The effects of multiple amino acid substitutions on the polypeptide backbone of tuna and horse cytochromes c

Abstract

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

Proton‐NMR studies of the effects of ionic strength and pH on the hyperfine‐shifted resonances and phenylalanine‐82 environment of three species of mitochondrial ferricytochrome c

“…X-ray crystallographic techniques and NMR studies have revealed that this residue is packed together with Thr9, Leu94 and Ile85 around the heme thioether-2 methyl group in tuna cytochrome c (Takano and Dickerson, 1981;Gao et al, 1989;see Fig. 1B).…”

“…Mobility of the side chain of Phe82 in ferricytochrome c is inferred from the fact that both ortho and both meta protons on opposite sides of the phenyl ring have the same chemical shifts (Gao et al, 1989;Moore and Williams, 1980;Boswell et al, 1980). The rotational flip-rate of Phe82 has been estimated to be lo3 s-' (Moore et al, 1982).…”

“…His33 of horse cytochrome c is situated near the C-terminus of the protein and is in van der Waal's contact with residue 102 of the C-terminal helix (Gao et al, 1989;Williams et al, 1985). This helix is made up of residues 88-104 in horse ferricytochrome c (Feng et al, 1989) and forms one edge of the heme crevice.…”

“…Third, we have tried to elucidate specific differences in the solution structures of ferricytochromes c from yeast, tuna and horse by a comparison of the above spectral changes for each of these three proteins. In some cases we have had to rely on the published crystal structure of tuna ferricytochrome c (Takano and Dickerson, 1981) and a detailed NMR comparison that has indicated very similar backbone conformations for tuna and horse ferricytochromes c (Gao et al, 1989) as well as the proton assignments obtained from the recently published 2D-NMR studies of horse ferricytochrome c (Feng et al, 1989) and yeast iso-1' ferricytochrome c (Gao et al, 1990).…”

Assignment of Pseudo‐contact‐shifted 1H NMR resonances in the EF site of Yb3 +‐substituted rabbit parvalbumin through a combination of 2D techniques and magnetic susceptibility tensor determination

“…A signals from non-coordinating residues, together with (iii) identification of the latter based on characteristic NOESY, COSY and TOCSY pattern^. '^-'^ On the other hand, the potential of pseudo-contact shifts [point (3) ] has not yet been fully exploited, except in the particular case of haeme proteins, where independent information on the magnetic susceptibility tensor is available." '2 When the magnetic susceptibility tensor is unknown, some success has been obtained only in the case of cobalt-substituted protein^,'^*'^ but again advantage was taken of the presence of strongly contact-shifted signals.…”