Site Directed Photoactive Residues

Caged Proteins by Introduction of Photoactive Residues via Site Directed, Unnatural Amino Acid Mutagenesis... 

Intramolecular electron transfer in cytochrome c has been investigated by attaching photoactive Ru complexes to the protein surface. Ru(bpy)2(C03) (bpy = 2,2 -bipyridine) has been shown to react with surface His residues to yield, after addition of excess imidazole (im), Ru(bpy)2(im)(His) +. The protein-bound Ru complexes are luminescent, but the excited states ( Ru ) are rather short lived (r 100 ns). When direct electron transfer from Ru to the heme cannot compete with excited-state decay, electron-transfer quenchers (e.g., Ru(NH3)6 + ) are added to the solution to intercept a small fraction (1-10%) of the excited molecules, yielding (with oxidative quenchers) Ru ". If, before laser excitation of the Ru site, the heme is reduced, then the Fe to Ru + reaction (ket) can be monitored by transient absorption spectroscopy. The ket values for five different modified cytochromes have been reported (Ru(His-33), 2.6(3) xlO Ru(His-39), 3.2(4) xlO Ru(His-62), 1.0(2) x 10 Ru(His-... 

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