Design of an artificial light-harvesting unit by protein engineering: Cytochrome b₅₆₂-green fluorescent protein chimera

We have generated a novel model protein for an artificial light-harvesting complex composed of two proteins, cytochrome b₅₆₂ (cytb₅₆₂) and enhanced green fluorescent protein (EGFP), in which two chromophores are fixed in each protein matrix. Cytb₅₆₂ was appended to the N-terminus of EGFP via a Gly-Ser linker and the resultant fusion protein was successfully expressed in Escherichia coli as a mixture of the apo- and the holo-forms as to the cytb₅₆₂ moiety. The fluorescence of EGFP was substantially quenched when the apo-form was reconstituted with hemin. Based on the fluorescence lifetime measurements, it appeared that light energy entrapped by EGFP is transferred to the heme of cytb₅₆₂ by resonance energy transfer (energy transfer yield: 65%). Spatial organization of two chromophores using small and stable protein matrices will be promising toward the construction of an artificial light-harvesting complex by protein engineering.