Cytochrome c553, a small heme protein that lacks misligation in its unfolded state, folds with rapid two-state kinetics

Cytochrome c553 (cyt c553) from Desulfovibrio vulgaris is a small helical heme protein that displays apparent two-state equilibrium-unfolding behavior. The covalently attached heme is low-spin, ligated by Met and His residues, in the native state but becomes high-spin upon unfolding at pH 7. Here, we show that in contrast to other c-type heme proteins, where misligations in the unfolded states are prominent, cyt c553 refolding kinetics at pH 7 proceeds rapidly without detectable intermediates. The extrapolated folding rate constant in water for oxidized cyt c553 matches exactly that predicted from the cyt c553 native-state topology: 5300 s−1(experimental) versus 5020 s−1 (predicted). We therefore conclude that the presence of the oxidized cofactor does not affect the intrinsic formation speed of the cyt c553 structural motif.