Strategies for the study of cytochrome c structure and function by site-directed mutagenesis

The class I cytochromes c have been extensively studied by biochemical and biophysical methods; however, many questions remain concerning the roles of specific amino acids in electron transfer and stability properties. The method of site-directed mutagenesis, which substitutes specific amino acid residues by genetic methods, is ideal for addressing these questions of cytochrome c structure and function. Practical considerations of mutational effects on protein processing and stability will be addressed. The criteria for the selection of mutation sites will be discussed. Examples of site-directed mutagenesis studies, which were designed to elucidate the factors controlling biological electron transfer, protein processing, and protein stability, are given.