Influence of the Interdomain Interface on Structural and Redox Properties of Multiheme Proteins.

Multiheme proteins are important in energy conversion and biogeochemical cycles of nitrogen and sulfur. A diheme cytochrome c 4 ( c 4 ) was used as a model to elucidate roles of the interdomain interface on properties of iron centers in its hemes A and B. Isolated monoheme domains c 4 -A and c 4 -B, together with the full-length diheme c 4 and its Met-to-His ligand variants, were characterized by a variety of spectroscopic and stability measurements. In both isolated domains, the heme iron is Met/His-ligated at pH 5.0, as in the full-length c 4 , but becomes His/His-ligated in c 4 -B at higher pH. Intradomain contacts in c 4 -A are minimally affected by the separation of c 4 -A and c 4 -B domains, and isolated c 4 -A is folded. In contrast, the isolated c 4 -B is partially unfolded, and the interface with c 4 -A guides folding of this domain. The c 4 -A and c 4 -B domains have the propensity to interact even without the polypeptide linker. Thermodynamic cycles have revealed properties of monomeric folded isolated domains, suggesting that ferrous (Fe II ), but not ferric (Fe III ) c 4 -A and c 4 -B, is stabilized by the interface. This study illustrates the effects of the interface on tuning structural and redox properties of multiheme proteins and enriches our understanding of redox-dependent complexation.