Fast Kinetic Methods with Photodiode Array Detection in the Study of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP+-Reductase

The primary function of Photosystem I (PSI)1 during photosynthesis is to provide reducing equivalents, in the form of NADPH, that will then be used in CO2 assimilation (Golbeck, 2006). In plants, this occurs via reduction of the soluble [2Fe-2S] Ferredoxin (Fd) by PSI. Subsequent reduction of NADP+ by Fdrd is catalysed by the FAD-dependent FerredoxinNADP+ reductase (FNR) (Eox/hq = -374 mV at pH 8.0 and 25oC) through the formation of a ternary complex (Arakaki et al., 1997; Nogues et al., 2004; Sancho et al., 1990). In most cyanobacteria and some algae under low iron conditions the FMN-dependent Flavodoxin (Fld), particularly its Fldsq/Fldhq couple (Eox/sq = -256 mV, Esq/hq = -445 mV at pH 8.0 and 25oC), substitutes for the Fdox/Fdrd pair in this reaction (Bottin & Lagoutte, 1992; Goni et al., 2009; Medina & Gomez-Moreno, 2004). Thus, two Fldhq molecules transfer two electrons to one FNRox that gets fully reduced after formation of the FNRsq intermediate. FNRhq transfers then both electrons simultaneously to NADP+ (Medina, 2009) (Figure 1). Additionally to their role in photosynthesis, Fld and FNR are ubiquitous flavoenzymes that deliver low midpoint potential electrons to redox-based metabolisms in plastids, mitochondria and bacteria in all kingdoms (Muller, 1991). They are also basic prototypes for a large family of di-flavin electron transferases that display common functional and structural properties, where the electron transfer (ET) flow supported by the Fld/FNR modules occurs in reverse direction to the photosynthesis (Brenner et al., 2008; Wolthers & Scrutton, 2004). The better understanding of the ET flavin-flavin mechanism in this system should witness a greater comprehension of the many physiological roles that Fld and FNR, either free or as modules in multidomain proteins, play. In these chains there are still many


Introduction
The primary function of Photosystem I (PSI) 1 during photosynthesis is to provide reducing equivalents, in the form of NADPH, that will then be used in CO 2 assimilation (Golbeck, 2006). In plants, this occurs via reduction of the soluble [2Fe-2S] Ferredoxin (Fd) by PSI. Subsequent reduction of NADP + by Fd rd is catalysed by the FAD-dependent Ferredoxin-NADP + reductase (FNR) (E ox/hq = -374 mV at pH 8.0 and 25ºC) through the formation of a ternary complex (Arakaki et al., 1997;Nogués et al., 2004;Sancho et al., 1990). In most cyanobacteria and some algae under low iron conditions the FMN-dependent Flavodoxin (Fld), particularly its Fld sq /Fld hq couple (E ox/sq = -256 mV, E sq/hq = -445 mV at pH 8.0 and 25ºC), substitutes for the Fd ox /Fd rd pair in this reaction (Bottin & Lagoutte, 1992;Goñi et al., 2009;. Thus, two Fld hq molecules transfer two electrons to one FNR ox that gets fully reduced after formation of the FNR sq intermediate. FNR hq transfers then both electrons simultaneously to NADP + (Medina, 2009) (Figure 1). Additionally to their role in photosynthesis, Fld and FNR are ubiquitous flavoenzymes that deliver low midpoint potential electrons to redox-based metabolisms in plastids, mitochondria and bacteria in all kingdoms (Müller, 1991). They are also basic prototypes for a large family of di-flavin electron transferases that display common functional and structural properties, where the electron transfer (ET) flow supported by the Fld/FNR modules occurs in reverse direction to the photosynthesis (Brenner et al., 2008;Wolthers & Scrutton, 2004). The better understanding of the ET flavin-flavin mechanism in this system should witness a greater comprehension of the many physiological roles that Fld and FNR, either free or as modules in multidomain proteins, play. In these chains there are still many open questions in understanding