Abstract
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.
Highlights
Water in and around proteins is recognized as being important to protein structure, function and stability (4 – 6)
The water molecule is adjacent to the heme and the hydrogen bonding network which is composed of conserved residues Asn52, Tyr67, and Thr78, which are hydrogen bonded in ferrocytochrome c to the Met80 sulfur which is one of the two heme iron ligands
A small proportion of the protein became oxidized during the thermal scans of the reduced proteins. This was detected in the differential scanning calorimetry (DSC) scan of ferrocytochrome c as a minor transition at the Tm of the oxidized protein
Summary
Wat166, invariant water 166 in the structure of yeast iso-1 cytochrome c; DSC, differential scanning calorimetry; ⌬Cp, change in molar heat capacity between native and unfolded states; Tm, midpoint for the transition; ⌬HvH, change in apparent (van’t Hoff) enthalpy at Tm; ⌬GD, change in Gibb’s free energy of denaturation ⌬SD, change in entropy of denaturation; ⌬SvH, change in apparent (van’t Hoff) entropy at Tm; ⌬HD, change in enthalpy of denaturation.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
