads coupled to CP47 antibody, the eluted material corresponded to a preparation of about 4 chlorophylls per 2 pheophytins and did not show any crossreaction with the antibody against CP47. The amount of CP47 protein in the 6-chlorophyll preparation as quantitated using Coomassie Blue staining or from gel blots was sufficient to account for most of the extra 2 chlorophylls. We conclude that D 1 -D2-cytochrome b559 preparations containing more than 4 chlorophylls per 2 pheophytins can be contaminated with small amounts of CP47-D1 -D2-Cyt b559 complex and that native photosystem I1 reaction centers contain 4 core chlorophylls per 2 pheophytins.

To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seedspecific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope "himet" (Hoffman et al., 1988, Plant Mol. Biol. 11, 717-729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent However, the protein was sufficintly destabilized to be degraded by vacuolar proteases.

Flavodoxin from the cyanobacterium Anabaena PCC 7119 has been shown to mediate, under illumination, the transfer of electrons from the thylakoidal membranes that were isolated from the same organism, to both the enzyme ferredoxin-NADP + reductase and cytochrome c. Chemical cross-linking of ferredoxin or flavodoxin to the photosynthetic membranes provides a preparation that is active in cytochrome c photoreduction without the addition of external protein carrier. NADP ÷ photoreduction, albeit diminished, was observed only after addition of exogenous electron carrier protein. Immunoblotting analysis of the chemical adduct reveals that flavodoxin binds to a 10 kDa polypeptide subunit in the cyanobacterial Photosystem I which appears to act as its physiological partner in the electron transfer process.

The reduced pyridine nucleotides NADH and NADPH play an essential role as cofactors of many enzymatic reactions. Many important chemical and pharmaceutical compounds can be synthesized through enzymatic reactions in which the reduced cofactors should be present in stoichiometric amounts. Due to the very high cost of these compounds, several methods have been designed for the regeneration of the reduced nucleotides at an industrial level.~-3 The simplest procedures described to produce large amounts of reduced pyridine nucleotides require the use of crude or purified preparations of the appropriate enzymes and the corresponding substrates. 4'5 This is very costly, since both the enzyme and the substrate are expensive. Moreover, the use of batch-type reactions adds some complications, since equilibrium is usually reached when only part of the reaction has taken place and also because the enzyme preparation cannot be used for further cycles. This problem can be overcome by the in situ regeneration of the reduced cofactor with the use of a sacrificial substrate as the ultimate source of reducing power, or by means of electrochemistry.

A covalent complex between ferredoxin-NADP ÷ reductase and fiavodoxin, two flavoproteins isolated from the nitrogen-fixing cyanobacterium Anabaena, has been formed by a cross-linking reaction with a water-soluble carbodiimide. The complex has a 1 : 1 stoichiometry and an absorption spectrum similar to that of the mixture of the free proteins. Both proteins can be detected immunochemically when they are in the complex, since they react with antisera raised against the isolated proteins. The complex is shown to be active in the NADPH-cytochrome c reductase reaction, although with a lower turnover number than FNR when ferredoxin is used as the electron carrier. Stopped-flow experiments have shown a lower rate of electron transfer (approx. 20-fold) from the semiquinone form of flavodoxin, when bound into the complex, to cytochrome c, as compared to the free protein. Anaerobic titration of the reduced complex with NADP + also indicates that there is transfer of electrons between the reductase and flavodoxin in the complex. Nevertheless, the covalent complex is found to be unable by itself to mediate the transfer of electrons from photosynthetic particles to NADP +. This indicates that the pathway for the entrance of electrons into the complex is partially or completely blocked depending on the degree of cross-linkage. However, some activity is shown when ferredoxin is added to the system. The covalent complex shows a limited ability to act as an electron transfer protein between photosynthetic membranes and either exogenous FNR or cytochrome c. The concentration of complex required in this case is also much higher than when free ferredoxin or flavodoxin are present. It is concluded that the covalent complex formed by ferredoxin-NADP + reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 can be used as a simplified model for the study of electron transfer reactions between two flavoproteins. It can not be used for the study of reactions involving reduction o

The oxidation-reduction potentials of ferredoxin-NADP' reductase and flavodoxin from the cyanobacterium Anuhaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/ flavodoxin semiquinone couple (E2) and the flavodoxin seniiquinone/hydroquinone couple (El) were - 21 2 mV and -436 mV, respectively. El was independent of pH above about pH 7, but changed by approximately -60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox-linked pK, at about 6.1, similar to those of certain other flavodoxins. E2 varied by - 50 mV/pH in the range pH 5 - 8. The redox potential for the two-electron reduction of ferredoxin-NADP' reductase was - 344 mV at pH 7 (A&, = - 30 mV/pH). In the 1 : 1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by + 8 mV and El was shifted by - 25 mV; the shift in potential for the reductase was + 4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by -21 mV and El shifted by + 20 mV; the reductase potential shifted by + 2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin-NADP' reductase from Anaharna vuriahilis was complexed with flavodoxin from Azotnbacter vinelandii. The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.

Flavodoxins are small flavoproteins which contain a single molecule of FMN as Prosthetic group.

The influence of electrostatic forces on the formation of, and electron transfer within, transient complexes between redox proteins was examined by comparing ionic strength effects on the kinetics of the electron transfer reaction between reduiced ferredoxins (Fd) and oxidized ferredoxin-NADP+ reductases (FNR) from Anabaena and from spinach, using lase flash photolysis techniques.

The 1:1 complex of ferredoxin (Fd) and Ferredoxin-NADP+ reductase (FNR) at low ionic strength has been a model for redox interactions within electrostatically stabilized complexes (1-3).