oligosaccharide portions of these molecules in Me2S0 and Me2NCH0. In these studies, the amide side chain (in GalNAcj3( 1-.3)Gal) was found to exist in two conformations basically involving torsions about the trans arrangement (T = 160° J. Am. Chem. SOC. 1991, 113, 6822-6831 and 60'). In this report we have shown by comparing experimental and simulated NOES from the NH proton that for the oligosaccharides LND-I and LNF-I only one of these two conformations agrees with experiment.Abstract: Conformational dynamics within the complex between Zn-substituted cytochrome c peroxidase (ZnCcP) and cytochrome c (Cc) has been studied by examining the quenching of the 'ZnP excited state by the ferriheme of Cc. The temperature and solvent dependence of the triplet-state quenching rate constants (k,) show that complexes of ZnCcP with a large set of Fe3+ cytochromes c undergo a transition between a low-temperature state that does not exhibit triplet quenching and a high-temperature state that does. Within the narrow transition range (220 K C T C 250 K), the decay traces for the [ZnCcP, Fe3+Cc] complexes are nonexponential, and outside of this range they are exponential. This behavior is displayed by complexes with Cc(Drosophi1a melanogaster), Cc(Candida krusei) and a suite of site-directed mutants of Cc(yeast iso-I) where position 82 contains either an aliphatic (Met, Ser, Leu, or Ile) or an aromatic (Phe) residue. Above 250 K, k, varies strongly among these complexes and decreases sharply with the concentration of cosolvent (EG = ethylene glycol), apparently because of increasing viscosity, while both the breadth of the transition range and its midpoint vary little within this class. MCD and optical spectra between ambient and 4 K rule out the trivial explanation that the transition might reflect changes in the coordination state, and the invariance of k, with a IO-fold increase in [Cc] shows that the proteins remain bound as a complex upon cooling. As the midpoint and breadth of the transition are unaffected by changes in percent EG, the transition does not appear to arise from a solventdriven process. Instead, we propose that, at ambient temperatures, the binding interface of the [CcP, Cc] complex undergoes rapid dynamic rearrangements between the subset of conformers that exhibit 3ZnP quenching and the subset that does not. Below the transition range, the complex exists in the latter form, and it is suggested that, upon heating, there is a cooperative loosening of the binding interactions within the interface. We present an heuristic description of the complex based on the statistical mechanical description of the cooperative helix-coil transition in poly(amino acids). In contrast, members of a second class of complexes, those with Cc(horse), Cc(tuna), and Cc(rat), have low quenching rate constants ( k , = 40 s-I at ambient) that decrease smoothly to k, = 0 s-] by 250 K. Furthermore, k, for these complexes shows little dependence upon either solvent or cytochrome, and the triplet decay traces remain exponential at all t...
The methionine 80 sulfur-heme iron bond of rat cytochrome c, whose stability is decreased by mutating the phylogenetically invariant residue proline 30 to alanine and increased when tyrosine 67 is changed to phenylalanine, recovers its wild-type characteristics when both substitutions are performed on the same molecule. Titrations with urea, analyzed according to the heteropolymer theory [Alonso, D. O. V., & Dill, K. A. (1991) Biochemistry 30, 5974-5985], indicate that both single mutations increase the solvent exposure of hydrophobic groups in the unfolded state, while in the double mutant this conformational perturbation disappears. Similar increases in solvent exposure of hydrophobic groups are observed when the sulfur-iron bond of the wild-type protein is broken by alkylation of the methionine sulfur, by high pH, or by binding the heme iron with cyanide. The compensatory effects of the two single mutations do not extend to the overall stability of the protein. The added loss of conformational stability due to the single mutations amounts to 7.3 kcal/mol out of the 9 kcal/mol representing the overall free energy of stabilization of the native conformation of the wild-type protein. The folded conformation of the doubly mutated protein is only 2 kcal/mol less stable than that of the wild type. These results indicate that the double mutant protein is able to retain the essential folding pattern of cytochrome c and the thermodynamic stability of the methionine sulfur-heme iron bond, in spite of structural differences that weaken the overall stability of the molecule.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.