Equilibrium unfolding of a small low‐potential cytochrome, cytochrome c 553 from Desulfovibrio vulgaris

To understand general aspects of stability and folding of c ‐type cytochromes, we have studied the folding characteristics of cytochrome c 553 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 c 553 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 c 553 unfolds by an apparent two‐state process. Reduced cytochrome c 553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c 553 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 c 553 become high‐spin. The lack of heme misligation in unfolded cytochrome c 553 implies that its unfolded structure is less constrained than those of cytochromes c with low‐spin, misligated hemes.