Role of the highly conserved tryptophan of cytochrome c in stability.

To test the importance of the highly conserved tryptophan to the structure and function of cytochrome c, we have prepared the mutant W67Y (tryptophan 67 substituted by tyrosine) of Rhodobacter capsulatus cytochrome c2. The mutation appears to have little effect on the redox potential or in vitro electron transfer properties. The presence of the W67Y near-infrared absorbance at 702 nm establishes that methionine is ligated to the heme group but the 6-nm red-shift in the absorbance maxima suggests that the environment of the heme iron-methionine sulfur bond is perturbed. Moreover, the pK for W67Y alkaline transition is decreased 0.9 pH units with respect to the wild-type, indicating that the heme iron-methionine sulfur bond of the mutant is destabilized approximately 1 kcal/mol. Based on guanidine-HCl denaturation monitored with the 220-nm circular dichroism signal, the W67Y conformational stability is decreased by 40% (2.5 kcal/mol) and 60% (3.0 kcal/mol) in the oxidized and reduced states, respectively. Collectively, these data suggest that tryptophan 67 of Rb. capsulatus cytochrome c2, or its equivalent in other species, is important to stability but is not critical to the time-averaged structure, redox potential, or function.