Conserved tryptophan in cytochrome c: importance of the unique side-chain features of the indole moiety

The absolute conservation of tryptophan at position 59 in cytochrome c is r elated to the unique chemical nature of its indole moiety. The indole side chain of Trp-59 possesses three salient features: bulk, hydrophobicity and the ability of its indole nitrogen to act as a hydrogen-bond donor. Crystal lographic evidence identifies the indole nitrogen of Trp-59 as having a sta bilizing hydrogen-bonding interaction with the buried carboxylate group of haem propionate 7. Side-chain bulk is also likely to be important because a Phe or Leu residue can replace Trp to give an at least partly functional p rotein, whereas the smaller Gly or Ser cannot. Semisynthetic analogues were designed to test the importance of the side-chain features of tryptophan b y using a recently developed method for stereoselective fragment religation in yeast cytochrome e. Three yeast iso-1 cytochrome c analogues were produ ced in which Trp-59 was replaced by a non-coded amino acid: p-iodophenylala nine, beta-(3-pyridyl)-alanine or beta-(2-naphthyl)-alanine. Replacement of Trp-59 with these noncoded amino acids allows the reasons for its conserva tion to be analysed, because they vary with respect to size, hydrophobicity and hydrogen-bond potential. Our results show that decreasing the bulk and hydrophobicity of the side chain at position 59 has a profound but differe nt impact on physicochemical and biological parameters from those of abolis hing hydrogen-bond donor potential. This suggests that Trp-59 has both a lo cal and a global stability effect by solvating a buried charge and by havin g a key role in the packing of the cytochrome c hydrophobic core.