Structure of yeast iso-1-cytochrome c in which arginine-38 is replaced by alanine-38

Abstract

Mitochondria1 cytochrome c is a monohaem electron-transfer protein of 103-1 14 amino acids. Arginine-38 is an unvaried, internally located residue that interacts with the buried haem prioponate-7 (HP-7) [ 11. Previous work with related bacterial cytochromes has led to proposals that arginine-38 reduces the pK, of HP-7 to less than 4.5 [2] and has an important role in the oxidation-state-linked protein conformational change [ 31. To test these proposals mutants of yeast iso-1-cytochrome c have been constructed in which arginine-38 has been replaced by lysine, histidine, asparagine, glutamine, leucine and alanine (R. L. Cutler, M. Smith & A. G. Mauk, unpublished work). In the present communication we report 'H n.m.r. spectroscopic data showing that replacement of arginine-38 by alanine-38 does not significantly affect the interaction of HP-7 with the neighbouring tryptophan-59.Samples of ferricytochrome c were prepared for n.m.r. as previously described [4]. Both proteins contained the substitutions cysteine-102 to threonine-102; they differed in residue 38, which was arginine or alanine. N.m.r. spectra were recorded with a JEOL GX-400 n.m.r. spectrometer. Spectra showing nuclear Overhauser enhancements (NOE) [5] and two-dimensional correlated spectroscopy (2D-COSY ) spectra [6] were obtained using the standard JEOL PLEXUS packages. Fig. 1 shows the NOE difference spectrum of the alanine-38 ferricytochrome obtained upon saturation of a HP-7 PCH resonance. This is very similar to the NOE difference spectrum obtained for the arginine-38 protein and for horse ferricytochrome c [4]. The pattern of NOES show that the conformation of HP-7 is not significantly altered by the replacement of arginine-38 by alanine-38. The peak at 7.6 p.p.m. comes from the tryptophan 59 residue which is hydrogen-bonded to HP-7 in wild-type yeast cytochrome c [l].Abbreviations used: HP-7, haem propionate-7, NOE, nuclear Overhauser enhancement; 2D-COSY, two-dimensional correlated spectroscopy.