Equilibrium unfolding of a small low-potential cytochrome, cytochrome c₅₅₃ from Desulfovibrio vulgaris

Abstract

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c₅₅₃ from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c₅₅₃ by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c₅₅₃ unfolds by an apparent two-state process. Reduced cytochrome c₅₅₃ is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c₅₅₃ is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c₅₅₃ become high-spin. The lack of heme misligation in unfolded cytochrome c₅₅₃ implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.