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

Abstract

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 (FeII), but not ferric (FeIII) 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.