Mapping the cytochrome c553 interacting site using 1H and 15N NMR

AbstractCytochrome c553 is the electron transfer partner of formate dehydrogenase and of [Fe]-hydrogenase, two metalloenzymes essential in the metabolism of sulfate reducing bacteria. These two enzymes contain a ‘ferredoxin-like’ domain which presents 30% identity with Desulfovibrio desulfuricans Norway ferredoxin I. This was chosen as a model for the ‘ferredoxin-like’ domain involved in the electron transfer reaction with cytochrome c553. 1D NMR titration of complex formation gave us the stoichiometry (1:1) and the dissociation constant of the complex (Kd ∼3×10−6 M). 2D heteronuclear NMR experiments were performed to analyze the 1H and 15N chemical shift variations that are induced by the protein-protein recognition. This is the first mapping of the interaction site on a c-type cytochrome, using heteronuclear NMR