title: A proton NMR study of the non-covalent complex of horse cytochrome c and yeast cytochrome-c peroxidase and its comparison with other interacting protein complexes.
abstract: Cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) forms a noncovalent 1:1 complex with horse cytochrome c in low ionic strength solution that is detectable by proton NMR spectroscopy. When the entire proton hyperfine-shifted spectrum is considered only five hyperfine resonances exhibit unambiguously detectable shifts: the heme 8-CH3 and 3-CH3 resonances, single proton resonances near 19 ppm and -4 ppm and the methionine-80 methyl group. These shifts are very similar to those observed for the covalently crosslinked complex of cytochrome-c peroxidase and horse cytochrome c, but different from those reported for cytochrome c complexes with flavodoxin and cytochrome b5. By comparison with the shifts reported for lysine-13-modified cytochrome c we conclude that the results reported here support the Poulos-Kraut proposed structure for the molecular redox complex between cytochrome-c peroxidase and cytochrome c. These results indicate that the principal site of interaction with cytochrome-c peroxidase is the exposed heme edge of horse cytochrome c, in proximity to lysine-13 and the heme pyrrole II. The noncovalent cytochrome-c peroxidase-cytochrome c complex exists in the rapid-exchange time limit even at 500 mHz proton frequency. Our data provide an improved estimate of the minimum off-rate for exchanging cytochrome c as 1133 (+/- 120) s-1 at 23 degrees C.
mesh_terms: D000818:Animals; D003196:Computer Graphics; D003574:Cytochrome c Group; D003578:Cytochrome-c Peroxidase; D006418:Heme; D006736:Horses; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010544:Peroxidases; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D003574:Cytochrome c Group; D006418:Heme; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase; D008239:Lysine