not only the ET mechanisms, but also the role that the flavin itself might play. Recent studies on the FNR:Fld interaction and ET indicate that the orientation driven by the alignment of the Fld molecular dipole moment with that of FNR contributes to the formation of a bunch of alternative binding modes competent for the efficient ET reaction (Frago et al., 2010;Goñi et al., 2009;Medina et al., 2008). FNR, Fld and NADP + are able to form a ternary complex, indicating that NADP + is able to occupy a site on FNR without displacing Fld Velázquez-Campoy et al., 2006). The two binding sites are not completely independent, and the overall reaction is expected to work in an ordered two-substrate process, with the pyridine nucleotide binding first, as reported for the Fd system (Batie & Kamin, 1984a, 1984b. Complex formation of Fd rd with FNR:NADP + was found to increase the ET rate by facilitating the rate-limiting step of the process, the dissociation of the product (Fd ox ) (Carrillo & Ceccarelli, 2003). Binding equilibrium and steady-state studies in WT and mutant proteins envisaged similar mechanisms for Fld, but the less specific FNR:Fld interaction might alter this behaviour (Medina, 2009). Fast kinetic stopped-flow methods have been used for the analysis of the mechanisms involving binding and ET between FNR and Fld. So far only single wavelength stoppedflow spectrophotometry studies (mainly at 600 nm) have been reported (Casaus et al., 2002;Goñi et al., 2008;Goñi et al., 2009;Nogués et al., 2003;Nogués et al., 2005), not involving the effects of NADP + , the ionic strength of the media, or the evaluation of the intermediate and final compounds of the equilibrium mixture. In this work, we use stopped-flow with photodiode array detection (PDA) to better evaluate the intermediate and final species in the equilibrium mixture during the ET process in the binary Fld:FNR and ternary Fld:FNR:NADP + systems.

Experimental methods improving the kinetic analysis of pre-steady-state electron transfer processes
ET has been analysed at different ionic strengths (I) and in both directions; the physiological photosynthetic ET, from Fld hq to FNR ox , and the reverse ET, from FNR hq to Fld ox , used to provide reducing equivalents to different metabolic pathways. Different Anabaena FNR and Fld variants have been used in the kinetic characterization of these ET processes using stopped-flow with photodiode detection.

Stopped-flow pre-steady-state kinetic measurements
Fast ET processes between Fld and FNR were followed by stopped-flow under anaerobic conditions in 50 mM Tris/HCl, pH 8.0 at 12ºC. Reactions were analysed by collecting multiple wavelength absorption data (360-700 nm) using an Applied Photophysics SX17.MV stopped-flow and a photodiode array detector (PDA) with the X-Scan software (App. Photo. Ltd.). Typically, 400 spectra per second were collected for each reaction. Anaerobic conditions were obtained by several cycles of evacuation and bubbling with O 2 -free argon. FNR hq and Fld hq samples were obtained by photoreduction in the presence of 1 mM EDTA and 5 µM 5-deazariboflavin . The use of PDA allowed detecting that under our experimental conditions the produced Fld hq samples usually contained a small amount of Fld sq (detected at 580 nm), taken into account in subsequent analysis. Unless otherwise stated, the mixing molar ratio was 1:1 with a final concentration of 10 µM for each protein. Molar ratios of 1:1, 1:2, 1:4 and 1:8 were also assayed for the reaction of FNR hq and Fld ox , with a final FNR hq concentration of 10 µM. Reactions were also analysed at different ionic strengths, obtained using variable NaCl concentrations ranging from 0 to 300 mM in 50 mM Tris/HCl, pH 8.0. The reaction between FNR ox and Fld hq was also studied in presence of ~300 µM NADP + at the indicated ionic strength conditions.

