Miksovska J, Schiffer M, Hanson D K, Sebban P
Centre de Génétique Moléculaire, bât. 24, Centre National de la Recherche Scientifique, 91198, Gif, France.
Proc Natl Acad Sci U S A. 1999 Dec 7;96(25):14348-53. doi: 10.1073/pnas.96.25.14348.
In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone Q(A)(-) and the secondary acceptor Q(B)(-) after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near Q(B); some of them carry additional mutations distant from the quinone sites (M231Arg --> Leu, M43Asn --> Asp, M5Asn --> Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala --> Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either Q(A)(-) or Q(B)(-), suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to Q(B) is evident from examination of the pattern of H(+)/Q(A)(-) proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q(-) species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.
在细菌光合反应中心，与两个醌分子不同还原态相关的质子化事件，构成了静电相互作用以及蛋白质内部因光生电荷引入而发生的不同动力学事件的内在探针。在pH 7.5条件下，已对来自转基因菌株和野生型的反应中心在第一次和第二次闪光后形成初级半醌Q(A)(-)和次级受体Q(B)(-)时的质子摄取动力学和化学计量进行了测量。修饰菌株在靠近Q(B)的L212Glu和/或L213Asp位点发生了突变；其中一些还携带了远离醌位点的额外突变（M231Arg→Leu、M43Asn→Asp、M5Asn→Asp），以补偿L213Asp的缺失。我们的数据表明，这些突变扰乱了蛋白质系统对半醌形成的反应、远距离补偿性突变如何恢复正常反应，以及酪氨酸残基（M247Ala→Tyr）在增加和加速质子摄取方面的活性。数据表明，在形成Q(A)(-)或Q(B)(-)时观察到的质子摄取动力学事件之间存在直接相关性，这表明相同的残基对任何一种半醌物种的产生都有反应。因此，通过检查H(+)/Q(A)(-)质子摄取模式，可以明显看出将第一个质子转移到Q(B)的效率。蛋白质复合物对电荷引入的这种离域反应是由一个相互作用网络协调的，该网络连接了Q(-)物种、可极化残基以及位于反应中心结构该区域的众多水分子。这可能是跨膜氧化还原蛋白将电子转移与质子摄取/释放反应偶联的一般特性。
