NMR ASSIGNMENT OF RHODOBACTER-CAPSULATUS FERRICYTOCHROME C', A 28-KDA PARAMAGNETIC HEME PROTEIN

The cytochromes c' are paramagnetic heme proteins generally consisting of two identical 14 kDa subunits. The H-1 and N-15 resonances of the ferricytochrome c' from the purple phototrophic bacterium Rhodobacter capsulatus have been extensively assigned by the TOCSY-HSQC, NOESY-HSQC, HSQC-NOESY-HSQC, and HNHA 3D heteronuclear experiments performed on an 8 mM sample labeled with N-15. In addition, the C-13(alpha) and (CO)-C-13 resonances were assigned by the HNCA and multiple-quantum HNCOCA 3D experiments performed on a 0.5 mM sample labeled with C-13 and N-15. Th, assignment of the backbone C-13 resonances was used to confirm the H-1 and N-15 assignments and to better define secondary structure. On the basis of medium-range NOEs, (3)J(HN alpha) coupling constants, and backbone C-13 chemical shifts, the secondary structure consists of four helices: helix-1 (3-29), helix-2 (33-49), helix-3 (78-97), and helix-4 (103-117). On the basis of long-range NOE contacts, the Rb. capsulatus ferricytochrome c' is a four-helix bundle protein in which consecutive helices are antiparallel with respect to one another.