Kinetic analysis of multiple-wavelength absorption data
Spectral intermediates formed during reactions were resolved by singular value decomposition (SVD) using Pro-Kineticist (App. Photo. Ltd.). Data collected were fit either to a single step, AB, to a two steps ABC, or to a three steps, ABC, model allowing estimation of the conversion rate constants (k AB , k BC , k CD ) (Tejero et al., 2007). Estimated errors in the determined rate constant values were 15%. It should be stressed that SVD analysis of PDA spectra over a selected time domain resolves the spectra in the minima number of spectral intermediates species that are formed during the reaction, reflecting a distribution of protein intermediates (reactants, complexes, products...) at a certain point along the reaction time course, and not discrete enzyme intermediates. Moreover, none of them represent individual species and, their spectra cannot be included as fixed values in the global-fitting. Consequently, a spectral intermediate, in particular one that is formed in the middle of a reaction sequence, is an equilibrium distribution of protein species that are formed in a resolvable kinetic phase. Model validity was assessed by lack of systematic deviation from the residual plot at different wavelengths, inspection of calculated spectra and consistence among the number of significant singular values with the fit model. Simulations using Pro-Kineticist were also performed in order to validate the determined ET kinetics constants for the direct and reverse processes.
2.4 Determination of the absorbance spectra of FNR sq and Fld sq , and of protein discrete species contained in the spectral intermediates Due to the high maximum level for Fld sq stabilisation, the spectrum from this species was easily determined from photoreduction experiments ( Fig. 2A) (Frago et al., 2007). The spectrum of FNR sq was determined by following the one-electron oxidation of FNR hq with ferricyanide in the spectral range between 360 and 700 nm (Batie & Kamin, 1984a). Molar ratios FNR hq :ferricyanide of 1:10, 1:15 and 1:20 were used, with a final FNR hq concentration of 10 µM (Fig. 2B). Analysis of the spectroscopic data along the time was performed by Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS). Intermediate A, B, C and D species were deconvoluted initially considering they are produced by the lineal combination of FNR ox , FNR sq , FNR hq , Fld ox , Fld sq and Fld hq spectra (Fig. 2), and having into account the mass balance for total FNR and Fld. Deviations from the linear combination of the different redox states of FNR and Fld were observed in all cases. Visible spectra of FNR-Fld reaction mixtures slightly differ from the lineal combination of the individual components due to modulation of the flavin spectroscopic properties when changing its environment polarity upon complex formation. This indicates contribution of FNR:Fld complexes to the spectra of intermediate and final species (Casaus et al., 2002;Martínez-Júlvez et al., 2001;Nogués et al., 2003). Nevertheless, simulations proved that the method was adequate to estimate the percentage of the different redox states of each protein.

Spectral evolution of the electron transfer from Fld hq to FNR ox
Mixing Fld hq with FNR ox at I ≤ 125 mM produced a slight increase in the global amount of neutral semiquinone species within the instrumental dead-time. Then, absorbance decreased in the region of the flavin band-I (458 and 464 nm), while additionally increased in the 507-650 nm range ( Fig. 3A and 3B). The overall reaction best fits a single ET step (described by an apparent k BC rate constant), without detection of a protein-protein interaction step (k AB ). Resolution of the spectroscopic properties of B was consistent with FNR ox and Fld hq as the main components, while those of the final species C indicated Fld accumulated mainly as Fld sq , while FNR consisted mainly of FNR ox (80%) in equilibrium with smaller amounts of FNR sq and FNR hq (10% each). Deviation of the lineal combination of the Kinetics of the absorbance changes at 458, 464, 507, 517 and 577 nm. (C). Absorbance spectra for the pre-steady-state kinetically distinguishable species obtained by global analysis.
Intermediate B and C species are shown in green and red lines, respectively. The inset shows the evolution of these species along the time.
different FNR and Fld redox spectra was observed for B and C (Fig. 2), indicating mutual modulation of the spectroscopic properties of each one of the flavins, and, therefore, of its environment, by the presence of the second flavoprotein. This indicates a number of molecules must be forming FNR:Fld interactions, which might even be in different oxidoreduction states.
Noticeably, increasing ionic strength up to 125 mM had a drastic deleterious impact in the ET apparent k BC rate constant (Fig. 4), consistent with the lack of interaction observed by difference spectroscopy when WT FNR ox was titrated with WT Fld ox at I = 125 mM (not shown). These results indicated a considerable decrease in the FNR:Fld affinity upon increasing the ionic strength of the media.  .
