Rational design of a protein-based molecular device consisting of blue fluorescent protein and zinc protoporphyrin IX incorporated into a cytochrome b₅₆₂ scaffold

To make a single molecular photo-device, it is essential to control the exact orientation of two types of proteins. We made a chimeric protein in which cytochrome b₅₆₂ was linked to the N-terminus of enhanced green fluorescent protein, cytb₅₆₂-EGFP. Within cytb₅₆₂-EGFP, the excitation energy of EGFP was transferred to the cytochrome b₅₆₂ cofactor fixed proximally to EGFP. Cytb₅₆₂-EGFP was engineered so that iron protoporphyrin IX was substituted by zinc protoporphyrin IX to make it a suitable cofactor for photo-induced electron transfer. The photosensitizer pigment was optimized and the EGFP was replaced by a blue fluorescent mutant that gave 15% higher energy transfer efficiency.