Electron-nuclear interactions in two prototypical [2Fe-2S] proteins: Selective (chiral) deuteration and analysis of H-1 and H-2 NMR signals from the alpha and beta hydrogens of cysteinyl residues that ligate the iron in the active sites of human ferredoxin and Anabaena 7120 vegetative ferredoxin

A vertebrate ferredoxin (human ferredoxin) and a plant-type ferredoxin (the ferredoxin from the vegetative form of Anabaena 7120) were labeled selecti vely with deuterium at their active site cysteines, The recombinant protein s were produced in Escherichia coli and labeled by replacing natural abunda nce cysteine in the defined culture medium with [H-2(alpha)]-cysteine, [H-2 (beta 2), H-2(beta 3)]-cysteine, or [H-2(beta 2)]-cystine. The chiral label ed cystine ([H-2(beta 2)]-cystine) was prepared by selective hydrogen excha nge catalyzed by cystathionine gamma-synthase, NMR spectra of these samples in their oxidized and reduced states support unambiguous identifications b y atom type of H-1 and H-2 NMR signals from the cysteine alpha and beta hyd rogens. These signals lie outside the normal diamagnetic spectral region as a result of interaction of the hydrogens with unpaired electron density fr om the iron-sulfur cluster, and their chemical shifts are highly dependent on local conformation at the active site. The very different chemical prope rties of the iron centers of plant-type and vertebrate ferredoxins reflect relatively small differences in the conformation of the iron-sulfur cluster ligands. (C) 2000 Academic Press.