Further increases in the ionic strength (I > 225 mM) produced the appearance of an additional final slow absorbance increase in the band-I, and, therefore, two ET steps described the process (Fig. 5). Thus, a slight absorbance bleaching of the band-I was observed from species B into C (as before described at lower I), but C further evolved with absorbance increases of this band and minor changes in the semiquinone one ( Fig. 5A and 5C). Resolution of the spectroscopic properties of B was also consistent with FNR ox and Fld hq as the main components, those of C indicated Fld sq and FNR ox in equilibrium with some amounts of FNR sq (10%), and FNR hq (20%), while D was mainly composed by Fld sq and FNR ox with 15% of Fld ox and FNR hq . The ionic strength additionally contributed to considerably diminish the apparent k BC ET rates (Fig. 4). ET from Fld hq to the FNR ox :NADP + preformed complex was consistent with two ET steps at all the assayed ionic strengths, and the presence of the pyridine nucleotide apparently modulated the electronic properties of the flavins (compare Fig. 6 with 3A and 3C). These observations correlated with the changes produced in the FNR spectrum upon NADP + complexation (Tejero et al., 2005). Despite the presence of NADP + barely affected apparent k BC between Fld and FNR (Table 1, Fig. 4), C showed increments in both the semiquinone and the band-I of the flavin with regard to B. The different spectroscopic properties of the intermediate species in the absence and presence of the nucleotide suggests that the nicotinamide portion of NADP + must contribute to the catalytically competent active site, probably by modulating the orientation and/or distance between the reacting flavins. This is in agreement with the negative cooperative effect observed for simultaneous binding of Fld and NADP + to FNR (Velázquez-Campoy et al., 2006). Additionally, C slowly evolved with a slight absorbance increase at the band-I of the flavin with minor changes in the semiquinone. These observations still indicate initial production of Fld sq and reduction of FNR ox , but suggest an additional step consistent with further FNR reoxidation. Analysis of the kinetic traces at 340 nm, where absorbance increase upon reduction of NADP + to NADPH is expected, shows that amplitudes are considerably larger for samples containing the coenzyme (not shown). This indicates the last detected step must be related with the hydride transfer from FNR hq to NADP + to produce its reduced form. Additionally, while the ionic strength of the media decreases the amplitudes at 340 nm for the reactions in the absence of NADP + , those in its presence are nearly unaffected (not shown). This process was also analysed for some FNR or Fld mutants previously produced and showing altered interaction or ET properties. This includes the FNR variant produced when Ala substituted for E301, a residue involved in the FNR catalytic mechanism as proton donor as well as for the stabilisation of reaction intermediates (Dumit et al., 2010;. Analysis of the reduction of this variant by WT Fld hq using the PDA detector showed that most of the ET took place within the instrumental dead time (not shown), confirming this is a very fast process as previously suggested . Moreover, in contrast to the WT process, no changes in rate constants or species were observed upon increasing the ionic strength of the media (Table 1). Thus, only information of the final spectra was obtained, being this similar to those for the WT reaction with main accumulation of Fld sq and FNR ox . In the presence of NADP + production of NADPH was observed at 340 nm with similar rates to those reported for the WT reaction. The process was similarly analysed for two Fld mutants for which the interaction step with FNR was reported to be modified by the introduction of multiple mutations (Goñi et al., 2009). Spectral evolution for the ET process between E16K/E61K Fld hq and FNR ox at low ionic strength (I = 25 mM) presented similar features to those above described for WT Fld (not shown). Despite a similar starting behaviour was observed for the reaction of E16K/E61K/D126K/D150K Fld hq , final absorbance increase and decrease in the flavin I and semiquinone bands, respectively, were observed (not shown). In both cases, but particularly with E16K/E61K/D126K/D150K Fld hq , evolution of the system was considerably hindered.  Fig. 7B). Surprisingly, C shows important amounts of the reduced species of both proteins and its additional transformation is consistent with Fld sq and FNR hq as the main products of the reaction. For both Fld mutants the increase in the ionic strength again had deleterious kinetic effects in the overall ET (

