The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c' from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four-helix bundle structure containing a covalently bound five-coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer-monomer transition according to the absence and presence of CO. Herein, domain-swapped dimeric AVCP was constructed and utilized to form a tetramer and high-order oligomers. The X-ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain-swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain-swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit-subunit interactions at the interfaces of the domain-swapped dimer and monomer subunits in the tetramer were also similar to the subunit-subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain-swapped dimer and two monomers upon CO binding. Without monomers, the domain-swapped dimers formed tetramers, hexamers, and higher-order oligomers in the absence of CO, whereas the oligomers dissociated to domain-swapped dimers in the presence of CO, demonstrating that the domain-swapped dimer maintains the CO-induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer-monomer transition protein.

The [Cr(edda or eddp)(N-N)]-type complexes (edda=ethylenediamine-N,N′-diacetate; eddp=ethylenediamine-N,N′-dipropionate), trans(O)-[Cr(edda)(phen)]Cl and csi(O)-[Cr(eddp)(phen)]Cl, were first prepared stereoselectively and characterized by UV-Vis absorption and IR spectra and X-ray crystal analysis. The optically active (−)CD510-[Cr(edda)(phen)]+ was readily racemized even in an acidic aqueous solution within a period of several hours.

Cytochrome c' (CP) is a gas-binding homo-dimeric heme protein. Mesophilic Allochromatium vinosum CP (AVCP) and thermophilic Hydrogenophilus thermoluteolus CP (PHCP) have high sequence and structure similarities. AVCP is known to exhibit a dimer-monomer transition upon CO binding/dissociation, whereas detailed CO-binding properties of PHCP remain unrevealed. Here, we found that the CO-binding affinity of wild-type PHCP is lower than that of AVCP, and the PHCP dimer does not dissociate to monomers under CO-saturated reduced conditions. The CO-binding affinity of PHCP increased by mutations in the subunit-subunit interface (F11T, T18F, or F71D). The T18F, F71D, and T18F/F71D PHCP variants exhibited similar dimer-monomer transitions upon CO binding/dissociation to that of AVCP, although the F11T variant did not. The simulated structures of the PHCP variants revealed that the T18F and F71D mutations caused rearrangement in the subunit-subunit interface, whereas the F11T mutation did not, indicating that the effective dimer-monomer transitions upon CO binding/dissociation are induced by the rearrangement in the subunit-subunit interface. The present results indicate that subunit-subunit interface mutation of oligomeric proteins is a useful approach in the adjustment of protein stability and ligand binding affinity, leading to a change in the quaternary structural property.

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