Shen J R, Vermaas W, Inoue Y
Solar Energy Research Group, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
J Biol Chem. 1995 Mar 24;270(12):6901-7. doi: 10.1074/jbc.270.12.6901.
The gene coding for cytochrome c-550 in Synechocystis sp. PCC 6803 was cloned based on the N-terminal sequence of the mature polypeptide. Using the most probable translation start codon, the gene is expected to code for 160 amino acid residues. This includes a cleavable N-terminal leader sequence of 25 residues. This leader sequence has an Arg-Asn-Arg sequence immediately before the cleavage site; this is characteristic for transit peptides in prokaryotes. Comparison of this sequence with the leader sequence of the photosystem II-associated extrinsic 33-kDa protein from the same cyanobacterium showed an identity of 13 out of 25 residues. These results suggest that after synthesis of the apoprotein, cytochrome c-550 is transported into the thylakoid lumen. Using the cloned gene, insertion and deletion mutants of Synechocystis sp. PCC 6803 were constructed. In the absence of cytochrome c-550, both mutants were capable of photoautotrophic growth but at a significantly reduced rate. Atrazine bindng and Western blot analysis showed that these mutants on a per-chlorophyll basis contained 53-67% of the amount of photosystem II as compared with wild type. The photosystem II-specific oxygen-evolving activity at saturating light intensity was reduced to about 40% of that in the wild type strain. Taken together, these results indicate that the cytochrome c-550 is transported into the thylakoid lumen and contributes to optimal functional stability of photosystem II in cyanobacteria. This supports our biochemical evidence that cytochrome c-550 is associated with the lumenal side of photosystem II as one of the extrinsic proteins enhancing oxygen evolution (Shen, J.-R., Ikeuchi, M., and Inoue, Y. (1992) FEBS Lett. 301, 145-149; Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832). Based on these results, the gene for cytochrome c-550 was named psbV. The possible evolutionary relationship among extrinsic proteins of the photosystem II donor side is discussed.
基于成熟多肽的N端序列，克隆了集胞藻PCC 6803中编码细胞色素c-550的基因。使用最可能的翻译起始密码子，该基因预计编码160个氨基酸残基。这包括一个由25个残基组成的可裂解N端前导序列。该前导序列在裂解位点之前紧邻一个精氨酸-天冬酰胺-精氨酸序列；这是原核生物中转肽的特征。将该序列与来自同一蓝细菌的光系统II相关外在33 kDa蛋白的前导序列进行比较，发现25个残基中有13个相同。这些结果表明，脱辅基蛋白合成后，细胞色素c-550被转运到类囊体腔中。利用克隆的基因，构建了集胞藻PCC 6803的插入和缺失突变体。在没有细胞色素c-550的情况下，两种突变体都能够进行光合自养生长，但速率显著降低。莠去津结合和蛋白质免疫印迹分析表明，与野生型相比，这些突变体以每叶绿素计含有的光系统II量为野生型的53 - 67%。在饱和光强下，光系统II特异性放氧活性降至野生型菌株的约40%。综上所述，这些结果表明细胞色素c-550被转运到类囊体腔中，并有助于蓝细菌中光系统II的最佳功能稳定性。这支持了我们的生化证据，即细胞色素c-550作为增强放氧的外在蛋白之一与光系统II的腔侧相关联（沈，J.-R.，池内，M.，和井上，Y.（1992）《欧洲生物化学学会联合会快报》301，145 - 149；沈，J.-R.，和井上，Y.（1993）《生物化学》32，1825 - 1832）。基于这些结果，细胞色素c-550的基因被命名为psbV。讨论了光系统II供体侧外在蛋白之间可能的进化关系。