Spectral evolution of the electron transfer from FNR hq to Fld ox
When FNR hq reacted with Fld ox an initial bleaching and displacement of the maximum from 464 nm (typical of Fld ox ) to 458 nm (maximum for FNR ox ) was observed, together with the appearance of a neutral semiquinone band (Fig. 8A). Then, absorbance increased in both the band-I and the semiquinone one ( Fig. 8A and 8B). During the overall process only minor absorbance changes were detected in the Fld ox/sq isosbestic point (517 nm) at all the ionic strengths assayed, but an absorbance bleaching was initially observed at the FNR ox/sq one (507 nm) (Fig. 8B). When globally analysed this reaction best fits to a three steps process (A  B  C  D) (Fig.  8C). Conversion of species A into B was relatively fast (k AB >50 s -1 ) with very little absorbance changes. Both species had FNR hq and Fld ox as the main components, but deviation of mathematical combination of individual redox spectra (Fig. 2) indicated their spectroscopic properties were modulated by the presence of each other. This suggests a number of FNR hq and Fld ox molecules must be forming a FNR hq :Fld ox complex. This step appears, therefore, rather indicative of a protein-protein interaction or reorganisation event than of an ET one. B evolved to C in a relatively slow process (Table 1), in which the intensity of the band-I of the flavin gets decrease by 20% and displaced to shorter wavelengths, whereas accumulation of semiquinone is produced (Fig. 8B). These observations are compatible with Fld ox being consumed and FNR sq , Fld sq and FNR ox as the main components of species C. On conversion of C into D, there is an increment in the band-I absorption intensity (with the maximum at 458 nm) and minor changes in the semiquinone band amplitude. This is consistent with FNR ox and Fld sq as the main components of D. The faster ET rates are observed at the lower ionic strength, but the ionic strength effect is much more moderated than for the reverse reaction (Fig. 9A). Reaction of FNR hq with Fld ox was also analysed at different protein-protein ratios showing similar behaviour and a linear dependence of k BC and k CD rates (Fig. 9B). . Ionic strength dependence of the apparent rate constants for the reduction of Fld ox by FNR hq . The reaction was fit to a two-step process, k BC (black) and k CD (green). (B). Fld ox concentration dependence of the apparent rate constants for the reduction of Fld ox by FNR hq at I = 25 mM.
Reactions of FNR hq with E16K/E61K or E16K/E61K/D126K/D150K Fld ox variants showed similar features to the WT Fld (Fig. 10). Transformation of A into B appeared slightly slower for E16K/E61K (k AB 11 s -1 ), while those species could not be resolved for E16K/E61K/D126K/D150K. These observations are in agreement with the 23-fold decrease and the absence of interaction reported for the complexes of E16K/E61K or E16K/E61K/D126K/D150K Fld ox , respectively, with FNR ox (Goñi et al., 2009). B evolved to C in an ET process only 2-fold slower than for WT Fld ox (Table 1) and similarly consistent with FNR sq and Fld sq as the main components of C. On conversion of C into D, absorbance increments are observed in the band-I and the semiquinone band of the flavin, particularly in the E16K/E61K/D126K/D150K Fld variant. This might be consistent with FNR sq deproportionation into FNR ox and FNR hq , with the FNR hq produced being able to reduce another Fld ox molecule to the semiquinone state. Again processes of E301A FNR ox with Fld hq resembled those for the native proteins, with rate constants just slightly decreased and in agreement with previous reported data .

