Studies of protein-protein association between yeast cytochrome c peroxidase and yeast iso-1 ferricytochrome c by hydrogen-deuterium exchange labeling and proton NMR spectroscopy.
Hydrogen-deuterium (H-D) exchange labeling and proton NMR have been applied to study the protein-protein association between cytochrome c peroxidase (CcP) and yeast iso-1 ferricytochrome c. Specifically, the exchange behavior of individual backbone amide protons of yeast iso-1 ferricytochrome c in both CcP-bound (i.e., complexed) and free (i.e., never in the complex) forms has been investigated and used in an attempt to map the binding site of CcP on yeast iso-1 ferricytochrome c when the noncovalent complex was formed in very low salt solution. The exchange rates of certain amino acid amide protons were significantly slowed down, by up to 40-fold, in the complex compared to the free form. The protected regions on iso-1 ferricytochrome c include parts of the 10's helix and the 70's helix surrounding the cytochrome c heme solvent-exposed edge (the so-called "front side" of iso-1 cytochrome c). These regions are very similar to the cytochrome c peroxidase binding interface on iso-1 ferricytochrome c that has been defined by X-ray crystallographic data. This further supports the direct involvement of the front side of iso-1 cytochrome c in binding with cytochrome c peroxidase. The results from our H-D exchange experiments also indicated that the amide proton exchange rates of Trp59, Asp60, and part of the 90's helix, all of which are located on the opposite side (the "back" side) of ferricytochrome c from the heme solvent-exposed edge, are also retarded upon complex formation.
Yi
Q
Q
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4630.
Erman
J E
JE
Satterlee
J D
JD
D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium; D006859:Hydrogen; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics
D000596:Amino Acids; D003574:Cytochrome c Group; D006859:Hydrogen; D003903:Deuterium; D003578:Cytochrome-c Peroxidase