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; 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 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; Goñi et al., 2009; Medina & Gómez-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 (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