H-1 AND C-13 NMR ASSIGNMENTS AND STRUCTURAL ASPECTS OF A FERROCYTOCHROME C-551 FROM THE PURPLE PHOTOTROPHIC BACTERIUM ECTOTHIORHODOSPIRA-HALOPHILA

Two-dimensional nuclear magnetic resonance was used to assign the H-1 and C-13 resonances of ferrocytochrome c-551 from Ectothiorhodospira halophila, a halophilic phototrophic purple bacterium. This 78-residue protein belongs to a small subgroup of class I cytochromes c together with the analogous cytochromes c-551 from E. halochloris and E. abdelmalekii. A nearly complete assignment of C-13 resonances was obtained at natural abundance using a gradient-enhanced H-1-detected heteronuclear single quantum coherence experiment (HSQC). This was found to be extremely useful for the unambigous assignment of side chain protons. The secondary structure of the protein was determined from analyses of short- and medium-range nuclear Overhauser enhancements (NOE), amide proton exchange and C-13 alpha chemical shifts. Three helices could be identified which are well conserved among the class I cytochromes c. There is some evidence for two other regions of less well defined helical structure. From a preliminary analysis of long-range NOE it is shown that in the E. halophila cytochrome c-551 the general cytochrome c fold is well conserved, including the three conserved helices (residues 2-8, 41-50, 63-76), the regions around the heme ligands (Cys14-Ser15-Ser16-Cys17-His18, Met55) and the Omega loop (residues 18-28). In addition, three variable segments of the protein are discussed in detail, one of those including a cis-proline, a feature so far unique in the cytochrome c family. Structural alignments of the E. halophila cytochrome c-551 with two other Pseudomonas cytochrome c(5) homologs (Azotobacter vinelandii cytochrome c(5) and Chlorobium limicola cytochrome c-555) are provided which are based on sequence similarities and secondary structure alignments.