Liu Xiaoying, Gong Xing, Hicks David B, Krulwich Terry A, Yu Linda, Yu Chang-An
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Biochemistry. 2007 Jan 9;46(1):306-13. doi: 10.1021/bi0619167.
Interaction between the cytochrome caa3 respiratory chain complex and F1F0-ATP synthase from extremely alkaliphilic Bacillus pseudofirmus OF4 has been hypothesized to be required for robust ATP synthesis by this alkaliphile under conditions of very low protonmotive force. Here, such an interaction was probed by differential scanning calorimetry (DSC) and by saturation transfer electron paramagnetic resonance (STEPR). When the two purified complexes were embedded in phospholipid vesicles individually [(caa3)PL, (F1F0)PL)] or in combination [(caa3 + F1F0)PL] and subjected to DSC analysis, they underwent exothermic thermodenaturation with transition temperatures at 69, 57, and 46/75 degrees C, respectively. The enthalpy change, deltaH (-8.8 kcal/mmol), of protein-phospholipid vesicles containing both cytochrome caa3 and F1F0 was smaller than that (-12.4 kcal/mmol) of a mixture of protein-phospholipid vesicles formed from each individual electron transfer complex [(caa3)PL + (F1F0)PL]. The rotational correlation time of spin-labeled caa3 (65 micros) in STEPR studies increased significantly when the complex was mixed with F1F0 prior to being embedded in phospholipid vesicles (270 micros). When the complexes were reconstituted separately and then mixed together, or either mitochondrial cytochrome bc1 or F1F0 was substituted for the alkaliphile F1F0, the correlation time was unchanged (65-70 micros). Varying the ratio of the two alkaliphile complexes in both the DSC and STEPR experiments indicated that the optimal stoichiometry is 1:1. These results demonstrate a physical interaction between the cytochrome caa3 and F1F0-ATP synthase from B. pseudofirmus OF4 in a reconstituted system. They support the suggestion that such an interaction between these complexes may contribute to sequestered proton transfers during alkaliphile oxidative phosphorylation at high pH.
据推测，极端嗜碱芽孢杆菌OF4的细胞色素caa3呼吸链复合物与F1F0 - ATP合酶之间的相互作用，是该嗜碱菌在极低质子动力条件下进行强大ATP合成所必需的。在此，通过差示扫描量热法（DSC）和饱和转移电子顺磁共振（STEPR）对这种相互作用进行了探究。当将两种纯化的复合物分别包埋在磷脂囊泡中[（caa3）PL、（F1F0）PL]或组合包埋[（caa3 + F1F0）PL]并进行DSC分析时，它们分别在69、57和46/75℃发生放热热变性。同时含有细胞色素caa3和F1F0的蛋白质 - 磷脂囊泡的焓变ΔH（-8.8 kcal/mmol），小于由每个单独的电子传递复合物形成的蛋白质 - 磷脂囊泡混合物[（caa3）PL +（F1F0）PL]的焓变（-12.4 kcal/mmol）。在STEPR研究中，当自旋标记的caa3复合物在包埋于磷脂囊泡之前与F1F0混合时，其旋转相关时间显著增加（从65微秒增加到270微秒）。当复合物分别重构然后混合在一起，或者用线粒体细胞色素bc1或F1F0替代嗜碱菌的F1F0时，相关时间不变（65 - 70微秒）。在DSC和STEPR实验中改变两种嗜碱菌复合物的比例表明，最佳化学计量比为1:1。这些结果证明了在重构系统中，嗜碱芽孢杆菌OF4的细胞色素caa3和F1F0 - ATP合酶之间存在物理相互作用。它们支持了这样一种观点，即这些复合物之间的这种相互作用可能有助于在高pH值下嗜碱菌氧化磷酸化过程中隔离质子转移。
