High-resolution 1H- and 15N-NMR studies of Rhodospirillum rubrum cytochrome c2.
Rhodospirillum rubrum cytochrome c2 was uniformly enriched in 15N and studied by 1H- and 15N-NMR spectroscopy. Relaxation and NOE data allowed determination of the rotational correlation time and indicated more rapid side-chain motion in the native protein and increased segmental motion in the base-denatured protein. The pi nitrogen of the ligand histidine and the indolic nitrogen of the invariant tryptophan both remain protonated and act as proton-donors in hydrogen bonds over a wide pH range and therefore do not contribute to pH-related changes in the midpoint potential. pK values identified by numerous methods in the ferrocytochrome at pH 6.9 and in the ferricytochrome at pH 6.2 arise from His-42. At pH values below the pK, the imidazolium group participates in a salt bridge or in a hydrogen bond with the carboxylate group of the inner propionate of the heme. Loss of the proton causes a local conformational change which alters the midpoint potential. The pK values of the amino terminus and lysines were also determined from pH titrations monitored by 15N-NMR. Similar titrations of the ferricytochrome monitored by 1H-NMR showed structural heterogeneity in that the resonance of heme ring methyl 8 split into a doublet as the pH was raised.
Smith
G M
GM
Department of Food Science & Technology, University of California, Davis.
Yu
L P
LP
D003574:Cytochrome c Group / Q000737:chemistry*; D045362:Cytochromes c2; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D012247:Rhodospirillum rubrum / Q000201:enzymology*
D003574:Cytochrome c Group; D045362:Cytochromes c2