Efficient Assembly of Cytochrome-Based Protoporphyrin IX Composite and Its Characterization as a Photosensitizer

Apo-<i>r</i>C₅₅₂ C14A/M69F, a heme-deficient mutant of cytochrome <i>c</i>₅₅₂ from <i>Thermus thermophilus</i> HB8, forms a thermally stable composite with protoporphyrin IX (PPIX). However, the apoprotein yield was compromised because of contamination of the purified protein with the holo-protein bearing a covalently attached heme moiety, impeding efficient composite preparation and subsequent studies on the composite. A newly prepared <i>r</i>C₅₅₂ mutant involving quadruple mutations, C11A, C14A, H15F, and M69F (<i>r</i>C₅₅₂-qm), addressed this problem while preserving the inherent thermal stability of a thermophilic bacterial protein. The results obtained for the R125A mutant of <i>r</i>C₅₅₂-qm corroborated the hypothesis that PPIX occupies the cavity of <i>r</i>C₅₅₂-qm with an orientation similar to that of heme <i>c</i> in the wild-type protein. The PPIX composite with <i>r</i>C₅₅₂-qm (PPIX@<i>r</i>C₅₅₂-qm) exhibited superior singlet oxygen (¹O₂) production activity compared to the PPIX composite with human serum albumin (PPIX@HSA). Stern-Volmer quenching analysis suggested that the enhanced ¹O₂ production of PPIX@<i>r</i>C₅₅₂-qm stems from the facilitated access of O₂ to photoexcited PPIX within PPIX@<i>r</i>C₅₅₂-qm relative to PPIX@HSA. Photoexcitation of PPIX@<i>r</i>C₅₅₂-qm induced the self-oxidation of PPIX in an aqueous medium, yielding a composite containing a particular chlorin derivative (photo-PPIX) as the degradation intermediate. The photo-PPIX@<i>r</i>C₅₅₂-qm composite was stable in the solution and showed ¹O₂ production activity upon exposure to red light because of the characteristic of an absorption band of the chlorin ring. This study proposes the <i>r</i>C₅₅₂-qm mutant as a platform for readily creating a stable cytochrome-based photosensitizer responsive to visible and red light in combination with readily available PPIX.