Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex ENZYME WITH ONE INACTIVE MONOMER IS FULLY ACTIVE BUT UNABLE TO ACTIVATE THE SECOND UBIQUINOL OXIDATION SITE IN RESPONSE TO LIGAND BINDING AT THE UBIQUINONE REDUCTION SITE
Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex ENZYME WITH ONE INACTIVE MONOMER IS FULLY ACTIVE BUT UNABLE TO ACTIVATE THE SECOND UBIQUINOL OXIDATION SITE IN RESPONSE TO LIGAND BINDING AT THE UBIQUINONE REDUCTION SITE
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DOI：
10.1074/jbc.m109.072959
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发表时间：
2010-01-01
影响因子：
4.8
通讯作者：
Trumpower, Bernard L.
中科院分区：
文献类型：
--
作者：
Castellani, Michela;Covian, Raul;Trumpower, Bernard L.
我们先前提出，二聚细胞色素bc（1）复合物对泛醇氧化和bc（1）单体之间的快速电子转移表现出半位点反应性（Covian，R.，Kleinschroth，T.，路德维希，B.，和特朗普，B. L.（2007）J.Biol.Chem.282，22289-22297）。在这里，我们证明了先前提出的一半的网站的反应性和单体间的电子转移，通过表征泛醇氧化的动力学在二聚体的bc（1）复杂的副球菌，其中包含一个或两个细胞色素B亚基失活Y147 S突变。在一个细胞色素B亚基中具有Y147 S突变的酶具有完全催化活性，而在两个细胞色素B亚基中具有Y147 S突变的酶的活性仅为具有完全野生型或异二聚体细胞色素B亚基的酶的活性的10-16%。在存在抗霉素的预稳态条件下，具有一个失活细胞色素B亚基的酶与具有两个野生型细胞色素B亚基的二聚体在泛醇还原细胞色素B和c（1）的速率和程度上也没有区别。然而，只有一个突变的细胞色素B亚基的酶没有表现出在低浓度的抗霉素的野生型二聚体酶中观察到的稳态速率的刺激，证实了泛醇氧化的半位点反应性可以在野生型二聚体中通过抑制剂与一个泛醌还原位点的结合来调节。
We previously proposed that the dimeric cytochrome bc(1) complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc(1) monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289-22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc(1) complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10-16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c(1) by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wildtype dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.