Uncompetitive substrate inhibition and noncompetitive inhibition by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-Nonyl-4-hydroxyquinoline-N-oxide (NQNO) is observed for the cytochrome bo(3) complex: Implications for a Q(H-2)-loop proton translocation mechanism
Uncompetitive substrate inhibition and noncompetitive inhibition by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-Nonyl-4-hydroxyquinoline-N-oxide (NQNO) is observed for the cytochrome bo(3) complex: Implications for a Q(H-2)-loop proton translocation mechanism
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DOI：
10.1021/bi961723r
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发表时间：
1997-01-28
期刊：
影响因子：
2.9
通讯作者：
Chan, SI
中科院分区：
文献类型：
--
作者：
Musser, SM;Stowell, MHB;Chan, SI
大肠杆菌的细胞色素bo（3）泛醌醇氧化酶复合物含有两个泛醌醇结合位点（UQ（H-2））。这些结合位点之一，泛醇氧化位点，显然与膜中的UQ（H-2）池处于动态平衡。第二个位点对泛醌（UQ）具有高亲和力，稳定半醌种类，并且物理上靠近酶的低自旋血红素B组分。已经提出，在该位点中的UQ分子在酶周转期间保持与酶的强结合，并作为辅助因子促进电子从底物泛醇转移到血红素B [Sato-Watanabe等（1994）J.Biol.Chem.269，28908-28912]。在本文中，稳态营业额的酶的存在和不存在的抑制剂（UHDBT和NQNO），似乎被认为是ubisemiquinone类似物的检查。结果发现，动力学占最好的非竞争性抑制剂结合模型。此外，在高浓度下，底物泛醇-1和泛醇-2以非竞争性方式抑制周转。总之，这些观察结果强烈表明，必须有至少两个UQ（H-2）结合位点，在周转条件下与UQ（H-2）库快速平衡。尽管这些数据不排除强结合的UQ分子起作用以促进电子转移到血红素B的可能性，但它们与如果两个UQ（H-2）结合位点在Q（H-2）环机制（类似于细胞色素bc（1）复合物的机制）中起作用时预期的行为更一致，如Musser和同事最初提出的[（1993）FEBS Lett. 327，131-136]。在该模型中，泛醇在一个位点被氧化，泛醌在第二个位点被还原。虽然血红素-铜泛醇和细胞色素c氧化酶复合物的结构相似性表明这两个酶家族通过类似机制转运质子的可能性，但目前的观察结果表明，血红素-铜泛醇氧化酶复合物的Q（H-2）环质子转运机制应进一步研究和实验测试。
The cytochrome bo(3) ubiquinol oxidase complex from Escherichia coli contains two binding sites for ubiquinone(ol) (UQ(H-2)). One of these binding sites, the ubiquinol oxidation site, is clearly in dynamic equilibrium with the UQ(H-2) pool in the membrane. The second site has a high affinity for ubiquinone (UQ), stabilizes a semiquinone species, and is located physically close to the low-spin heme b component of the enzyme. The UQ molecule in this site has been proposed to remain strongly bound to the enzyme during enzyme turnover and to act as a cofactor facilitating the transfer of electrons from the substrate ubiquinol to heme b [Sato-Watanabe et al. (1994) J. Biol. Chem. 269, 28908-28912]. In this paper, the steady-state turnover of the enzyme is examined in the presence and absence of inhibitors (UHDBT and NQNO) that appear to be recognized as ubisemiquinone analogs. It is found that the kinetics are accounted for best by a noncompetitive inhibitor binding model. Furthermore, at high concentrations, the substrates ubiquinol-1 and ubiquinol-2 inhibit turnover in an uncompetitive fashion. Together, these observations strongly suggest that there must be at least two UQ(H-2) binding sites that are in rapid equilibrium with the UQ(H-2) pool under turnover conditions. Although these data do not rule out the possibility that a strongly bound UQ molecule functions to facilitate electron transfer to heme b, they are more consistent with the behavior expected if the two UQ(H-2) binding sites were to function in a Q(H-2)-loop mechanism (similar to that of the cytochrome bc(1) complex) as originally proposed by Musser and co-workers [(1993) FEBS Lett. 327, 131-136]. In this model, ubiquinol is oxidized at one site and ubiquinone is reduced at the second site. While the structural similarities of the heme-copper ubiquinol and cytochrome c oxidase complexes suggest the possibility that these two families of enzymes translocate protons by similar mechanisms, the current observations indicate that the Q(H-2)-loop proton translocation mechanism for the heme-copper ubiquinol oxidase complexes should be further investigated and experimentally tested.