THE NATURE OF THE THERMAL-EQUILIBRIUM AFFECTING THE IRON COORDINATIONOF FERRIC CYTOCHROME-C

In cytochrome c, ligation of the heme iron by the methionine-80 sulfur plays a major role in determining the structure and the thermodynamic stability of the protein. In the ferric state, this bond is reversibl y broken by moderately acid or alkaline pH's (pK's 2.5 and 9.4, respec tively) and by exogenous ligands. NMR studies of horse ferricytochrome c in which the Met-65 and Met-80 methyl groups were chemically enrich ed with C-13 demonstrate that, at 59 degrees C, a temperature at which the protein is still folded, the sulfur-iron bond is already partiall y broken. This structural change corresponds to the reversible disappe arance upon moderate heating of the 695 nm band, characteristic of the sulfur-iron coordination of this protein. The thermal effect results from a shift in the alkaline pK from 9.4 at 25 degrees C to 8.2 at 59 degrees C. The exchange rate from iron-bound to free methionine-80 at 59 degrees C is 1.8 s(-1), as measured by saturation transfer experime nts. The free and bound methionine-80 epsilon-methyl groups in the H-1 spectrum are assigned as (1.87, 2.25) and -21.43, respectively; in th e C-13 spectrum they are assigned as 15.6 and 12.8, respectively(all t hese values are in ppm from 3-(trimethylsilyl)propionic-2,2,3,3-d(4) a cid, sodium salt).