Wang Ruili, Sivakumar Velautham, Li Yajing, Redding Kevin, Hastings Gary
Department of Physics and Astronomy, Georgia State University, Atlanta, Georgia 30303, USA.
Biochemistry. 2003 Aug 26;42(33):9889-97. doi: 10.1021/bi034230c.
Site-directed mutagenesis in combination with Fourier transform infrared difference spectroscopy has been used to study how hydrogen bonding modulates the electronic and physical organization of P700, the primary electron donor in photosystem I. Wild-type PS I particles from Chlamydomonas reinhardtii and a mutant in which ThrA739 is changed to alanine [TA(A739) mutant] were studied. ThrA739 is thought to provide a hydrogen bond to the chlorophyll-a' molecule of P700 (the two chlorophylls of P700 (P700(+)) will be called P(A) and P(B) (P(A)(+) and P(B)(+))). The mutation considerably alters the (P700(+)-P700) FTIR difference spectra. However, we were able to describe all of the mutation induced changes in the difference spectra in terms of difference band assignments that were proposed recently (Hastings, G., Ramesh, V. M., Wang, R., Sivakumar, V. and Webber, A. (2001) Biochemistry 40, 12943-12949). Upon comparison of mutant and wild type (P700(+)-P700) FTIR difference spectra, it is shown that (1) the 13(3) ester carbonyl modes of P(A) and P(B) are unaltered upon mutation of ThrA739 to alanine. (2) The 13(3) ester carbonyl modes of P(A)(+)/P(B)(+) upshift/downshift upon mutation. These oppositely directed shifts indicate that the mutation modifies the charge distribution over the pigments in the P700(+) state, with charge on P(B) being relocated onto P(A). We also show that the 13(1) keto carbonyl mode of P(B)/P(B)(+) is unaltered/downshifted upon mutation, as is expected for the above-described mutation induced charge redistribution in P700(+). Although the 13(3) ester carbonyl modes of the chlorophylls of P700 in the ground state are unaltered upon mutation, the 13(1) keto carbonyl mode of P(A) upshifts upon mutation, as does the 13(1) keto carbonyl mode of P(A)(+). For P700 in the ground state, bands that we associate with HisA676/HisB656 upshift/downshift upon mutation. For the P700(+) state, bands that we associate with HisA676/HisB656 also upshift/downshift upon mutation. These observations are also consistent with the notion that the mutation leads to the charge on P(B)(+) being relocated onto P(A)(+). In addition, we suggest that a hydrogen bond to the 13(1) keto carbonyl of P(A) is still present in the TA(A739) mutant, probably mediated through an introduced water molecule.
定点诱变结合傅里叶变换红外差光谱已被用于研究氢键如何调节光系统I中主要电子供体P700的电子和物理组织。研究了莱茵衣藻的野生型PS I颗粒以及苏氨酸A739突变为丙氨酸的突变体[TA(A739)突变体]。苏氨酸A739被认为为P700的叶绿素-a'分子提供氢键（P700的两个叶绿素（P700(+)）将被称为P(A)和P(B)（P(A)(+)和P(B)(+)））。该突变极大地改变了(P700(+)-P700)傅里叶变换红外差光谱。然而，我们能够根据最近提出的差谱带归属来描述差光谱中所有由突变引起的变化（黑斯廷斯，G.，拉梅什，V. M.，王，R.，西瓦库马尔，V.和韦伯，A.（2001年）《生物化学》40，12943 - 12949）。通过比较突变体和野生型的(P700(+)-P700)傅里叶变换红外差光谱，结果表明：(1) 将苏氨酸A739突变为丙氨酸后，P(A)和P(B)的13(3)酯羰基模式未改变。(2) 突变后，P(A)(+)/P(B)(+)的13(3)酯羰基模式上移/下移。这些相反方向的移动表明该突变改变了P700(+)状态下色素上的电荷分布，P(B)上的电荷重新分布到了P(A)上。我们还表明，P(B)/P(B)(+)的13(1)酮羰基模式在突变后未改变/下移，这与上述P700(+)中由突变引起的电荷重新分布预期一致。尽管基态下P700的叶绿素的13(3)酯羰基模式在突变后未改变，但P(A)的13(1)酮羰基模式在突变后上移，P(A)(+)的13(1)酮羰基模式也是如此。对于基态下的P700，我们认为与组氨酸A676/组氨酸B656相关的谱带在突变后上移/下移。对于P700(+)状态，我们认为与组氨酸A676/组氨酸B656相关的谱带在突变后也上移/下移。这些观察结果也与突变导致P(B)(+)上的电荷重新分布到P(A)(+)上的观点一致。此外，我们认为TA(A739)突变体中仍然存在与P(A)的13(1)酮羰基的氢键，可能是通过引入的水分子介导的。
