The absolute conservation of tryptophan at position 59 in cytochrome c is related 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. Crystallographic evidence identifies the indole nitrogen of Trp-59 as having a stabilizing 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 protein, whereas the smaller Gly or Ser cannot. Semisynthetic analogues were designed to test the importance of the side-chain features of tryptophan by using a recently developed method for stereoselective fragment religation in yeast cytochrome c. Three yeast iso-1 cytochrome c analogues were produced in which Trp-59 was replaced by a non-coded amino acid: p-iodophenylalanine, beta-(3-pyridyl)-alanine or beta-(2-naphthyl)-alanine. Replacement of Trp-59 with these non-coded amino acids allows the reasons for its conservation 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 different impact on physicochemical and biological parameters from those of abolishing hydrogen-bond donor potential. This suggests that Trp-59 has both a local and a global stability effect by solvating a buried charge and by having a key role in the packing of the cytochrome c hydrophobic core.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Black KM, Clark-Lewis I, Wallace CJ

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