NMR IS A UNIQUE AND NECESSARY STEP IN THE INVESTIGATION OF IRON SULFUR PROTEINS - THE HIPIP FROM R-GELATINOSUS AS AN EXAMPLE

H-1 NOE and 2D NMR studies have been performed on oxidized and reduced HiPIP from Rhodocyclus gelatinosus (formerly Rhodopseudomonas gelatinosa). By saturating the beta-CH2 Protons of the cysteines in the oxidized form which are hyperfine shifted, NOEs are measured on signals which have been then assigned to specific residues through 2D NMR. In this way the signals of the cysteines have been specifically assigned. Since we had already related the shifts of the beta-CH2 Protons to the oxidation state of each iron, we succeeded in connecting the individual cysteines with the oxidation state of each iron. Support for the assignment comes from 2D measurements of the reduced protein which is less paramagnetic. Signals in the oxidized and reduced species can be related through EXSY. These results represent a unique and successful strategy to frame an iron sulfur cluster within a protein, with the iron ions labelled according to their oxidation number.