The cytochromes c provide a wide range of natural and mutant homologous proteins which may be used to study structure/function relationships in biological electron-transfer reactions. A description of the cytochrome c structure has been provided by high-resolution X-ray crystallography for the cytochromes from tuna, bonito, rice and yeast (Saccharomyces iso-1). Correlation of these structures with NMR parameters is necessary to confirm the structure of the protein in solution and to permit the routine characterisation of cytochromes c with novel sequences. We have previously reported a method based on the analysis of pseudocontact shifts which allowed us to compare the conformations of some amino acid side chains of tuna cytochrome c in solution and in the crystalline state. Here we report a comparison of the conformations of the polypeptide backbone of cytochromes c in proteins from tuna and horse, using the chemical shifts of the amide NH and C,H protons. It is found that the backbone conformation and hydrogen-bond network is closely conserved between these proteins, despite 19 amino acid substitutions. Appreciable differences occur in two regions, around Asn 31 and at the beginning of the 60s helix. Evidence for some rotational or translational motion of the C-terminal helix is also presented.Cytochromes c are small electron-transfer proteins ( M , ~1 2 5 0 0 ; 103-113 residues) which are present in the intermembrane space of all eukaryotic mitochondria where they fulfil a variety of functions. While their primary role is that of an electron-transfer protein supplying electrons to the terminal oxidase of the mitochondrial respiratory chain, they are also involved in other electron-transfer reactions, for example as electron acceptors from sulphite oxidase or as electron donors to cytochrome c peroxidase [l].Currently, amino acid sequences have been determined for cytochromes c from more than 90 different species [2]. The range of sequence similarities is impressively large, varying from 100% (e.g. man vs chimp) to about 50% (e.g. tuna vs rice) while only 25 residues are conserved in all cytochromes c. In many cases these differences in primary structure can be recognised in biological assays. Two separate types of molecular recognition have been exploited in these studies. Firstly the reactions between a single oxidase and a number of cytochromes c from different species have been investigated and some kinetic and thermodynamic parameters have been defined [3-51. From the data obtained it is clear that the overall thermodynamics of the reaction are essentially unaltered by the variation in the amino acid sequence, while the kinetics of the electron-transfer reaction are more substantially affected. The latter may reflect changes in the cytoCorrespondence to