The substitution of proline 35 by alanine in Rhodobacter capsulatus cytochrome c2 affects the overall protein stability but not the alkaline transition

It was shown by Koshy et al. [1990, Proc. Natl Acad. Sci USA, 87, 8697-8701; 1994, Biochem. J., 299, 347-350] that the substitution of proline 30by alanine (P30A) of Drosophila melanogaster and rat cytochromes cexhibited decreased stabilities in both the heme iron-methionine sulfur(Fe-S) bond and overall protein conformation. Now we have found that thestability properties of the equivalent mutant of Rhodobacter capsulatuscytochrome c2 (P35A) are somewhat different. Based on optical and NMRspectroscopies, the Rb.capsulatus P35A alkaline transition (pKalk) wasfound to be unchanged with respect to the wild type, suggesting that themutation in Rb.capsulatus cytochrome c2 has little effect on the stabilityof the Fe-S bond. However, Rb.capsulatus conformational stability was foundto be decreased by 1.6 kcal/mol in the oxidized state. The difference inthe stability properties of the equivalent proline to alanine substitutionsin various species underscores the importance of studying mutations in morethan one species before drawing generalizations about the role of conservedresidues in protein structure and function.