Electron-Nuclear Interactions in Two Prototypical [2Fe–2S] Proteins: Selective (Chiral) Deuteration and Analysis of 1H and 2H NMR Signals from the Alpha and Beta Hydrogens of Cysteinyl Residues That Ligate the Iron in the Active Sites of Human Ferredoxin

A vertebrate ferredoxin (human ferredoxin) and a plant-type ferredoxin (the ferredoxin from the vegetative form of Anabaena 7120) were labeled selectively with deuterium at their active site cysteines. The recombinant proteins were produced in Escherichia coli and labeled by replacing natural abundance cysteine in the defined culture medium with [2Hα]-cysteine, [2Hβ2, 2Hβ3]-cysteine, or [2Hβ2]-cystine. The chiral labeled cystine ([2Hβ2]-cystine) was prepared by selective hydrogen exchange catalyzed by cystathionine γ-synthase. NMR spectra of these samples in their oxidized and reduced states support unambiguous identifications by atom type of 1H and 2H NMR signals from the cysteine alpha and beta hydrogens. These signals lie outside the normal diamagnetic spectral region as a result of interaction of the hydrogens with unpaired electron density from the iron–sulfur cluster, and their chemical shifts are highly dependent on local conformation at the active site. The very different chemical properties of the iron centers of plant-type and vertebrate ferredoxins reflect relatively small differences in the conformation of the iron–sulfur cluster ligands.