Hydrogen bonds between nitrogen donors and the semiquinone in the Qi-site of the bc1 complex
Hydrogen bonds between nitrogen donors and the semiquinone in the Qi-site of the bc1 complex
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DOI：
10.1074/jbc.m702333200
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发表时间：
2007-08-31
影响因子：
4.8
通讯作者：
Crofts, Antony R.
中科院分区：
文献类型：
--
作者：
DikanovO, Sergei A.;Hollan, J. Todd;Crofts, Antony R.
稳定在类球红细菌bc（1）复合物Q（i）-位点的泛半醌与来自局部蛋白质环境的氮形成氢键，初步鉴定为His-217的环N。用X波段（近似9.7GHz）和S波段（3.4GHz）脉冲EPR谱研究了N-14和N-15的相互作用。S带光谱的应用使我们能够确定参与氢键形成的14 N的完整核四极张量，并将其明确地分配给His-217的N（π）。该张量与其他醌加工位点中的半醌和N-δ之间的H-键相比具有明显的特征。用15 N进行的实验表明，His-217的N（N+）是泛醌环境中唯一携带任何可观的未配对自旋密度的氮，并且允许计算与His-217的N（N+）的各向同性和各向异性耦合。根据这些数据，我们可以估计转移到氮的2s和2 p轨道上的未成对自旋密度以及从氮到羰基氧的距离为2.38 +/- 0.13 A。其它蛋白质氮与半醌的超精细偶联均小于0.1MHz。这并不排除Asn-221的氮作为半醌的甲氧基氧的可能的氢键供体。突变株N221 T、N221 H、N221 I、N221 S、N221 P和N221 D的动力学实验支持该残基的机械作用，所有这些突变株均表现出一定的抑制作用，但保留了部分周转。
The ubisemiquinone stabilized at the Q(i)-site of the bc(1) complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of N-14 and N-15 have been studied by X-band (similar to 9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the N (epsilon) of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and N-delta in other quinone-processing sites. The experiments with 15N showed that the N (epsilon) of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the N (epsilon) of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38 +/- 0.13 A. The hyperfine coupling of other protein nitrogens with semiquinone is < 0.1MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. Amechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.