CHANGES IN GLOBAL STABILITY AND LOCAL-STRUCTURE OF CYTOCHROME-C UPON SUBSTITUTING PHENYLALANINE-82 WITH TYROSINE

We have examined the F82Y;C102T variant of Saccharomyces cerevisiae is o-1-cytochrome c using high-resolution proton nuclear magnetic resonan ce spectroscopy, chemical denaturation, and differential scanning calo rimetry. Comparison of proton chemical shifts, paramagnetic shifts, an d nuclear Overhauser effects indicates structural changes are localize d to the vicinity of position 82. One alteration involves the rearrang ement of the side chain of leucine-85. Using many more proton assignme nts than were available in the initial report [G. J. Pielak, R. A. Atk inson, J. Boyd, and R. J. P. Williams, Eur. J. Biochem. 177, 179-185 ( 1988)], a second alteration involving an interaction between arginine- 13 and tyrosine-82 is observed. The interaction appears to involve a h ydrogen bond with the eta-protons of arginine's guanido group acting a s donor and tyrosine's phenolic eta-oxygen as acceptor. In spite of th is potentially-stabilizing interaction, the free energy of denaturatio n decreases by approximately 2.4 kcal mol-1. Results are discussed wit h respect to alterations in the native and denatured states.