Insights into the electron transfer processes in the Fld:FNR system
The physiological ET from WT Fld hq to WT FNR ox was reported as a very fast process difficult to follow by single-wavelength stopped-flow methods. Nevertheless, this methodology resulted useful to study the processes for some FNR or Fld mutants with altered interaction or ET properties, but interpretation of the data was usually confuse due to the spectral similarities between the different oxido-reduction states of both proteins (Fig.  2) (Casaus et al., 2002;Goñi et al., 2008;Goñi et al., 2009;Medina et al., 1998;Nogués et al., 2003;Nogués et al., 2005). Analysis of mutants suggested that the reaction might take place in two steps interpreted as formation of the semiquinones of both proteins followed by further reduction of FNR sq to the fully reduced state, with further accumulation of Fld sq at the end of the reaction. Therefore, a similar behaviour was assumed for the WT system. In this work, we have taken advantage of stopped-flow with PDA detection to better evaluate the intermediate and final species in the equilibrium mixture during these ET processes for the reactions using the WT proteins and some of their mutants. Analysis of the process for the reduction of WT FNR ox by WT Fld hq , under similar conditions to those so far reported at single-wavelengths, indicates that even using PDA it is difficult to extract conclusions for this ET process. The data suggest a very fast interaction (or collisional) step unable to be observed (k AB ), followed by an ET step than cannot be resolved from the subsequent equilibration to finally produce Fld sq and FNR ox (process (1) in Scheme 1). However, despite FNR sq is hardly detected along the reaction, both semiquinones, Fld sq and FNR sq , must be initially produced. Moreover, FNR sq does not www.intechopen.com appear to be further reduced to FNR hq , and apparently quickly relaxes to FNR ox . Surprisingly, such behaviour was observed even when the overall ET reaction results slowed down by increasing the ionic strength. Thus, fast relaxation of the equilibrium distribution after the initial ET is produced with the consequent accumulation of FNR ox and Fld sq . Previous fast kinetic studies using laser flash photolysis indicated that ET from WT FNR sq to WT Fld ox is an extremely fast process (k obs ~ 7000 s -1 ), suggesting the produced FNR sq will quickly react with any traces of Fld ox producing Fld sq and FNR ox (Medina et al., 1992). Moreover, the proper nature of the PDA experiment might also contribute to this effect, since the high intensity of the lamp simultaneously exciting a wavelength range might produce side energy transfer reactions. (2) Scheme 1. Reaction pathways describing the processes observed for the reaction of FNR ox with Fld hq in the WT system (1) and with some of the Fld hq mutants (2).
When reduction of E301A FNR ox by Fld hq was analysed a very similar behaviour to the WT one described the process, again suggesting quick FNR sq deproportionation. When using the same methodology to analyse the process for two Fld mutants, with interaction and ET parameters considerably hindered (Goñi et al., 2009), intermediates and products of the reaction were in agreement with the mechanism previously proposed using singlewavelength detection and with the final production of FNR hq and Fld sq under the assayed conditions (Scheme 1 reaction (2)). The PDA analysis additionally allowed improving the determination of the ET rates (Table 1). In these cases the initially produced FNR sq appears unable to quickly react with traces of the Fld ox mutants, preventing the quick relaxation after the initial ET. This effect can be explained since the E ox/sq for these Fld variants is more negative than in WT Fld, getting closer to the FNR E ox/sq and making ET from FNR sq to Fld ox less favourable from the thermodynamic point of view than in the WT reaction (Table 2). Therefore, our observations suggest that the stopped-flow methodology, independently of the detector, does not allow to identify the initial acceptance by WT or E301A FNR ox of a single electron from WT Fld hq , since under the experimental conditions (even upon increasing the ionic strength of the media) the subsequent relaxation of the putatively initially produced FNR sq is faster than the initial ET process. Moreover, the products of this relaxation consist of a mixture of species that might not have physiological relevance within the Anabaena cell, where FNR sq must be able to accept electrons from a second Fld hq molecule.
The reverse ET reaction, FNR hq with Fld ox was reported as a slow process (when compared with the photosynthetic one) taking place in two sequential ET steps; production of both flavoprotein semiquinones (reaction (3) in Scheme 2), followed by the reduction of a second Fld ox molecule by the FNR sq produced in the first step (reaction (4) in Scheme 2) (Casaus et al., 2002;Medina et al., 1998;Nogués et al., 2005). The spectral evolutions acquired using the PDA detector confirm such mechanism, and allowed to improve the assignment of intermediates and the major contribution of apparent rate constants to particular steps of the process.
(4) k B C + Scheme 2. Reaction pathways describing the processes observed for the reaction of the FNR hq variants with the Fld ox variants.
