Changing the invariant proline-30 of rat and Drosophila melanogaster cytochromes c to alanine or valine destabilizes the heme crevice more than the overall conformation.

Abstract

Drosophila melanogaster and rat cytochromes c in which proline-30 was converted to alanine or valine were expressed in a strain of baker's yeast, Saccharomyces cerevisiae, where they sustained aerobic growth. The mutations had no significant effect on the spectra or redox potentials but altered drastically the stability of the bond between the methionine-80 sulfur and the heme iron, as judged by four criteria: (i) the alkaline pKa values of the 695-nm band of the ferric form of the mutant proteins decreased by almost 1 pH unit as compared to the wild types; (ii) the acid pKa values increased by 0.5 to 1.2 pH units; (iii) the 695-nm band half-disappeared at temperatures 10-20TC lower in the mutant proteins than in the wild types; and (iv) the 695-nm band of the mutant proteins was susceptible to concentrations of urea that had little influence on their overall structure. The valinesubstituted rat cytochrome c had properties intermediate between those of the wild type and the alanine mutant. The destabilized coordinative bond is located in space a long distance from the mutation site. It is suggested that the mutations weaken the hydrogen bond between the carbonyl of residue 30 and the imino group of the imidazole of histidine-18, modifying the bonding of the heme iron by that imidazole, which, in turn, through a trans effect, weakens the bond between the heme iron and the other axial ligand, the sulfur of methionine-80. Alternatively, the effect of the mutations may be propagated allosterically along the peptide chain.

Lack of major involvement of human uroplakin genes in vesicoureteral reflux: Implications for disease heterogeneity

“…The main conclusion from these results is that some UP alterations, such as the ones we identified here, may play a role, albeit a relatively minor one, in VUR (see below). Specifically, the proline to alanine change in UP III could lead to the loss of a "proline-kink" thus resulting in a major change in protein conformation and possibly protein stability [36][37][38]. Although these results indicate that UP alterations can play a minor role in VUR, perhaps the most significant and somewhat surprising conclusion from our data is that major UP mutations are not found in patients with primary VUR.…”

Increasing the redox potential of isoform 1 of yeast cytochrome c through the modification of select haem interactions

“…Some success was demonstrated by the increased stability of the Y67F mutant [12][13][14][15], but not with different Pro$! mutants [16], which lead to haem-crevice destabilization.…”

Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochromec

“…23–29 Destabilization of the heme coordination loop is the likely explanation for the decreased pK a of the alkaline transition for several mutants of cyt c that target this loop, as well as for the presence of Lys-ligated conformers for cyt c in the presence of urea. 4 Mechanistic studies have suggested that deprotonation of a yet unknown “trigger” group precedes ligand replacement.…”