A series of familiar hemoproteins such as myoglobin, horseradish peroxidase, cytochrome P450 possess a cofactor, protoporphyrin IX iron complex (heme b), which is bound in the heme pocket via Fe–axial ligand coordination and non-covalent interaction with large affinities. The cofactor is removable from the heme pocket under acidic conditions to yield a corresponding apoprotein. Furthermore, the protein reconstitution is enabled by incorporating an artificial cofactor into the apoprotein. Over the last decade, we have focused on the heme-substitution with several porphyrinoid metal complexes.¹⁾ In addition, we have introduced a heme moiety onto the hemoprotein surface via a covalent linkage to construct a supramolecular protein assembly via interprotein heme–heme pocket interaction. Here, we present a topic of hemoprotein assembly formed by the reconstitutional method.

A hexameric tyrosine-coordinated heme protein (HTHP) containing photosensitizer molecules was prepared to construct a biomolecule having engineered photochemical properties.²⁾ Zn protoporphyrin IX (ZnPP) or Zn chlorin e6 (ZnCe6) was inserted into the apoprotein of HTHP to yield the reconstituted protein, which maintained the intrinsic hexameric structure of HTHP. Femtosecond transient absorption measurements by a flash photolysis system suggest the occurrence of rapid singlet–singlet annihilation within a few picoseconds for each protein completely reconstituted with the photosensitizer molecules. This finding supports the fact that the photo-induced energy migration occurs within the protein with the zinc complex. The fluorescence quenching efficiency determined by the addition of methyl viologen as an electron acceptor for the completely reconstituted protein with ZnPP is 2.3 fold-higher than that of the partially photosensitizer-inserted protein where only one molecular of ZnPP is inserted into the hexamer. This indicates that the energy migration occurs among the photosensitizers bound within the protein matrix. These findings are expected to lead to development of new protein-based artificial light harvesting systems.

Furthermore, our group recently tried to introduce five fluorescein molecules and one Texas Red on the surface of the cysteine-containing HTHP mutant with ZnPP. Upon the irradiation of fluorescein at 593 nm, the energy transfer from fluorescein was observed by monitoring the emission of Texas Red at 615 nm.³⁾ We found two pathways of the energy transfer; one is the direct energy transfer from fluorescein to Texas Red and another is the energy transfer via ZnPP. The ratio of the pathways is 39 : 61, indicating that the energy is collected at the Texas Red molecule as a funnel-like bottom for light-harvesting. This system will serve as a new model to demonstrate a light-harvesting event.

REFERENCES 1) Hayashi, T. In Handbook of Porphyrin Science; Kadish, K. M.; Smith, K. M.; Guilard, R., Eds.; World Scientific Publishing: Singapore, 2010; Vol. 5, pp 1–69. 2) Oohora, K.; Mashima, T.; Ohkubo, K.; Fukuzumi, S.; Hayashi, T. Chem. Commun. 2015, 51, 11138. 3) Mashima, T.; Oohora, K.; Hayashi, T. Phys. Chem. Chem. Phys. 2017 in press.