A new strategy for semisynthesis of a photoactivatable redox protein is described. Three protohemin molecules with ruthenium tris(2,2′-bipyridine) attached by different spacers were synthesized. The Ru(bpy)3-protohemins were incorporated into the heme crevice of apomyoglobin (apo-Mb) to yield semisynthetic Mbs carrying Ru(bpy)3 as a photosensitizer (Ru(bpy)3-Mb). The photoactivation properties and the reaction mechanisms of Ru(bpy)3-Mbs were investigated by steady-state photoirradiation and laser flash photolysis. The photoactivation of Ru(bpy)3-Mbs was spectrophotometrically demonstrated by comparison with an intermolecular control, namely an equimolar mixture of Ru(bpy)3 and native Mb. The spacer structure considerably influenced net activation efficiency over a wide pH range as measured by steady-state visible light irradiation and quantum yield. Laser flash photolysis yielded the rate of the photoinduced electron transfer (ET) from the lifetime of the excited Ru(bpy)3 (ket = 4.4 × 107 s-1 for Mb(1b) and ket = 3.7 × 107 s-1 for Mb(1c)) and the back ET rate (kback = (2.0-3.7) × 107 s-1 for Mb(1b) and kback = (1.4-2.4) × 107 s-1 for Mb(1c)) from the decay of the transient absorption. These data consistently explained the results of the net photoreaction as follows. (i) The intermolecular control system was less photoactivated because little ET occurred from the excited state of Ru(bpy)3 to Mb. (ii) The short lifetime of the charge-separated state after photoinduced ET greatly decreased the photoactivation efficiency of Ru(bpy)3-Mb with the shortest spacer. (iii) The photochemical and photophysical data of the other two Ru(bpy)3-Mb derivatives (the net photoreaction, quantum yield, and ET/back ET rates) were essentially identical, indicating that flexible spacers consisting of oxyethylene units do not rigidly fix the distance between Ru(bpy)3 and the heme center of Mb. In addition, Ru(bpy)3-Mbs were highly photoactivated under aerobic conditions in a manner similar to that under anaerobic conditions, although O2 usually quenches the photoexcited state of Ru(bpy)3. This was probably due to the accelerated intramolecular ET from *Ru(bpy)3 to heme, not to O2 in Ru(bpy)3-Mbs. We therefore showed that visible light affects the content of O2-bound Mb even in air.
All Science Journal Classification (ASJC) codes
- Physical and Theoretical Chemistry
- Inorganic Chemistry