Bidirectional Photoinduced Electron Transfer in Ruthenium(II)-Tris-bipyridyl-Modified PpcA, a Multi-heme c‑Type Cytochrome from Geobacter sulfurreducens

PpcA, a tri-heme cytochrome c 7 from Geobacter sulfurreducens, was investigated as a model for photosensitizer-initiated electron transfer within a multi-heme “molecular wire” protein architecture. Escherichia coli expression of PpcA was found to be tolerant of cysteine site-directed mutagenesis, demonstrated by the successful expression of natively folded proteins bearing cysteine mutations at a series of sites selected to vary characteristically with respect to the three -CXXCH- heme binding domains. The introduced cysteines readily reacted with Ru­(II)-(2,2′-bpy)2(4-bromomethyl-4′-methyl-2,2′-bipyridine) to form covalently linked constructs that support both photo-oxidative and photo-reductive quenching of the photosensitizer excited state, depending upon the initial heme redox state. Excited-state electron-transfer times were found to vary from 6 × 10–12 to 4 × 10–8 s, correlated with the distance and pathways for electron transfer. The fastest rate is more than 103-fold faster than previously reported for photosensitizer–redox protein constructs using amino acid residue linking. Clear evidence for inter-heme electron transfer within the multi-heme protein is not detected within the lifetimes of the charge-separated states. These results demonstrate an opportunity to develop multi-heme c-cytochromes for investigation of electron transfer in protein “molecular wires” and to serve as frameworks for metalloprotein designs that support multiple-electron-transfer redox chemistry.