Paddock M L, Flores M, Isaacson R, Shepherd J N, Okamura M Y
Department of Physics, University of California, San Diego, La Jolla, CA 92093, USA.
Appl Magn Reson. 2010 Jan 1;37(1-4):39. doi: 10.1007/s00723-009-0042-2.
In photosynthetic bacteria, light-induced electron transfer takes place in a protein called the reaction center (RC) leading to the reduction of a bound ubiquinone molecule, Q(B), coupled with proton binding from solution. We used electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) to study the magnetic properties of the protonated semiquinone, an intermediate proposed to play a role in proton coupled electron transfer to Q(B). To stabilize the protonated semiquinone state, we used a ubiquinone derivative, rhodoquinone, which as a semiquinone is more easily protonated than ubisemiquinone. To reduce this low-potential quinone we used mutant RCs modified to directly reduce the quinone in the Q(B) site via B-branch electron transfer (Paddock et al. in Biochemistry 44:6920-6928, 2005). EPR and ENDOR signals were observed upon illumination of mutant RCs in the presence of rhodoquinone. The EPR signals had g values characteristic of rhodosemiquinone (g(x) = 2.0057, g(y) = 2.0048, g(z) ∼ 2.0018) at pH 9.5 and were changed at pH 4.5. The ENDOR spectrum showed couplings due to solvent exchangeable protons typical of hydrogen bonds similar to, but different from, those found for ubisemiquinone. This approach should be useful in future magnetic resonance studies of the protonated semiquinone.
在光合细菌中，光诱导电子转移发生在一种称为反应中心（RC）的蛋白质中，导致结合的泛醌分子Q(B)还原，并伴随着从溶液中结合质子。我们使用电子顺磁共振（EPR）和电子-核双共振（ENDOR）来研究质子化半醌的磁性，质子化半醌是一种被认为在质子耦合电子转移到Q(B)过程中起作用的中间体。为了稳定质子化半醌状态，我们使用了一种泛醌衍生物——玫红醌，它作为半醌比泛半醌更容易质子化。为了还原这种低电位醌，我们使用了经过修饰的突变反应中心，通过B分支电子转移直接还原Q(B)位点的醌（Paddock等人，《生物化学》44:6920 - 6928，2005）。在玫红醌存在的情况下，光照突变反应中心时观察到了EPR和ENDOR信号。在pH 9.5时，EPR信号具有玫红半醌的特征g值（g(x) = 2.0057，g(y) = 2.0048，g(z) ∼ 2.0018），在pH 4.5时发生变化。ENDOR光谱显示了与氢键典型的溶剂可交换质子的耦合，类似于但不同于泛半醌的情况。这种方法在未来质子化半醌的磁共振研究中应该会很有用。
