Zhang H, Carrell C J, Huang D, Sled V, Ohnishi T, Smith J L, Cramer W A
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392, USA.
J Biol Chem. 1996 Dec 6;271(49):31360-6. doi: 10.1074/jbc.271.49.31360.
A soluble, 139-residue COOH-terminal polypeptide fragment of the Rieske iron-sulfur protein of the cytochrome b6f complex from spinach chloroplasts was obtained by limited proteolysis of the complex and a two-step chromatography purification protocol. The purified Rieske iron-sulfur protein fragment was characterized by: (i) a single NH2-terminal sequence, NH2-Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of the intact Rieske protein; (ii) a single molecular weight species determined by mass spectrometry with a molecular weight of 14,620 +/- 2 without the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with redox- and pH-dependent maxima and minima; and (iv) a reduced-oxidized optical difference spectrum characterized by DeltaepsilonmM = 3.8 mM-1 cm-1 for DeltaA at 394 versus 409 nm, which was used to determine the midpoint oxidation-reduction potential, which is +359 +/- 7 mV at 25 degrees C from pH 5.5-6.5, and +319 +/- 2 mV at pH 7, with an apparent pKox = 6.5 +/- 0.2 for the oxidized protein. The EPR spectrum measured at 17 K was characterized by the g values, gz = 2.03 and gy = 1.90, and a broad band centered at gx approximately 1.74, very similar or identical to those of the Rieske cluster in the b6f complex, implying that the environment of the [2Fe-2S] cluster is similar to that in the complex. Midpoint potential determination by low temperature EPR yielded a redox midpoint potential (Em) of +365-375 mV of the soluble Rieske fragment at pH 6 and 7 and an Em of +295-300 mV of the Rieske cluster in the cytochrome b6f complex at pH 6 and 7. The Em difference implies that the environment of the cluster in the soluble Rieske fragment is slightly more polar than that of the cluster in the intact complex. Single crystals of the Rieske polypeptide were obtained that are capable of x-ray diffraction to atomic resolution (<2.5 A), contain one molecule per asymmetric unit, a solvent content of approximately 30%, and belong to the triclinic space group P1 with cell dimensions, a = 29.1 A, b = 31.9 A, c = 35.8 A, alpha = 95.6 degrees, beta = 107.1 degrees, gamma = 117.3 degrees.
通过对菠菜叶绿体细胞色素b6f复合物的 Rieske 铁硫蛋白进行有限蛋白酶解，并采用两步色谱纯化方案，获得了一种可溶性的、由139个氨基酸残基组成的该蛋白的COOH末端多肽片段。纯化后的 Rieske 铁硫蛋白片段具有以下特征：(i) 单一的NH2末端序列，NH2-Phe-Val-Pro-Pro-Gly-Gly，从完整 Rieske 蛋白的第41位残基开始；(ii) 通过质谱测定的单一分子量物种，在没有[2Fe-2S]簇的情况下分子量为14,620±2；(iii) 具有氧化还原和pH依赖性最大值和最小值的光吸收光谱；(iv) 还原-氧化光差光谱，其特征在于在394与409nm处，ΔA的ΔεmM = 3.8 mM-1 cm-1，用于确定氧化还原中点电位，在25℃、pH 5.5 - 6.5时为+359±7 mV，在pH 7时为+319±2 mV，氧化蛋白的表观pKox = 6.5±0.2。在17K下测量的EPR光谱的特征在于g值，gz = 2.03和gy = 1.90，以及以gx约1.74为中心的宽带，与b6f复合物中 Rieske 簇的非常相似或相同，这意味着[2Fe-2S]簇的环境与复合物中的相似。通过低温EPR测定中点电位，在pH 6和7时，可溶性 Rieske 片段的氧化还原中点电位(Em)为+365 - 375 mV，在pH 6和7时，细胞色素b6f复合物中 Rieske 簇的Em为+295 - 300 mV。Em差异表明可溶性 Rieske 片段中簇的环境比完整复合物中簇的环境极性稍大。获得了 Rieske 多肽的单晶，其能够进行X射线衍射至原子分辨率(<2.5 Å)，每个不对称单元包含一个分子，溶剂含量约为30%，属于三斜晶系空间群P1，晶胞参数为a = 29.1 Å，b = 31.9 Å，c = 35.8 Å，α = 95.6°，β = 107.1°，γ = 117.3°。
