I. Introduction

The earth's primary energy conversion of sunlight into biomass is oxygeneic photosynthesis, driven by large protein-cofactor complexes in the plasma membrane of photosynthetic bacteria and in thylakoid membranes within chloroplasts of plants. These complexes, photosystem II and photosystem I, capture light energy and act sequentially to raise the energy of electrons. These electrons are utilized in electron transfer chains to generate a proton gradient across the membrane as well as NADPH. The electromotive force of the proton gradient is used by ATP Synthase to synthesize ATP. The NADPH is produced in the last steps of electron transfer as ferredoxin (Fd) transfers electrons to ferredoxin-NADP⁺ oxidoreductase (FNR) which then reduces NADP⁺ to NADPH. Together, the ATP and the NADPH provide energy to drive the light-independent Calvin Cycle, which fixes carbon from CO₂ into organic compounds. See Figure 1 for a schematic of the light-dependent processes.

Shown at left is the complex between ferredoxin (Fd) and ferredoxin-NADP⁺ oxidoreductase (FNR) from maize [Kurisu, et al., 2001]. Fd acquires electrons from Photosystem I and transfers these to the FNR prosthetic group, FAD. FAD in FNR acquires electrons sequentially from two Fds, moving from an oxidized state through a semiquinone to a fully reduced state, which then transfers two electrons to NADP⁺, reducing it to NADPH.

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It is possible that electrons are transferred directly from Fd to FNR since the distance from the FAD prosthetic group in FNR to the Fe atom that serves as the redox active site of the 2Fe-2S cluster in Fd is only ~6 Angstroms (Å) and the distance from FAD to the Fe-ligating sulfur atom of cys44 in Fd is only ~4 Å.

The interface between FNR and Fd buries approximately 800 Angstroms² of each molecule in a concave-convex complementarity.

A hydrophobic environment near the electron transferring cofactors (2Fe-2S and FAD) is provided by 5 Fd and 4 FNR residues.

Examining FNR from the cyanobacterium Anabaena, amino terminal and carboxy terminal domains are observed to bind FAD and NADP, respectively (Tejero, et al., 2005). A glutamate in the active site of FNR has been implicated in proper binding of the nicotinamide ring of NADP(H) and in electron transfer between FNR and Fd (Aliverti, et al., 1998). This glutamate shifts position upon binding of Fd by FNR, an important aspect of an induced fit conformational change in the active site of FNR (see below).

The juxtaposition of FAD and NADP, sandwiched in the deep cleft separating the amino terminal and carboxy terminal domains is readily observed by rendering the surface of Anabaena FNR.

Returning now to the maize FNR-Fd complex, a number of salt bridges between charged residues on Fd and FNR contribute to complex stability and orientation of the two proteins.

Of particular note is the pair of intermolecular salt bridges observed for Glu29 and Arg40 of maize Fd, as noted previously. In unbound spinach ferredoxin, these residues are involved in an intramolecular salt bridge (Binda, et al., 1998) and this bridge may have been conserved among Fds from cyanobacteria to higher plants (Knaff, 1996, cited in Kurisu, et al., 2001). If this intramolecular salt bridge exists in unbound maize Fd, the switch to intermolecular electrostatic bonds with residues from FNR upon binding may result in a significant shift of the redox potential of Fd, as has been observed for spinach Fd (a shift of -90 mV) [Batie and Kamin, 1981].

Significant FNR conformational shifts are observed upon Fd binding (induced fit), as can be observed by morphing PDB 1GAW (free FNR) with PDB 1GAQ (FNR bound to Fd). The following three simulations, each containing seven repeats of the morph, were generated using the Yale Morph Server at the Database of Macromolecular Movements, maintained by the Gerstein lab. Although the starting and ending structures are genuine models based on crystallographic data (PDB ID's 1GAW and 1GAQ), the intermediate transitions are based on linear interpolation and some energy minimization and are only possible structures. Only FNR is represented in the morphs, i.e. Fd and FNR cofactors (FAD, NADP) are not shown.

• An overview of the induced fit changes that occur upon FNR binding to Fd.
  
  
• There is a pronounced movement of a loop structure in the FAD binding (amino terminal) domain of FNR, allowing Lys 88 and Lys 91 of FNR to to move to a position that promotes salt bridge formation with Fd residues (see button 6, above).
  
  
• The induced fit conformational change in FNR moves glu312, mentioned above, into the hydrogen bonding vicinity of ser96. In the free state, the h-bond acceptor oxygen of glu312 is 5.4 Å from the h-bond donor oxygen of Ser 96 but, upon complex formation, the two atoms are separated by ~3.5 Å. This may optimize the positioning of NADP⁺ for a productive interaction with FAD.

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Aliverti, A., et al.. (1998). J. Biol. Chem. 273, 34008–34015.

Batie, C.J. & Kamin, H. J.. (1981). Journal of Biol. Chem. 256, 7756–7763.

Binda, C., Coda, A., Aliverti, A., Zanetti, G. & Mattevi, A. Acta Crystallogr. D 54, 1353–1358 (1998).

Genji Kurisu, Masami Kusunoki, Etsuko Katoh, Toshimasa Yamazaki, Keizo Teshima, Yayoi Onda, Yoko Kimata-Ariga and Toshiharu Hase. (2001). Structure of the electron transfer complex between ferredoxin and ferredoxin-NADP⁺ reductase. Nature Structural Biology 8: 117-121.

Knaff, D.B. (1996). In Photosynthesis; the light reactions. Ort, D.R. & Yocum, C.F., eds. pp. 333–336. Kluwer Academic Publishers, Dordrecht.

Laurence Serre, Frederic M. D. Vellieux, Milagros Medina, Carlos Gomez-Moreno, Juan C. Fontecilla-Camps and Michel Frey. (1996). X-ray Strucutre of the Ferredoxin: NADP⁺ Reductase from the Cyanobacterium Anabaena PCC 7119 at 1.8 Å Resolution, and Crystallographic Studies of NADP⁺ Binding at 2.25 Å Resolution. J. Mol. Biol. 263 (1996): 20-39.

Tejero J, Pérez-Dorado I, Maya C, Martínez-Júlvez M, Sanz-Aparicio J, Gómez-Moreno C, Hermoso JA, Medina M.. (2005). C-terminal tyrosine of ferredoxin-NADP+ reductase in hydride transfer processes with NAD(P)+/H. Biochemistry 44(41):13477-90.

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