A cross-domain charge interaction governs the activity of NO synthase
A cross-domain charge interaction governs the activity of NO synthase
复制标题
DOI：
10.1074/jbc.ra117.000635
复制
发表时间：
2018-03-23
影响因子：
4.8
通讯作者：
Stuehr, Dennis J.
中科院分区：
文献类型：
--
作者：
Haque, Mohammad Mahfuzul;Tejero, Jesus;Stuehr, Dennis J.
一氧化氮合酶（NOS）酶在催化过程中进行域间电子转移反应，这可能依赖于域-域界面处的互补电荷相互作用。我们以前的研究结果和计算机生成的域对接模型的指导下，我们评估的重要性，跨域电荷相互作用的FMN血红素电子转移神经元NOS（nNOS）。我们逆转了在FMN结构域上形成电负性三联体的三个残基（Glu-762、Glu-816和Glu-819）的电荷，然后单独逆转了加氧酶结构域（NOSoxy）上的三个正电性残基（Lys-423、Lys-620和Lys-660）的电荷，以潜在地恢复与三联体的跨结构域电荷相互作用，但极性相反。三联体的电荷反转完全消除了nNOS中的血红素还原和NO合成。这些功能部分恢复的电荷逆转在加氧酶残基赖氨酸-423，但不是在赖氨酸-620或赖氨酸-660。血红素还原的完全恢复可能是无声的FMN中点电位的伴随变化，使电子转移到血红素pherically不利。我们的研究结果提供了直接的证据表明，跨域电荷配对所需的FMN血红素电子转移nNOS。在位置423处的电荷反转以拯救功能的独特能力指示其参与与FMN域三联体的基本跨域电荷相互作用。这支持了我们的结构域对接模型，并表明它可能描绘了nNOS催化过程中形成的生产性电子转移复合物。
NO synthase (NOS) enzymes perform interdomain electron transfer reactions during catalysis that may rely on complementary charge interactions at domain-domain interfaces. Guided by our previous results and a computer-generated domain-docking model, we assessed the importance of cross-domain charge interactions in the FMN-to-heme electron transfer in neuronal NOS(nNOS). We reversed the charge of three residues (Glu-762, Glu-816, and Glu-819) that form an electronegative triad on the FMN domain and then individually reversed the charges of three electropositive residues (Lys-423, Lys-620, and Lys-660) on the oxygenase domain (NOSoxy), to potentially restore a cross-domain charge interaction with the triad, but in reversed polarity. Charge reversal of the triad completely eliminated heme reduction and NO synthesis in nNOS. These functions were partly restored by the charge reversal at oxygenase residue Lys-423, but not at Lys-620 or Lys-660. Full recovery of heme reduction was probably muted by an accompanying change in FMN midpoint potential that made electron transfer to the heme thermodynamically unfavorable. Our results provide direct evidence that cross-domain charge pairing is required for the FMN-to-heme electron transfer in nNOS. The unique ability of charge reversal at position 423 to rescue function indicates that it participates in an essential cross-domain charge interaction with the FMN domain triad. This supports our domain-docking model and suggests that it may depict a productive electron transfer complex formed during nNOS catalysis.