ASSIGNMENT OF THE C-13 AND (CO)-C-13 RESONANCES FOR RHODOBACTER-CAPSULATUS FERROCYTOCHROME C(2) USING DOUBLE-RESONANCE AND TRIPLE-RESONANCE NMR-SPECTROSCOPY

Rhodobacter capsulatus cytochrome c(2) uniformly labelled with C-13/N-15 has been prepared. The C-13 resonances of the reduced state, including those of the carbonyl and heme C-13, have been assigned using a combination of various two- and three-dimensional correlated NMR experiments. Assignment of the sidechain C-13 resonances facilitated correction of a small number of previously misassigned sidechain H-1 and led to the additional assignment of 32 H-1. It was found that C-13 alpha and (CO)-C-13 secondary shifts were better indicators of secondary structure than H-1 alpha and C-13 beta secondary shifts. Moreover, it was demonstrated that, despite the significant ring current effects present in heme proteins, C-13 alpha and (CO)-C-13 secondary shifts can be employed to accurately identify secondary structure in heme proteins, independently of NOE experiments.