SEQUENCE-SPECIFIC ASSIGNMENT OF LIGAND CYSTEINE PROTONS OF OXIDIZED, RECOMBINANT HIPIP-I FROM ECTOTHIORHODOSPIRA-HALOPHILA

The H-1 NMR spectra of the oxidized high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila have been investigated, a nd the sequence-specific assignment of the protons of the cluster-boun d cysteines has been performed. The spectrum of this protein is differ ent from those of the six other proteins investigated to date in that at least one hyperfine shifted signal is missing. It is demonstrated t hat the ''missing'' signals of the beta CH2 protons of Cys 66 are unde r the diamagnetic envelope at 2.60 and 6.30 ppm (at 288 K). The hyperf ine shifts of these protons and those of the beta CH2 protons of Cys 3 6 are consistent with the proposition that the oxidized cluster is in equilibrium between two electronic species. These species differ with respect to their valence distribution within the protein frame and are present in a molar ratio of about 4:1 at room temperature. An empiric al relationship between the hyperfine shifts of the beta CH2 protons o f Cys 36 and 66 and the percentage of the two electronic species is pr oposed.