Rational Design of Domain‐Swapping‐Based c‐Type Cytochrome Heterodimers by Using Chimeric Proteins

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three‐dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c‐type cytochrome‐based chimeric proteins have been constructed and the domains swapped. According to X‐ray crystallographic analysis, the two chimeric proteins formed a domain‐swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H2O coordinated hemes was formed. Binding of an oxygen molecule to the His/H2O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe−O2 stretching band was observed at 580 cm−1 for the reduced/oxygenated heterodimer (at 554 cm−1 under an 18O2 atmosphere). These results show that domain swapping is a useful method to design multiheme proteins. Swap shop: A new methodology is applied in the construction of c‐type cytochrome (cyt) heterodimers through the domain swapping of two chimeric proteins, in which the N‐terminal regions of Pseudomonas aeruginosa cyt c551 and Hydrogenobacter thermophilus cyt c552 are exchanged with each other. The result is a heterodimer with different active sites.