Radical Mediated Rapid <i>In Vitro</i> Formation of <i>c</i>-Type Cytochrome.

A cytochrome <i>c</i>₅₅₂ mutant from <i>Thermus thermophilus</i> HB8 (<i>rC</i>₅₅₂ C14A) was reported, where the polypeptide with replaced Cys14 by alanine, overexpressed in the cytosol of <i>E. coli</i>. The apo-form of the C14A mutant (apo-C14A) without the original prosthetic group was obtained by simple chemical treatments that retained compact conformation amenable to reconstitution with heme <i>b</i> and zinc(II)-protoporphyrin(IX), gradually followed by spontaneous formation of a covalent bond between the polypeptide and porphyrin ring in the reconstituted apo-C14A. Further analysis suggested that the residual Cys11 and vinyl group of the porphyrin ring linked through the thiol-ene reaction promoted by light under ambient conditions. In this study, we describe the kinetic improvement of the covalent bond formation in accordance with the mechanism of the photoinduced thiol-ene reaction, which involves a thiyl radical as a reaction intermediate. Adding a radical generator to the reconstituted C14A mutant with either heme-<i>b</i> or zinc(II) porphyrin accelerated the bond-forming reaction, which supported the involvement of a radical species in the reaction. Partial observation of the reconstituted C14A in a dimer form and detection of sulfuryl radical by EPR spectroscopy indicated a thiyl radical on Cys11, a unique cysteinyl residue in <i>rC</i>₅₅₂ C14A. The covalent bond forming mediated by the radical generator was also adaptable to the reconstituted apo-C14A with manganese(II)-protoporphyrin(IX), which also exhibits light-mediated covalent linkage formation. Therefore, the radical generator extends the versatility of producing <i>c</i>-type-like cytochrome starting from a metallo-protoporphyrin(IX) and the apo-C14A instantaneously.