IDENTIFICATION OF THE IRON IONS OF HIGH-POTENTIAL IRON PROTEIN FROM CHROMATIUM-VINOSUM WITHIN THE PROTEIN FRAME THROUGH 2-DIMENSIONAL NMR EXPERIMENTS

2D NMR experiments performed on both the oxidized and reduced form of the high potential iron protein (HiPIP) from Chromatium vinosum, a paramagnetic iron sulfur protein for which the crystal structure is known in both oxidation states, allowed us to detect a number of scalar and dipolar connectivities of the isotropically shifted signals. On this basis it was possible to firmly identify the signals of the beta-CH2 and alpha-CH protons of the cluster-liganded cysteines and perform their sequence-specific assignments. The assignments mainly rely on the observation of NOESY cross peaks from beta or alpha-Cys protons to protons assigned to the few aromatic residues surrounding the cluster. This is the first sequence-specific assignment of Cys beta-CH2 protons for a Fe4S4 cluster. In the light of existing experimental evidence from Mossbauer data and of the theoretical model describing the magnetic coupling of the metal centers in the oxidized form, the present assignment establishes which iron ions of the oxidized cluster are in a pure ferric state and which are in a mixed valence state. These findings may be relevant as far as the actual mechanism of electron transfer is concerned. In addition, information is obtained on the angular dependence of the beta-CH2 hyperfine shifts in iron sulfur systems.