Krabben L, Schlodder E, Jordan R, Carbonera D, Giacometti G, Lee H, Webber A N, Lubitz W
Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Strasse des 17, Juni 135, D-10623 Berlin, Germany.
Biochemistry. 2000 Oct 24;39(42):13012-25. doi: 10.1021/bi001200q.
Two histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+)()/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Käss, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+)()/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+)(*), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656).
两个组氨酸为构成光系统I（PSI）初级电子供体（P700）的两个叶绿素a（Chl a）分子提供轴向配体。通过定点诱变，将绿藻莱茵衣藻中蛋白质亚基PsaA中的组氨酸676（His(A676)）和亚基PsaB中的组氨酸656（His(B656)）分别替换为非极性、不带电荷的极性、酸性和碱性氨基酸残基。只有用不带电荷的极性残基进行替换时，才会导致PSI在类囊体膜中大量积累。分离出这些PSI复合物，并对初级供体的物理性质进行了表征。在所有突变体中，P700(+)()/P700的中点电位均升高（最高可达140 mV），并且在替换His(B656)时，中点电位表现出对残基大小和极化率的依赖性。在光减去暗吸收光谱中，PsaB中的所有突变在室温下于665 nm处均表现出一条额外的漂白带，这与用天冬酰胺替换His(B656)时已发表的光谱[Webber, A. N., Su Hui, Bingham, S. E., Käss, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857 - 12863]相似。His(A676)的替换在680 nm左右表现出一个额外的肩峰。在P700(+)()/P700的低温吸收差异光谱中，观察到PsaB中His(B656)突变导致主漂白带蓝移2 nm，并且在660 nm左右的光谱特征发生了一些变化。PsaA中的类似替换仅表现出主漂白带的移动。在低温（T = 2 K）下(3)P700/P700吸收差异光谱中也发现了类似的突变效应。突变的PSI复合物中(3)P700的零场分裂参数没有显著变化。通过ENDOR光谱测定，只有在替换His(B656)时，P700(+)(*)的电子自旋密度分布才会改变。在所有测量中，有两个普遍的观察结果。（i）His(B656)的替换对P700物理性质的影响比His(A676)的突变要强得多。（ii）His(B656)与谷氨酰胺的交换在光谱或中点电位上引起的变化最小，而其他替换对P700的光谱特征表现出更强但非常相似的影响。这些数据提供了令人信服的证据，即阳离子自由基和P700三重态中的未成对电子主要定域在由His(B656)轴向配位的二聚体的Chl a上。
