Hamachi Itaru, Tanaka Shigeaki, Tsukiji Shinya, Shinkai Seiji, Oishi Shigero
Department of Chemistry & Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka 812-81, Japan, and Department of Chemistry, School of Science, Kitasato University, Sagamihara, Kanagawa 228, Japan.
Inorg Chem. 1998 Aug 24;37(17):4380-4388. doi: 10.1021/ic971507m.
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) (k(et) = 4.4 x 10(7) s(-)(1) for Mb(1b) and k(et) = 3.7 x 10(7) s(-)(1) for Mb(1c)) and the back ET rate (k(back) = (2.0-3.7) x 10(7) s(-)(1) for Mb(1b) and k(back) = (1.4-2.4) x 10(7) 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 O(2) 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 O(2) in Ru(bpy)(3)-Mbs. We therefore showed that visible light affects the content of O(2)-bound Mb even in air.
描述了一种光可激活氧化还原蛋白半合成的新策略。合成了三个通过不同间隔基连接三（2,2'-联吡啶）钌的原血红素分子。将Ru(bpy)(3)-原血红素掺入脱辅基肌红蛋白（脱辅基-Mb）的血红素裂隙中，以产生携带Ru(bpy)(3)作为光敏剂的半合成肌红蛋白（Ru(bpy)(3)-Mb）。通过稳态光照射和激光闪光光解研究了Ru(bpy)(3)-Mb的光激活特性和反应机制。通过与分子间对照物（即Ru(bpy)(3)和天然肌红蛋白的等摩尔混合物）比较，用分光光度法证明了Ru(bpy)(3)-Mb的光激活。通过稳态可见光照射和量子产率测量，间隔基结构在很宽的pH范围内对净激活效率有很大影响。激光闪光光解从激发态Ru(bpy)(3)的寿命得出光诱导电子转移（ET）速率（Mb(1b)的k(et) = 4.4×10(7) s(-1)，Mb(1c)的k(et) = 3.7×10(7) s(-1)）以及从瞬态吸收衰减得出的反向ET速率（Mb(1b)的k(back) = (2.0 - 3.7)×10(7) s(-1)，Mb(1c)的k(back) = (1.4 - 2.4)×10(7) s(-1)）。这些数据一致地解释了净光反应的结果如下。(i) 分子间对照系统光激活程度较低，因为从Ru(bpy)(3)的激发态到Mb几乎没有发生ET。(ii) 光诱导ET后电荷分离态的短寿命极大地降低了具有最短间隔基的Ru(bpy)(3)-Mb的光激活效率。(iii) 另外两种Ru(bpy)(3)-Mb衍生物的光化学和光物理数据（净光反应、量子产率以及ET/反向ET速率）基本相同，表明由氧化乙烯单元组成的柔性间隔基不会刚性固定Ru(bpy)(3)与Mb的血红素中心之间的距离。此外，尽管O(2)通常会淬灭Ru(bpy)(3)的光激发态，但Ru(bpy)(3)-Mb在有氧条件下以与厌氧条件下类似的方式被高度光激活。这可能是由于从Ru(bpy)(3)到血红素的分子内ET加速，而不是由于Ru(bpy)(3)-Mb中的O(2)。因此，我们表明可见光即使在空气中也会影响与O(2)结合的Mb的含量。
