Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel.
J Am Chem Soc. 2011 Mar 9;133(9):3043-55. doi: 10.1021/ja109688w. Epub 2011 Feb 10.
cd(1) nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d(1)-heme cofactor, found only in this class of enzymes, where the substrate, nitrite, binds and is converted to NO. For a long time, it was believed that NO must be released from the ferric d(1)-heme to avoid enzyme inhibition by the formation of ferrous-nitroso complex, which was considered as a dead-end product. However, recently an enhanced rate of NO dissociation from the ferrous form, not observed in standard b-type hemes, has been reported and attributed to the unique d(1)-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzolà, F. J. Biol. Chem. 2007, 282, 14761-14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d(1)-heme NO complex from Pseudomonas aeruginosa cd(1) NIR and two mutants Y10F and H369A/H327A in solution, searching for the unique properties that are responsible for the relatively fast release. There are three residues at the "distal" side of the heme (Tyr(10), His(327), and His(369)), and in this work we focus on the identification and characterization of possible H-bonds they can form with the NO, thereby affecting the stability of the complex. For this purpose, we have used high field pulse electron-nuclear double resonance (ENDOR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the ENDOR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d(1)-heme complex of cd(1) NIR forms H-bonds with Tyr(10) and His(369), whereas the second conserved histidine, His(327), appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr(10) is removed from the NO. We have also observed a larger solvent accessibility to the distal pocket in the mutants as compared to the wild-type. Moreover, it was shown that the H-bonding network within the active site is dynamic and that a change in the protonation state of one of the residues does affect the strength and position of the H-bonds formed by the others. In the Y10F mutant, His(369) is closer to the NO, whereas mutation of both distal histidines displaces Tyr(10), removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis are discussed.
cd(1) 亚硝酸盐还原酶（NIR）是一种关键的酶，参与反硝化过程，将亚硝酸盐还原为一氧化氮（NO）。它包含一种专门的 d(1)-血红素辅因子，仅存在于这类酶中，亚硝酸盐作为底物结合并转化为 NO。长期以来，人们一直认为必须从高铁 d(1)-血红素中释放 NO，以避免亚铁-亚硝酰复合物的形成导致酶抑制，因为亚铁-亚硝酰复合物被认为是一种无出路的产物。然而，最近报道了一种从亚铁形式中增强释放 NO 的速率，这在标准的 b 型血红素中观察不到，这归因于独特的 d(1)-血红素结构（Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzolà, F. J. Biol. Chem. 2007, 282, 14761-14767）。在这里，我们报告了在溶液中对铜绿假单胞菌 cd(1) NIR 及其两个突变体 Y10F 和 H369A/H327A 的亚铁 d(1)-血红素-NO 配合物的空间和电子结构的详细研究，寻找负责相对快速释放的独特性质。血红素的“远端”侧有三个残基（Tyr(10)、His(327)和 His(369)），在这项工作中，我们专注于鉴定和表征它们可能与 NO 形成的氢键，从而影响配合物的稳定性。为此，我们使用了高磁场脉冲电子-核双共振（ENDOR）与密度泛函理论（DFT）计算相结合。DFT 计算对于根据几何结构分配和解释 ENDOR 光谱是必不可少的。我们表明，cd(1) NIR 的亚硝酰基 d(1)-血红素配合物中的 NO 与 Tyr(10)和 His(369)形成氢键，而第二个保守的组氨酸 His(327)似乎较少参与 NO 氢键形成。这与晶体结构形成对比，晶体结构表明 Tyr(10)被从 NO 中移除。我们还观察到突变体中远端口袋的溶剂可及性比野生型更大。此外，还表明活性位点内的氢键网络是动态的，一个残基的质子化状态的变化确实会影响由其他残基形成的氢键的强度和位置。在 Y10F 突变体中，His(369)更接近 NO，而两个远端组氨酸的突变则使 Tyr(10)移位，从而使其失去氢键。讨论了所发现的氢键网络在配合物稳定性和催化方面的意义。
