Structure-function studies of [2Fe-2S] ferredoxins.
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).
Holden
H M
HM
Institute for Enzyme Research, University of Wisconsin-Madison 53705-4098.
Jacobson
B L
BL
Hurley
J K
JK
Tollin
G
G
Oh
B H
BH
Skjeldal
L
L
Chae
Y K
YK
Cheng
H
H
Xia
B
B
Markley
J L
JL
D000595:Amino Acid Sequence; D017033:Anabaena; D000818:Animals; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
C081714:2Fe-2S ferredoxin; D005288:Ferredoxins