Thus, for the process with the E16K/E61K Fld variant, the step corresponding to complex formation-reorganisation was erroneously related with an ET step in a previous study. At the lowest ionic strength assayed our data indicate that E16K/E61K Fld ox and, particularly, E16K/E61K/D126/D150K Fld ox are still able to accept electrons from FNR ox with apparent rates that only decreased by 2-fold and with final production of FNR ox occurring in higher degree that in the WT system (Table 1, Fig. 8 and 10). Their slightly more negative E ox/sq values (Table 2) makes them poorer electron acceptors from FNR hq than WT Fld and might explain the small differences in rates (Goñi et al., 2009). Additionally, E301A FNR hq is also able to pass electrons to Fld ox with a rate 5-fold slower than WT (Table 1). This behaviour might be related with the very low stability of the semiquinone form in this FNR mutant (Table 2), that makes the formation of this intermediate state non-favourable .
A biphasic dependence of the observed rate constants on the protein concentration has been reported for the ET reaction from Fd to FNR (Fig. 4), and associated with the appearance of www.intechopen.com an optimum ionic strength value and with the electrostatic stabilisation at low ionic strengths of non-optimal orientations within the intermediate ET complex. Thus, specific electrostatic and hydrophobic interactions play an important role in these association and dissociation processes, as well as in the rearrangement of the complex (Faro et al., 2002a;Hurley et al., 2006;Martínez-Júlvez et al., 1998;Martínez-Júlvez et al., 1999;Martínez-Júlvez et al., 2001;Morales et al., 2000;Nogués et al., 2003). Despite some residues on the FNR surface are critical for the interaction with Fld and it is accepted that FNR interacts using the same region with Fld and Fd (Hurley et al., 2002;Martínez-Júlvez et al., 1999), the bell-shaped profile for the ionic strength dependence is not reproduced for ET reactions between FNR and Fld (Fig. 4). A strong deleterious influence of the ionic strength is observed on the overall ET process between Fld and FNR, particularly in the photosynthetic direction ( Fig. 4 and 9A). This suggests re-arrangement of the initial FNR:Fld interaction either does not take place or does not increase the efficiency of the system, while at lower ionic strength the electrostatic interactions contribute to produce more efficient orientations between the flavin cofactors. Biochemical and docking studies suggested that the FNR:Fld interaction does not rely on a precise complementary surface of the reacting molecules. Thus, WT Fld might adopt different orientations on the FNR surface without significantly altering the relative disposition and contact between the FMN and FAD groups of Fld and FNR and, therefore, the distance between their methyl groups ( Fig. 11A) (Goñi et al., 2009;Medina et al., 2008;Medina, 2009). Those studies suggested the molecular dipole moment alignment as one of the major determinants for the efficiency of this system (Fig. 11B). However, kinetic parameters reported to the date for these processes were only obtained at low ionic strength (0.03 M), conditions far away from those found in the thylakoid (0.15-0.3 M (Durán et al., 2006)). Our data suggest that at physiological ionic strengths the ET efficiency, particularly in the photosynthetic direction, will be considerably hindered with regard to the data reported in vitro at low ionic strengths. The ionic strength will shield the dipole moment alignment contribution, making it just one additional contribution to proteins encounter. Among those contributions we might include electrostatic and hydrophobic interactions imposed by the thylakoid membrane, physical diffusional parameters and molecular crowding inside the cell. It is also worth to note that increasing the ionic strength makes reduction of FNR by Fld hq only 4-8 times faster than the reverse process (Compare Fig. 4A and 9A). Thus, shielding the effect of the dipole moment appears to have a larger impact in producing the competent ET orientation between the redox centres in the FNR ox :Fld hq complex than in the FNR hq :Fld ox one. In other words, it reduces the probability of obtaining the best FNR ox :Fld hq orientations for ET. The Fld mutants here studied, particularly E16K/E61K/D126/D150K, have lost the ability to efficiently reduce FNR (Table 1). More positive E sq/hq values (Table 2) might somehow contribute to this behaviour, but previous studies suggested the introduced mutations induced changes in the Fld electrostatic potential surfaces, as well as in the orientation and magnitude of the Fld molecular dipole moment (Goñi et al., 2009). Despite the thermodynamic parameters favour the process, the observed reaction might only correlate with a collisional-type reaction. Therefore, it could exit the possibility that steering of the dipole moment contribution might produce a positive effect on the overall ET process.
