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 c₄ (c₄) 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 c₄-A and c₄-B, together with the full-length diheme c₄ 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 c₄, but becomes His/His-ligated in c₄-B at higher pH. Intradomain contacts in c₄-A are minimally affected by the separation of c₄-A and c₄-B domains, and isolated c₄-A is folded. In contrast, the isolated c₄-B is partially unfolded, and the interface with c₄-A guides folding of this domain. The c₄-A and c₄-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) c₄-A and c₄-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.