Baniulis Danas, Zhang Huamin, Zakharova Taisiya, Hasan S Saif, Cramer William A
Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, USA.
Methods Mol Biol. 2011;684:65-77. doi: 10.1007/978-1-60761-925-3_7.
The cytochrome b6f complex from the filamentous cyanobacteria (Mastigocladus laminosus, Nostoc sp. PCC 7120) and spinach chloroplasts has been purified as a homo-dimer. Electrospray ionization mass spectroscopy showed the monomer to contain eight and nine subunits, respectively, and dimeric masses of 217.1, 214.2, and 286.5 kDa for M. laminosus, Nostoc, and the complex from spinach. The core subunits containing or interacting with redox-active prosthetic groups are petA (cytochrome f), B (cytochrome b6, C (Rieske iron-sulfur protein), D (subunit IV), with protein molecular weights of 31.8-32.3, 24.7-24.9, 18.9-19.3, and 17.3-17.5 kDa, and four small 3.2-4.2 kDa polypeptides petG, L, M, and N. A ninth polypeptide, the 35 kDa petH (FNR) polypeptide in the spinach complex, was identified as ferredoxin:NADP reductase (FNR), which binds to the complex tightly at a stoichiometry of approx 0.8/cytf. The spinach complex contains diaphorase activity diagnostic of FNR and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the cytochrome b6f complex. The purified cytochrome b6f complex contains stoichiometrically bound chlorophyll a and β-carotene at a ratio of approximately one molecule of each per cytochrome f. It also contains bound lipid and detergent, indicating seven lipid-binding sites per monomer. Highly purified complexes are active for approximately 1 week after isolation, transferring 200-300 electrons/cytf s. The M. laminosus complex was shown to be subject to proteolysis and associated loss of activity if incubated for more than 1 week at room temperature. The Nostoc complex is more resistant to proteolysis. Addition of pure synthetic lipid to the cyanobacterial complex, which is mostly delipidated by the isolation procedure, allows rapid formation of large (≥0.2 mm) crystals suitable for X-ray diffraction analysis and structure determination. The crystals made from the cyanobacterial complex diffract to 3.0 Å with R values of 0.222 and 0.230 for M. laminosus and Nostoc, respectively. It has not yet been possible to obtain crystals of the b6f complex from any plant source, specifically spinach or pea, perhaps because of incomplete binding of FNR or other peripheral polypeptides. Well diffracting crystals have been obtained from the green alga, Chlamydomonas reinhardtii (ref. 10).
来自丝状蓝细菌（层理鞭枝藻、集胞藻属PCC 7120）和菠菜叶绿体的细胞色素b6f复合物已被纯化为同型二聚体。电喷雾电离质谱显示，单体分别含有8个和9个亚基，层理鞭枝藻、集胞藻和菠菜复合物的二聚体质量分别为217.1、214.2和286.5 kDa。含有氧化还原活性辅基或与之相互作用的核心亚基是petA（细胞色素f）、B（细胞色素b6）、C（ Rieske铁硫蛋白）、D（亚基IV），蛋白质分子量分别为31.8 - 32.3、24.7 - 24.9、18.9 - 19.3和17.3 - 17.5 kDa，还有4个3.2 - 4.2 kDa的小多肽petG、L、M和N。菠菜复合物中的第九个多肽，即35 kDa的petH（FNR）多肽，被鉴定为铁氧还蛋白：NADP还原酶（FNR），它以约0.8/cytf的化学计量比紧密结合到复合物上。菠菜复合物具有诊断FNR的双氢酶活性，并且在促进铁氧还蛋白依赖的电子从NADPH转移到细胞色素b6f复合物方面具有活性。纯化的细胞色素b6f复合物化学计量地结合有叶绿素a和β - 胡萝卜素，其比例约为每个细胞色素f各一个分子。它还含有结合的脂质和去污剂，表明每个单体有7个脂质结合位点。高度纯化的复合物在分离后约1周内具有活性，转移200 - 300个电子/cytf·s。如果在室温下孵育超过1周，层理鞭枝藻复合物会被蛋白酶解并伴随活性丧失。集胞藻复合物对蛋白酶解更具抗性。向主要因分离过程而脱脂的蓝细菌复合物中添加纯合成脂质，可快速形成适合X射线衍射分析和结构测定的大晶体（≥0.2 mm）。由蓝细菌复合物制成的晶体衍射到3.0 Å，层理鞭枝藻和集胞藻的R值分别为0.222和0.230。尚未能够从任何植物来源，特别是菠菜或豌豆中获得b6f复合物的晶体，这可能是因为FNR或其他外周多肽的结合不完全。已从绿藻莱茵衣藻中获得了衍射良好的晶体（参考文献10）。
