The raised midpoint potential of the [2Fe2S] cluster of cytochrome bc1 is mediated by both the Qo site occupants and the head domain position of the Fe-S protein subunit
The raised midpoint potential of the [2Fe2S] cluster of cytochrome bc1 is mediated by both the Qo site occupants and the head domain position of the Fe-S protein subunit
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DOI：
10.1021/bi035938u
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发表时间：
2004-03-02
期刊：
影响因子：
2.9
通讯作者：
Daldal, F
中科院分区：
文献类型：
--
作者：
Cooley, JW;Roberts, AG;Daldal, F
我们之前报道过，在含有 bc(1) 复合物亚基的铁硫 (Fe-S) 铰链区具有丙氨酸插入 (+nAla 突变体) 的荚膜红杆菌突变株，其 [2Fe2S] 簇的氧化还原中点电位 (E-m) 增加。这些菌株中 Em 的改变，包含远离金属结合位点的突变，暗示金属中心的局部环境通过该亚基与氢醌氧化 (Q(o)) 位点相互作用的变化而间接改变 [Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) J. Biol.化学。 277 3464-3470]。随后，E-m 变化被认为主要是由于还原的 [2Fe2S] 簇和 Q(o) 位点泛醌 (Q) 之间更强或更稳定的氢键 [Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002) Biochemistry 41, 14372-14382]。为了进一步研究这个问题，通过电子顺磁共振（EPR）光谱监测Fe-S蛋白-Q相互作用，结果表明野生型和突变体蛋白与Q的相互作用是相似的。此外，当 Q(pool) 被化学耗尽时，突变体 bc(1) 复合物中 [2Fe2S] 簇的 E-m 仍然比经过类似处理的天然酶更正（例如，+2Ala 突变体的 [2Fe2S] E-m 比野生型高 55 mV）。这些数据表明，+nAla 突变体中 [2Fe2S] 簇的 E 增加部分是由于簇与 Q 的相互作用，部分是由于与 Q 氢键无关的其他因素。其中一个因素是突变蛋白 Q(0) 位点上 Fe-S 与天然酶不同位置的可能性，通过使用 EPR 光谱确定膜中 [2Fe2S] 簇的方向来解决。对于+2Ala 突变体，在没有抑制剂的情况下，[2Fe2S] 簇的方向与天然酶中看到的不同。然而，当两个样品暴露于豆黄素或粘噻唑时，+2AIa突变体簇与天然酶具有相似的方向。此外，+2Ala 突变体的 Q(池) 提取膜表现出较少的总体方向，与野生型菌株的 Q 耗尽膜相比，主要方向与 +2Ala 突变体的非 Q 耗尽膜中观察到的更相似。因此，独立于 Q(o) 位点居民且源自 +nAla 突变体中新观察到的 [2Fe2S] 簇方向的附加组分也有助于其 [2Fe2S] 簇中点电位的增加。虽然这些成分的分子基础仍有待确定。讨论了这些发现在 Q(o) 位点催化方面的显着意义。
We have previously reported that mutant strains of Rhodobacter capsulatus that have alanine insertions (+nAla mutants) in the hinge region of the iron sulfur (Fe-S) containing subunit of the bc(1) complex have increased redox midpoint potentials (E-m) for their [2Fe2S] clusters. The alteration of the Em in these strains, which contain mutations far from the metal binding site, implied that the local environment of the metal center is indirectly altered by a change in the interaction of this subunit with the hydroquinone oxidizing (Q(o)) site [Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) J. Biol. Chem. 277 3464-3470]. Subsequently, the E-m changes have been proposed to be predominantly due to a stronger or more stabilized hydrogen bonding between the reduced [2Fe2S] cluster and the Q(o) site inhabitant ubiquinone (Q) [Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002) Biochemistry 41, 14372-14382]. To further investigate this issue, Fe-S protein-Q interactions were monitored by electron paramagnetic resonance (EPR) spectroscopy and the findings indicated that the wild type and mutant proteins interactions with Q are similar. Moreover, when the Q(pool) was chemically depleted, the E-m of the [2Fe2S] cluster in mutant bc(1) complexes remained more positive than a similarly treated native enzyme (e.g., the [2Fe2S] E-m of the +2Ala mutant was 55 mV more positive than the wild type). These data suggest that the increased E of the [2Fe2S] cluster in the +nAla mutants is in part due to the cluster's interaction with Q, and in part to additional factors that are independent of hydrogen bonding to Q. One such factor, the possibility of a different position of the Fe-S at the Q(0) site of the mutant proteins versus the native enzyme, was addressed by determining the orientation of the [2Fe2S] cluster in the membrane using EPR spectroscopy. In the case of the +2Ala mutant, the [2Fe2S] cluster orientation in the absence of inhibitor is different than that seen in the native enzyme. However, the +2AIa mutant cluster shared a similar orientation with the native enzyme when both samples were exposed to either stigmatellin or myxothiazol. In addition, Q(pool) extracted membranes of +2Ala mutant exhibited fewer overall orientations, with the predominant one being more similar to that observed in the non-Q-depleted membranes of the +2Ala mutant than the Q-depleted membranes of a wild-type strain. Therefore, additional component(s) that are independent of Q(o) site inhabitants and that originate from the newly observed orientations of the [2Fe2S] clusters in the +nAla mutants also contribute to the increased midpoint potentials of their [2Fe2S] clusters. While the molecular basis of these components remains to be determined. salient implications of these findings in terms of Q(o) site catalysis are discussed.