Structure and stability effects of the mutation of glycine 34 to serine in Rhodobacter capsulatus cytochrome c(2).
Gly 34 and the adjacent Pro 35 of Rhodobacter capsulatus cytochrome c(2) (or Gly 29 and Pro 30 in vertebrate cytochrome c) are highly conserved side chains among the class I c-type cytochromes. The mutation of Gly 34 to Ser in Rb. capsulatus cytochrome c(2) has been characterized in terms of physicochemical properties and NMR in both redox states. A comparison of the wild-type cytochrome c(2), the G34S mutation, and the P35A mutation is presented in the context of differences in chemical shifts, the differences in NOE patterns, and structural changes resulting from oxidation of the reduced cytochrome. G34S is substantially destabilized relative to wild-type (2.2 kcal/mol in the oxidized state) but similarly destabilized relative to P35A. Nevertheless, differences in terms of the impact of the mutations on specific structural regions are found when comparing G34S and P35A. Although available data indicates that the overall secondary structure of G34S and wild-type cytochrome c(2) are similar, a number of both perturbations of hydrogen bond networks and interactions with internal waters are found. Thus, the impact of the mutation at position 35 is propagated throughout the cytochrome but with alterations at defined sites within the molecule. Interestingly, we find that the substitution of serine at position 34 results in a perturbation of the heme beta meso and the methyl-5 protons. This suggests that the hydroxyl and beta carbon are positioned away from the solvent and toward the heme. This has the consequence of preferentially stabilizing the oxidized state in G34S, thus, altering hydrogen bond networks which involve the heme propionate, internal waters, and key amino acid side chains. The results presented provide important new insights into the stability and solution structure of the cytochrome c(2).
Zhao
D
D
Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
Hutton
H M
HM
Meyer
T E
TE
Walker
F A
FA
MacKenzie
N E
NE
Cusanovich
M A
MA
D019943:Amino Acid Substitution; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045362:Cytochromes c2; D004795:Enzyme Stability / Q000235:genetics; D005998:Glycine; D017354:Point Mutation; D016354:Rhodobacter capsulatus / Q000378:metabolism*; D012694:Serine; D013329:Structure-Activity Relationship
D003574:Cytochrome c Group; D012694:Serine; D045362:Cytochromes c2; D005998:Glycine