Structure and stability effects of the mutation of glycine 34 to serine inRhodobacter capsulatus cytochrome c(2)

Gly 34 and the adjacent Pro 35 of Rhodobacter capsulatus cytochrome c(2) (o r Gly 29 and Pro 30 in vertebrate cytochrome c) are highly conserved side c hains among the class I c-type cytochromes. The mutation of Gly 34 to Ser i n Rb. capsulatus cytochrome c(2) has been characterized in terms of physico chemical 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 p atterns, and structural changes resulting from oxidation of the reduced cyt ochrome. G34S is substantially destabilized relative to wild-type (2.2 kcal /mol in the oxidized state) but similarly destabilized relative to P35A. Ne vertheless, differences in teres of the impact of the mutations on specific structural regions are found when comparing G34S and P35A. Although availa ble data indicates that the overall secondary structure of G34S and wild-ty pe cytochrome c(2) are similar, a number of both perturbations of hydrogen bond networks and interactions with internal waters are found. Thus, the im pact 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 perturb ation of the heme beta meso and the methyl-5 protons. This suggests that th e hydroxyl and beta carbon an positioned away from the solvent and toward t he heme. This has the consequence of preferentially stabilizing the oxidize d state in G34S, thus, altering hydrogen bond networks which involve the he me propionate, internal waters, and key amino acid side chains. The results presented provide important new insights into the stability and solution s tructure of the cytochromes c(2).