The role of a conserved water molecule in the redox-dependent thermal stability of iso-1-cytochrome c.
Eukaryotic cytochromes c contain a buried water molecule (Wat166) next to the heme that is associated through a network of hydrogen bonds to three invariant residues: tyrosine 67, asparagine 52, and threonine 78. Single-site mutations to two of these residues (Y67F, N52I, N52A) and the double-site mutation (Y67F/N52I) were introduced into Saccharomyces cerevisiae iso-1-cytochrome c to disrupt the hydrogen bonding network associated with Wat166. The N52I and Y67F/N52I mutations lead to a loss of Wat166 while N52A and Y67F modifications lead to the addition of a new water molecule (Wat166) at an adjacent site (Berghuis, A. M., Guillemette, J. G., McLendon, G., Sherman, F., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 236, 786-799; Berghuis, A. M., Guillemette, J. G., Smith, M., and Brayer, G. D. (1994) J. Mol. Biol. 235, 1326-1341; Rafferty, S. P., Guillemette, J. G., Berghuis, A. M., Smith, M., Brayer, G. D., and Mauk, A. G. (1996) Biochemistry, 35, 10784-10792). We used differential scanning calorimetry (DSC) to determine the change in heat capacity (DeltaCp) and the temperature dependent enthalpy (DeltaHvH) for the thermal denaturation of both the oxidized and reduced forms of the iso-1 cytochrome c variants. The relative stabilities were expressed as the difference in the free energy of denaturation (DeltaGD) between the wild type and mutant proteins in both redox states. The disruption of the hydrogen bonding network results in increased stability for all of the mutant proteins in both redox states with the exception of the reduced Y67F variant which has approximately the same stability as the reduced wild type protein. For the oxidized proteins, DeltaGD values of 1.3, 4.1, 1.5, and 5.8 kcal/mol were determined for N52A, N52I, Y67F, and Y67F/N52I, respectively. The oxidized proteins were 8.2-11.5 kcal/mol less stable than the reduced proteins due to a redox-dependent increase in the entropy of unfolding.
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Department of Chemistry and the Guelph-Waterloo Centre for Graduate Work in Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1. gguillem@chemistry.watstar.uwaterloo.ca
Berghuis
A M
AM
Frey
H E
HE
Lepock
J R
JR
Guillemette
J G
JG
D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D004795:Enzyme Stability; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics; D014867:Water / Q000737:chemistry*
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D014867:Water; D045304:Cytochromes c