Luminescent ruthenium(II)- and rhenium(I)-diimine wires bind nitric oxide synthase
Luminescent ruthenium(II)- and rhenium(I)-diimine wires bind nitric oxide synthase
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DOI：
10.1021/ja046971m
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发表时间：
2005-04-13
影响因子：
15
通讯作者：
Gray, HB
中科院分区：
文献类型：
--
作者：
Dunn, AR;Belliston-Bittner, W;Gray, HB
Ru（II）-和Re（I）-二亚胺丝结合至诱导型一氧化氮合酶（iNOSoxy）的加氧酶结构域。在钌线中，[Ru（L）2L ']（2+），L'是将4，4 '-二甲基联吡啶连接到大体积疏水基团（金刚烷，1）、血红素配体（咪唑，2）或F（3）的全氟化联苯桥。2结合在鼠iNOSoxy截短突变体Delta 65和Delta 114的活性位点中，如通过血红素Soret从422至426 nm的位移所证明的。1和3也结合Delta 65和Delta 114，如双相发光衰减动力学所证明的。然而，血红素吸收光谱在1或3的存在下不改变，并且Ru-线结合不受四氢生物蝶呤或精氨酸的存在的影响。这些数据表明，1和3可能反而结合到酶的远端侧的疏水表面补丁认为与NOS还原酶模块相互作用。具有与Ru-二亚胺线的性质类似的性质的络合物可以通过防止从还原酶模块到加氧酶结构域的电子转移来提供NOS抑制的有效手段。铼-二亚胺线[Re（CO）（3）L1 L1 ']（+），其中L-1是4，7-二甲基菲咯啉，L-1'是连接铼-连接的咪唑与远端咪唑（F（8）bp-im）（4）或F（F（9）bp）（5）的全氟化联苯桥，也与Delta 114形成络合物。4的结合将Delta 114血红素Soret移动到426 nm，表明末端咪唑连接血红素铁。稳态发光测量确定4：Delta 114解离常数为100 +/- 80 nM。重新布线5以5 +/-2 μ M的K-d结合Delta 114，导致水从血红素铁中部分置换。我们发现4和5都结合在NOS活性位点，这表明NOS抑制剂的新设计。重要的是，我们已经证明了时间分辨FET测量在表征小分子：蛋白质相互作用中的作用，否则将难以观察。
Ru(II)- and Re(I)-diimine wires bind to the oxygenase domain of inducible nitric oxide synthase (iNOSoxy). In the ruthenium wires, [Ru(L)2L'](2+), L' is a perfluorinated biphenyl bridge connecting 4,4'-dimethylbipyridine to a bulky hydrophobic group (adamantane, 1), a heme ligand (imidazole, 2), or F (3). 2 binds in the active site of the murine iNOSoxy truncation mutants Delta 65 and Delta 114, as demonstrated by a shift in the heme Soret from 422 to 426 nm. 1 and 3 also bind Delta 65 and Delta 114, as evidenced by biphasic luminescence decay kinetics. However, the heme absorption spectrum is not altered in the presence of 1 or 3, and Ru-wire binding is not affected by the presence of tetrahydrobiopterin or arginine. These data suggest that 1 and 3 may instead bind to the distal side of the enzyme at the hydrophobic surface patch thought to interact with the NOS reductase module. Complexes with properties similar to those of the Ru-diimine wires may provide an effective means of NOS inhibition by preventing electron transfer from the reductase module to the oxygenase domain. Rhenium-diimine wires, [Re(CO)(3)L1L1'](+), where L-1 is 4,7-dimethylphenanthroline and L-1' is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole to a distal imidazole (F(8)bp-im) (4) or F (F(9)bp) (5), also form complexes with Delta 114. Binding of 4 shifts the Delta 114 heme Soret to 426 nm, demonstrating that the terminal imidazole ligates the heme iron. Steady-state luminescence measurements establish that the 4:Delta 114 dissociation constant is 100 +/- 80 nM. Re-wire 5 binds Delta 114 with a K-d of 5 +/- 2 mu M, causing partial displacement of water from the heme iron. Our finding that both 4 and 5 bind in the NOS active site suggests novel designs for NOS inhibitors. Importantly, we have demonstrated the power of time-resolved FET measurements in the characterization of small molecule:protein interactions that otherwise would be difficult to observe.