马心肌红蛋白远端口袋组氨酸残基将亚硝酸盐的 O 结合模式导向血红素铁。
The distal pocket histidine residue in horse heart myoglobin directs the O-binding mode of nitrite to the heme iron.
机构信息
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.
出版信息
J Am Chem Soc. 2009 Dec 23;131(50):18119-28. doi: 10.1021/ja904726q.
It is now well-established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example, the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO(2)(-)) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMb(III) (i.e., ferric Mb(III)) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMb(III) H64V (2.0 A resolution) and its nitrite ion adduct metMb(III) H64V-nitrite (1.95 A resolution), and metMb(III) H64V/V67R (1.9 A resolution) and its nitrite ion adduct metMb(III) H64V/V67R-nitrite (2.0 A resolution). These are compared to the known structures of wild-type (wt) hh metMb(III) and its nitrite ion adduct hh metMb(III)-nitrite, which binds NO(2)(-) via an O-atom in a trans-FeONO configuration. Unlike wt metMb(III), no axial H(2)O is evident in either of the metMb(III) mutant structures. In the ferric H64V-nitrite structure, replacement of the distal His residue with Val alters the binding mode of nitrite from the nitrito (O-binding) form in the wild-type protein to a weakly bound nitro (N-binding) form. Reintroducing a H-bonding residue in the H64V/V67R double mutant restores the O-binding mode of nitrite. We have also examined the effects of these mutations on reactivities of the metMb(III)s with cysteine as a reducing agent and of the (ferrous) Mb(II)s with nitrite ion under anaerobic conditions. The Mb(II)s were generated by reduction of the Mb(III) precursors in a second-order reaction with cysteine, the rate constants for this step following the order H64V/V67R > H64V >> wt. The rate constants for the oxidation of the Mb(II)s by nitrite (giving NO as the other product) follow the order wt > H64V/V67R >> H64V and suggest a significant role of the distal pocket H-bonding residue in nitrite reduction.
现在已经证实，哺乳动物血红素蛋白与各种氮氧化物反应，这些反应可能在哺乳动物生理学中发挥重要作用。例如，亚铁血红素蛋白肌红蛋白（Mb）在缺氧条件下已被证明能将亚硝酸盐（NO2(-)）还原为一氧化氮（NO）。我们在这里证明，马心 metMb(III)（即三价铁 Mb(III)）的远端口袋组氨酸残基（His64）对亚硝酸盐离子与铁中心配位的方式有显著影响。确定了突变蛋白 metMb(III) H64V（2.0Å分辨率）及其亚硝酸盐离子加合物 metMb(III) H64V-nitrite（1.95Å分辨率）以及 metMb(III) H64V/V67R（1.9Å分辨率）及其亚硝酸盐离子加合物 metMb(III) H64V/V67R-nitrite（2.0Å分辨率）的晶体结构。这些结构与已知的野生型（wt）hh metMb(III)及其亚硝酸盐离子加合物 hh metMb(III)-nitrite的结构进行了比较，后者通过 trans-FeONO 构型中的一个 O 原子与 NO2(-)结合。与 wt metMb(III)不同，在任何一种 metMb(III)突变体结构中都没有明显的轴向 H2O。在 ferric H64V-nitrite 结构中，用 Val 取代远端 His 残基改变了亚硝酸盐的结合模式，从野生型蛋白中的亚硝酰（O 结合）形式转变为弱结合的硝酰（N 结合）形式。在 H64V/V67R 双突变体中重新引入一个氢键供体残基，恢复了亚硝酸盐的 O 结合模式。我们还研究了这些突变对 metMb(III)与半胱氨酸作为还原剂的反应性以及（亚铁）Mb(II)与亚硝酸盐离子在厌氧条件下的反应性的影响。Mb(II)是通过半胱氨酸与 Mb(III)前体的二级反应还原生成的，该步骤的速率常数遵循 H64V/V67R > H64V >> wt 的顺序。亚硝酸盐氧化 Mb(II)的速率常数（生成的另一个产物是 NO）遵循 wt > H64V/V67R >> H64V 的顺序，并表明远端口袋氢键供体残基在亚硝酸盐还原中起着重要作用。
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