Multiheme proteins are important in energy conversion and biogeochemical cycles of nitrogen and sulfur. Redox properties of these assemblies depend not only on the ligands to the heme iron and how multiple hemes are arranged in the protein but also on the protein matrix in between. While some multiheme proteins have no distinct interdomain boundaries, others have domains separated in modules, with clearly defined interfaces. A diheme cytochrome c4 (c4) was used as a model to elucidate the role of the interdomain interface on redox properties of the heme iron centers. Isolated monoheme domains c4-A and c4-B and multiple variants of the full-length proteins were studied by spectroscopic techniques, stability measurements, and MD simulations. Analyses of the isolated domains and similar monoheme proteins suggest that monoheme proteins arrange their polypeptides to enclose the heme edge. If this is not possible, the proteins dimerize and form an interface centered at heme propionates. Indeed, both c4-A and c4-B fragments dimerize in this way but the formation of oligomeric species is avoided in the full-length diheme protein. Contacts at the interface and surface charges of the domains tune reduction potentials of the heme centers. We have used phylogenetic analysis to identify polypeptide sequences that track the evolutionary relationship between two structurally-similar c4 proteins, one with apparent potentials of the two hemes being the same and the other with them being different by more than 100 mV. Analyses of potentials and redox-dependent stability of these proteins rationalize sequence changes that equalize potentials and increase cooperativity between the two metal centers. The study reveals factors that tune reduction potentials and cooperativity of metal centers in multiheme assemblies, important for multielectron delivery, and enriches understanding of redox-dependent complexation, important for unidirectional electron transfer.

Influence of the neighboring metal center and the interdomain interface on redox properties of cytochrome c4

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