Schröter T, Hatzfeld O M, Gemeinhardt S, Korn M, Friedrich T, Ludwig B, Link T A
Molekulare Genetik, Institut für Biochemie, Biozentrum, J.W. Goethe-Universität Frankfurt/Main, Germany.
Eur J Biochem. 1998 Jul 1;255(1):100-6. doi: 10.1046/j.1432-1327.1998.2550100.x.
Two mutations (S157A and Y159F) in the Rieske iron-sulfur subunit of the ubihydroquinone-cytochrome c oxidoreductase from Paracoccus denitrificans have been characterized with respect to the protein and [2Fe-2S] cluster stability, the enzyme activity and the redox potential of the [2Fe-2S] cluster. In the structure of the water-soluble fragment of the Rieske iron-sulfur protein of the bovine heart mitochondrial bc1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the [2Fe-2S] cluster: Ser163 O gamma-S-1 and Tyr165 O eta-Cys139 S gamma [Iwata, S., Saynovits, M., Link, T. A. & Michel, H. (1996) Structure 4, 567-579]. They are conserved in all known Rieske iron-sulfur proteins from bc1 complexes including that from P. denitrificans. Both amino acid exchanges, introduced as separate point mutations on plasmids containing the fbc operon, lead to a fully assembled three-subunit complex in P. denitrificans, with a metal and heme content as well as subunit composition indistinguishable from the parental strain. The purified complexes show decreased turnover numbers and redox potentials of the Rieske cluster. Whereas the turnover number of the bc1 complex isolated from the parental strain was 145 s(-1), the turnover numbers for the mutants S157A and Y159F were 10 s(-1) and 52 s(-1), respectively, corresponding to 7% and 36% activity, respectively. The midpoint potential Em of the Rieske cluster at pH 6 and 5 degrees C was +360 mV for the bc1 complex from the parental strain; the values in the mutant complexes were +316 mV (Y159F) and +265 mV (S157A). Shifts of the g values in the electron paramagnetic resonance spectra indicate an altered electron distribution in the mutants compared to in the wild-type protein.
已对反硝化副球菌泛醌 - 细胞色素c氧化还原酶的 Rieske 铁硫亚基中的两个突变（S157A 和 Y159F）在蛋白质和[2Fe - 2S]簇稳定性、酶活性以及[2Fe - 2S]簇的氧化还原电位方面进行了表征。在牛心线粒体 bc1 复合物的 Rieske 铁硫蛋白水溶性片段的结构中，这两个残基（牛序列中的 S163 和 Y165）与[2Fe - 2S]簇中的硫原子形成以下氢键：Ser163 Oγ - S - 1 和 Tyr165 Oη - Cys139 Sγ [岩田，S.，赛诺维茨，M.，林克，T. A. & 米歇尔，H.（1996 年）《结构》4，567 - 579]。它们在所有已知的来自 bc1 复合物的 Rieske 铁硫蛋白中都是保守的，包括来自反硝化副球菌的蛋白。在含有 fbc 操纵子的质粒上作为单独的点突变引入的这两个氨基酸交换，导致反硝化副球菌中形成完全组装的三亚基复合物，其金属和血红素含量以及亚基组成与亲本菌株无法区分。纯化的复合物显示 Rieske 簇的周转数和氧化还原电位降低。从亲本菌株分离的 bc1 复合物的周转数为 145 s⁻¹，而突变体 S157A 和 Y159F 的周转数分别为 10 s⁻¹ 和 52 s⁻¹，分别对应 7%和 36%的活性。亲本菌株的 bc1 复合物在 pH 6 和 5℃时 Rieske 簇的中点电位 Em 为 +360 mV；突变体复合物中的值分别为 +316 mV（Y159F）和 +265 mV（S157A）。电子顺磁共振光谱中 g 值的变化表明与野生型蛋白质相比，突变体中的电子分布发生了改变。