However, our analysis also shows that the increasing of the ionic strength again had a deleterious effect in the ET processes from these Fld hq mutants. Thus, electrostatic and hydrophobic interactions and the dipole moment still must contribute to the formation of productive interactions between both proteins at physiological ionic strengths. In vivo the presence of other proteins competing for Fld hq might also result in changes to electron channelling into distinct pathways. When going to physiological conditions Fld interaction with FNR is confirmed to be less specific than that of Fd. Subtle changes at the isoalloxazine environment influence the Fld binding abilities and modulate the ET processes by producing different orientations and distances between the redox centres. Therefore, ET reactions involving Fld might not have as much specific interaction requirements as other reactions involving protein-protein interactions. Thus, the bound state could be formed by dynamic ensembles instead of single conformations as has already been proposed for this system (Fig. 11) (Goñi et al., 2009;Medina et al., 2008) and also observed in other ET systems (Crowley & Carrondo, 2004;Worrall et al., 2003). This further confirms previous studies suggesting that Fld interacts with different structural partners through non-specific interactions, which in turn decreased the potential efficiency in ET that could achieve if unique and more favourable orientations were produced with a reduced number of partners. Heterogeneity of ET kinetics is an intrinsic property of Fld oxido-reduction processes, and can be most probably ascribed to different conformations of FNR:Fld complexes Sétif, 2001). During Fld-dependent photosynthetic ET the Fld molecule must pivot between its docking sites in PSI and in FNR. Formation of transient complexes of Fld with FNR in vivo is useful during this process, though not critical, for promoting efficient reduction of Fld and FNR and for avoiding reduction of oxygen by the donor redox centres (Goñi et al., 2008;Goñi et al., 2009;Hurley et al., 2006;Sétif, 2001Sétif, , 2006. www.intechopen.com

Conclusion
Single-wavelength fast kinetic stopped-flow methods have been widely used for the analysis of the mechanisms involving transient binding and ET between Fld and FNR. However, this methodology did not allow to un-ambiguously identifying the intermediate and final compounds of the reactions. PDA detection combined with fast kinetic stoppedflow methods results useful to better understand the mechanisms involving transient binding and ET between Fld and FNR. Despite the high similarity among the spectra for the same redox states within both proteins, the methodology here used allowed identifying the composition of the intermediate species and final species of the reactions, as long as the kinetics fits in the measurable instrumental time. The mechanism of these inter-flavin ET reactions is revisited, evaluating the evolution of the reaction along the time within a wavelength spectral range by using a PDA detector. Additionally, our analysis of the dependence of the inter-flavin ET mechanism on the ionic strength suggest that, under physiological conditions, the electrostatic alignment contributes to the overall orientation but it is not anymore the major determinant of the orientation of Fld on the protein partner surface. Additionally, the presence of the coenzyme reveals a complex modulation of the process.

Acknowledgment
This work has been supported by Ministerio de Ciencia e Innovación, Spain (Grant BIO2010-14983 to M.M.). We thank to Dr. G. Goñi for the production of the Fld mutants and Dr. R. Tauler for his help in initial spectral deconvolution of intermediate species. Photosynthesis is one of the most important reactions on Earth. It is a scientific field that is the topic of many research groups. This book is aimed at providing the fundamental aspects of photosynthesis, and the results collected from different research groups. There are three sections in this book: light and photosynthesis, the path of carbon in photosynthesis, and special topics in photosynthesis. In each section important topics in the subject are discussed and (or) reviewed by experts in each book chapter.

How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following: Ana Serrano and Milagros Medina (2012). Fast Kinetic Methods with Photodiode Array Detection in the Study of the Interaction and Electron Transfer Between Flavodoxin and Ferredoxin NADP+-Reductase, Advances in Photosynthesis -Fundamental Aspects, Dr Mohammad Najafpour (Ed.), ISBN: 978-953-307-928-8, InTech, Available from: http://www.intechopen.com/books/advances-in-photosynthesis-fundamental-aspects/revisitingthe-mechanism-of-interaction-and-electron-transfer-between-flavodoxin-and-ferredoxin-nadp