Mizusawa Naoki, Yamanari Toshihiro, Kimura Yukihiro, Ishii Asako, Nakazawa Shigeaki, Ono Taka-aki
Laboratory for Photo-Biology (1), RIKEN Photodynamics Research Center, The Institute of Physical and Chemical Research (RIKEN), 519-1399 Aoba, Aramaki, Aoba, Sendai 980-0845, Japan.
Biochemistry. 2004 Nov 23;43(46):14644-52. doi: 10.1021/bi0486076.
A free alpha-COO(-) in the C-terminal alanine-344 (Ala344) in the D1 protein of photosystem II is thought to be responsible for ligating the Mn cluster. The effects of the side group of the C-terminus of the D1 protein on the functional and structural properties of the oxygen-evolving complex (OEC) were comprehensively studied by replacing Ala344 with glycine (Gly), valine (Val), aspartate (Asp), or asparagine (Asn). All the mutants grew photoautotrophically under low-light conditions with lower O(2) evolution activity depending on the mutants when compared with the activity of the control wild type. The Gly-, Asp-, and Asn-substituted mutants did not grow under high-light conditions, while the Val-substituted mutant grew even under the high-light conditions. S(2)-state thermoluminescence bands appeared at slightly elevated temperatures when compared with those of the wild type in the Asp- and Gly-substituted mutants, but at almost normal temperatures in the Val- and Asn-substituted mutants. The oxygen-evolving core particles isolated from the mutants showed little change in protein composition. The Gly-, Asp-, and Asn-substituted core particles exhibited low-temperature electron spin resonance (ESR) spectra with reduced S(2) multiline and enhanced g = 4.1 ESR signals, while the Val-substituted particles showed a spectrum similar to that of the control particles. Mid-frequency Fourier transform infrared difference spectra showed distinctive changes in several bands arising from the putative carboxylate ligands for the Mn cluster in all substituted particles, but the bands for the putative C-terminal alpha-carboxylate did not seem to change in the substituted spectra. The changes induced by the Asp and Asn substitution resembled each other except for the amide I region, and showed some similarity to those induced by the Gly substitution in the symmetric carboxylate stretching region. The results were interpreted to mean that similar types of changes of the carboxylate ligands are induced by these substitutions. The band from a putative histidine ligand for the Mn cluster was similarly affected in the Gly-, Asp-, and Asn-substituted spectra, but not in the Val-substituted spectrum. Notably, marked changes in the amide I, amide II, and carboxylate bands were observed in the Val-substituted spectrum, which was different from the Gly-, Asp-, and Asn-substituted spectra. The results indicated that the structural perturbations induced by the Val substitution include large changes of the protein backbone and are considerably different from those induced by the other substitutions. Possible amino acid ligands participating in the changes deduced by Ala344 replacement in the D1 C-terminal and the effects of the changes of the side group on these ligands were considered on the basis of the available X-ray model of the OEC.
光系统II的D1蛋白C端丙氨酸-344（Ala344）中的游离α-COO(-) 被认为负责连接锰簇。通过用甘氨酸（Gly）、缬氨酸（Val）、天冬氨酸（Asp）或天冬酰胺（Asn）取代Ala344，全面研究了D1蛋白C端侧基对放氧复合体（OEC）功能和结构特性的影响。与对照野生型的活性相比，所有突变体在低光条件下均能进行光合自养生长，但放氧活性较低，且因突变体而异。甘氨酸、天冬氨酸和天冬酰胺取代的突变体在高光条件下不能生长，而缬氨酸取代的突变体即使在高光条件下也能生长。与野生型相比，天冬氨酸和甘氨酸取代的突变体中，S(2) 态热释光带在略高的温度下出现，而缬氨酸和天冬酰胺取代的突变体中则在几乎正常的温度下出现。从突变体中分离出的放氧核心颗粒的蛋白质组成几乎没有变化。甘氨酸、天冬氨酸和天冬酰胺取代的核心颗粒表现出低温电子自旋共振（ESR）光谱，其S(2) 多线减少，g = 4.1 ESR信号增强，而缬氨酸取代的颗粒表现出与对照颗粒相似的光谱。中频傅里叶变换红外差谱显示，在所有取代颗粒中，来自假定的锰簇羧酸盐配体的几个谱带发生了明显变化，但在取代光谱中，假定的C端α-羧酸盐的谱带似乎没有变化。除酰胺I区域外，天冬氨酸和天冬酰胺取代引起的变化彼此相似，并且在对称羧酸盐拉伸区域与甘氨酸取代引起的变化有一些相似之处。结果表明，这些取代诱导了类似类型的羧酸盐配体变化。在甘氨酸、天冬氨酸和天冬酰胺取代的光谱中，来自假定的锰簇组氨酸配体的谱带受到类似影响，但在缬氨酸取代的光谱中则没有。值得注意的是，在缬氨酸取代的光谱中观察到酰胺I、酰胺II和羧酸盐谱带的明显变化，这与甘氨酸、天冬氨酸和天冬酰胺取代的光谱不同。结果表明，缬氨酸取代引起的结构扰动包括蛋白质主链的巨大变化，与其他取代引起的扰动有很大不同。基于现有的OEC X射线模型，考虑了可能参与D1 C端Ala344取代所推断变化的氨基酸配体，以及侧基变化对这些配体的影响。
