Witt Heike, Schlodder Eberhard, Teutloff Christian, Niklas Jens, Bordignon Enrica, Carbonera Donatella, Kohler Simon, Labahn Andreas, Lubitz Wolfgang
Max-Volmer-Laboratorium für Biophysikalische Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany.
Biochemistry. 2002 Jul 9;41(27):8557-69. doi: 10.1021/bi025822i.
The primary electron donor P700 of photosystem I is a dimer comprised of chlorophyll a (P(B)) and chlorophyll a' (P(A)). P(A) is involved in a hydrogen bond network with several surrounding amino acid residues and a nearby water molecule. To investigate the influence of hydrogen bond interactions on the properties of P700, the threonine at position A739, which donates a putative hydrogen bond to the 13(1)-keto group of P(A), was replaced with valine, histidine, and tyrosine in Chlamydomonas reinhardtii using site-directed mutagenesis. Growth of the mutants was not impaired. (i) The (P700(+)* - P700) FTIR difference spectra of the mutants lack a negative band at 1634 cm(-1) observed in the wild-type spectrum and instead exhibit a new negative band between 1658 and 1672 cm(-1) depending on the mutation. This band can therefore be assigned to the 13(1)-keto group of P(A) which is upshifted to higher frequencies upon removal of the hydrogen bond. (ii) The main bleaching band in the Q(y)() region of the (P700(+)* - P700) and ((3)P700 - P700) absorption difference spectra is blue shifted for the mutants by approximately 6 nm compared to that of the wild type. A blue shift is also observed for the main bleaching in the Soret region. (iii) The (P700(+)* - P700) CD difference spectrum of the wild type reveals two bands at 694 nm (positive CD) and 680 nm (negative CD) of approximately equal area. For each mutant, these two components are blue-shifted to the same extent. The results strongly suggest that a blue shift of the Q(y) absorption band of P(A) is responsible for a blue shift of the exciton bands. (iv) Redox titrations yielded a decrease in the midpoint potential for the oxidation of P700 by 32 mV for the exchange of Thr against Val. (v) ENDOR spectroscopy shows that the hfc of the methyl protons at position 12 of the spin-carrying Chl P(B) is decreased due to the removal of the hydrogen bond to P(A). This indicates a redistribution of spin density in P700(+)* compared to that in the wild type. This gives evidence for an electronic coupling between the two halves of the dimer in the wild type and mutants.
光系统I的初级电子供体P700是一个由叶绿素a（P(B)）和叶绿素a'（P(A)）组成的二聚体。P(A)参与了与周围几个氨基酸残基和一个附近水分子形成的氢键网络。为了研究氢键相互作用对P700性质的影响，利用定点诱变技术，将莱茵衣藻中向P(A)的13(1)-酮基提供推定氢键的A739位苏氨酸分别替换为缬氨酸、组氨酸和酪氨酸。突变体的生长未受影响。(i) 突变体的（P700(+)* - P700）傅里叶变换红外（FTIR）差谱在1634 cm(-1)处没有野生型光谱中观察到的负带，而是根据突变情况在1658至1672 cm(-1)之间出现一个新的负带。因此，该带可归属于P(A)的13(1)-酮基，其在氢键去除后向更高频率位移。(ii) 突变体的（P700(+)* - P700）和（(3)P700 - P700）吸收差谱的Q(y)()区域中的主要漂白带相对于野生型蓝移了约6 nm。在索雷特区域的主要漂白也观察到蓝移。(iii) 野生型的（P700(+)* - P700）圆二色（CD）差谱在694 nm（正CD）和680 nm（负CD）处显示出两个面积大致相等的带。对于每个突变体，这两个组分以相同程度蓝移。结果强烈表明，P(A)的Q(y)吸收带的蓝移是激子带蓝移的原因。(iv) 氧化还原滴定表明，用缬氨酸替换苏氨酸后，P700氧化的中点电位降低了32 mV。(v) 电子核双共振（ENDOR）光谱表明，由于与P(A)的氢键去除，携带自旋的叶绿素P(B)第12位甲基质子的超精细偶合（hfc）降低。这表明与野生型相比，P700(+)*中的自旋密度重新分布。这为野生型和突变体中二聚体两半之间的电子耦合提供了证据。
