Structure-function studies of [2Fe-2S] ferredoxins

Abstract

The ability to overexpress [2Fe-2S] ferredoxins in Escherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegatative and heterocyst forms of Anabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in in these proteins in their oxidized and reduced states has been studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partner Anabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).

Mutations of Glu92 in Ferredoxin I from Spinach Leaves Produce Proteins Fully Functional in Electron Transfer but Less Efficient in Supporting NADP+ Photoreduction

“…First, the cross-linking of Fd I to FNR was not prevented by the elimination of the carboxyl group at position 92, but its rate was decreased in the case of Fd-E92K in keeping with the lower affinity for FNR of this mutant as measured by the Kd of the complex (Table 3). Second, the role for the sidechain of Glu92 according to the three-dimensional structure of the cyanobacterial ferredoxin (Holden et al, 1994) should be to stabilize the [2Fe-2S] binding loop through hydrogen bonding to the Ser45 side-chain. Indeed, beside a modification of the redox potential of the iron-sulfur cluster in the Glu92 mutants, small changes in their EPR spectra were also observed , suggesting a perturbation of the iron-sulfur cluster environment.…”

“…In the chloroplast-type ferredoxins, the acidic cluster is fairly well conserved: Glu92 (spinach protein sequence numbering) is strictly conserved ; Glu93 is conservatively substituted by Asp in two cases or changed to Gln; Glu94 is more variable, but in such a way that an acidic residue is always present at the positions 93 or 94 (SwissProt Data Bank). Whereas ferredoxins from higher plants have not been structurally characterised, the three-dimensional structure of ferredoxins from cyanobacteria has been determined both by X-ray crystallography and NMR (Holden et al, 1994). Recently, sitedirected mutagenesis studies on cyanobacterial ferredoxins have been reported (Holden et al, 1994;Schmitz et al, 1993).…”

“…Whereas ferredoxins from higher plants have not been structurally characterised, the three-dimensional structure of ferredoxins from cyanobacteria has been determined both by X-ray crystallography and NMR (Holden et al, 1994). Recently, sitedirected mutagenesis studies on cyanobacterial ferredoxins have been reported (Holden et al, 1994;Schmitz et al, 1993). Specifically, the mutants at position 94, which is the equivalent of Glu92 of the spinach Fd I, were found unable to transfer electrons to ferredoxin -NADP' reductase when reduced by flash photolysis (Hurley et al, 1993.…”

Mutations of surface residues in Anabaena vegetative and heterocyst ferredoxin that affect thermodynamic stability as determined by guanidine hydrochloride denaturation

“…These H bonds are part of an evolutionarily conserved hydrogen bonding pattern believed to stabilize the [2Fe-2S] binding loop by tethering it to the short C-terminal a-helix (Holden et al, 1994). Preliminary X-ray crystallographic data on the E94K mutant (B.L.…”

“…The wt stability of Vfd mutant E95K can be rationalized in terms of what is known about the environment of this residue. Because the electron density from the E95 side chain is at the protein surface and is not well defined in the electron density map derived from X-ray analysis (Holden et al, 1994), this residue is solvent exposed and appears not to participate in specific interactions with the rest of the protein. …”