Assignment of N-15 chemical shifts and N-15 relaxation measurements for oxidized and reduced iso-1-cytochrome c

A protocol for complete isotopic labeling of iso-1-cytochrome c from the eu karyote Saccharomyces cerevisiae is reported. Assignments are reported for the vast majority of the N-15 amide resonances in both oxidized and reduced states. N-15 heteronuclear relaxation experiments were collected to study the picosecond-nanosecond backbone dynamics of this protein. Relaxation rat es were computed and fit to spectral density functions by a model-free anal ysis. Backbone amides in the overlapping loop B/C region are the most flexi ble on the picosecond-nanosecond time scale in both forms of the protein. T he results show that, on average, the protein backbone is slightly more dyn amic in the oxidized than the reduced state, though not significantly so. E xchange terms, which suggest significant motion on a time scale at least an order of magnitude slower than the overall correlation time of 5.2 ns, wer e required for only two residues in the reduced state and 27 residues in th e oxidized state. When analyzed on a per-residue basis, the lower order par ameters found in the oxidized state were scattered throughout the protein, with a few continuous segments found in loop C and the C-terminal helix, su ggesting greater flexibility of these regions in the oxidized state. The re sults provide-dynamic interpretations for previously presented structural a nd functional data, including redox-dependent changes that occur in the pro tein. The way is now paved for extensive dynamic analysis of variant cytoch romes c.