Characterization of pH-dependent conformational heterogeneity in Rhodospirillum rubrum cytochrome c2 using 15N and 1H NMR.
The 15N-enriched ferricytochrome c2 from Rhodospirillum rubrum has been studied by 15N and 1H NMR spectroscopy as a function of pH. The 15N resonances of the heme and ligand tau nitrogen are broadened beyond detection because of paramagnetic relaxation. The 15N resonance of the ligand histidine phi nitrogen was unambiguously identified at 184 ppm (pH 5.6). The 15N resonances of the single nonligand histidine are observed only at low pH, as in the ferrocytochrome because of the severe broadening caused by tautomerization. The dependence of the 15N and 1H spectra of the ferricytochrome on pH indicated that the ligand histidine tau NH does not dissociate in the neutral pH range and is involved in a hydrogen bond, similar to that in the reduced state. Because neither deprotonated nor non-hydrogen-bonded forms of the ligand histidine are observed in the spectra of either oxidation state, the participation of such forms in producing heterogeneous populations having different electronic g tensors is ruled out. Transitions having pKa's of 6.2, 8.6, and 9.2 are observed in the ferricytochrome. The localized conformational change around the omega loops is observed in the neutral pH range, as in the ferrocytochrome. Structural heterogeneity leads to multiple resonances of the heme ring methyl at position 8. The exchange rate between the conformations is temperature dependent. The transition with a pKa of 6.2 is assigned to the His-42 imidazole group. The displacement of the ligand methionine, which occurs with a pKa of 9.2, causes gross conformational change near the heme center.(ABSTRACT TRUNCATED AT 250 WORDS)
Yu
L P
LP
Department of Food Science and Technology, University of California, Davis 95616.
Smith
G M
GM
D003574:Cytochrome c Group / Q000378:metabolism*; D045362:Cytochromes c2; D006639:Histidine; D006859:Hydrogen; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D012247:Rhodospirillum rubrum / Q000378:metabolism*
D003574:Cytochrome c Group; D009587:Nitrogen Isotopes; D006639:Histidine; D006859:Hydrogen; D045362:Cytochromes c2