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

Abstract

Cytochrome c 553 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 c 553 . 1D NMR titration of complex formation gave us the stoichiometry (1:1) and the dissociation constant of the complex (K d V V3U U10 36 M). 2D heteronuclear NMR experiments were performed to analyze the 1 H and 15 N 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.z 1999 Federation of European Biochemical Societies.

Heteronuclear NMR and Soft Docking: An Experimental Approach for a Structural Model of the Cytochrome c553−Ferredoxin Complex

“…The experimental approach uses the NMR data on the mapping of the interaction site on cytochrome c 553 (23) and on the ferredoxin (the present work) to limit the set of solutions. Nineteen NMR constraints (twelve on the cytochrome and seven on the ferredoxin) were used to score the 1000 docking solutions according to their compatibility with this experimental data.…”

“…In the other two solutions, this tyrosine residue is positioned away from the complex interface and makes intramolecular hydrogen bonds within the cytochrome molecule. Moreover, Ser9, the amino acid in the cytochrome, which is the most perturbed in the heteronuclear NMR experiments (23), is at the center of the interaction site in solution 2. For these reasons, we think that solution 2 is the best solution (Figure 7A).…”

Structure/Function Relationships of [NiFe]- and [FeFe]-Hydrogenases

“…This complex is very similar to the one between cytochrome c 553 and a two-[Fe 4 S 4 ] ferredoxin that is homologous to the hydrogenase ferredoxin-like domain. In that case, NMR shift information could be used for both redox partners because of their small sizes …”

Structural Model of the Fe-Hydrogenase/Cytochromec 553 Complex Combining Transverse Relaxation-optimized Spectroscopy Experiments and Soft Docking Calculations

“…These NMR data were used as a filter to choose the most accurate structure obtained from the ab initio docking solutions proposed by the software BiGGER (15). We have recently demonstrated (13,16) that the use of NMR filters necessitates the labeling of both proteins so that NMR constraints are applied on both partners of the complex. In the present work, the labeling of hydrogenase is not possible because of the poor expression of the enzyme in the sulfatereducing bacteria.…”