Noguchi Takumi, Nojiri Masaki, Takei Ken-ichi, Odaka Masafumi, Kamiya Nobuo
Institute of Materials Science, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan.
Biochemistry. 2003 Oct 14;42(40):11642-50. doi: 10.1021/bi035260i.
Nitrile hydratase (NHase) from Rhodococcus N-771, which catalyzes hydration of nitriles to the corresponding amides, exhibits novel photosensitivity; in the dark, it is in the inactive form that binds an endogenous nitric oxide (NO) molecule at the non-heme iron center, and photodissociation of the NO activates the enzyme. NHase is also known to have a unique active site structure. Two cysteine ligands to the iron center, alphaCys112 and alphaCys114, are post-translationally modified to sulfinic acid (Cys-SO(2)H) and sulfenic acid (Cys-SOH), respectively, which are thought to play a crucial role in the catalytic reaction. Here, we have determined the protonation structures of these Cys-SO(2)H and Cys-SOH groups using Fourier transform infrared (FTIR) spectroscopy in combination with density functional theory (DFT) calculations. The light-induced FTIR difference spectrum of NHase between the dark inactive and light active forms exhibited two prominent signals at (1154-1148)/1126 and (1040-1034)/1019 cm(-1), which downshifted to 1141/1114 and 1026/1012 cm(-1), respectively, in the uniformly (34)S-labeled NHase. In addition, a minor signal at 915/908 cm(-1) also showed a considerable downshift upon (34)S labeling. These (34)S-sensitive signals were basically conserved in D(2)O buffer with only slight shifts. Vibrational frequencies of methanesulfenic acid (CH(3)SOH) and methanesulfinic acid (CH(3)SO(2)H), simple model compounds of Cys-SOH and Cys-SO(2)H, respectively, were calculated using the DFT method in both the protonated and deprotonated forms and in metal complexes. Comparison of the calculated frequencies and isotope shifts with the observed ones provided the assignment of the two major signals around 1140 and 1030 cm(-1) to the asymmetric and symmetric SO(2) stretching vibrations, respectively, of the S-bonded Cys-SO(2)(-) complex, and the assignment of the minor signal around 910 cm(-1) most likely to the SO stretch of the S-bonded Cys-SO(-) complex. These assignments and the small frequency shifts upon deuteration are consistent with the view that the deprotonated alphaCys112-SO(2)(-) and alphaCys114-SO(-) are hydrogen-bonded with the protons from betaArg56 and/or betaArg141, forming a reactive cavity at the interface of the alpha and beta subunits. There is further speculation that either of these groups is hydrogen bonded to a reactant water molecule, increasing its basicity to facilitate the nucleophilic attack on the nitrile substrate bound to the iron center.
来自红球菌N - 771的腈水合酶（NHase）可催化腈水合生成相应的酰胺，它具有独特的光敏性；在黑暗中，它处于无活性形式，在非血红素铁中心结合一个内源性一氧化氮（NO）分子，NO的光解离会激活该酶。已知NHase还具有独特的活性位点结构。铁中心的两个半胱氨酸配体，αCys112和αCys114，分别在翻译后被修饰为亚磺酸（Cys - SO₂H）和次磺酸（Cys - SOH），这被认为在催化反应中起关键作用。在此，我们结合密度泛函理论（DFT）计算，使用傅里叶变换红外（FTIR）光谱法确定了这些Cys - SO₂H和Cys - SOH基团的质子化结构。NHase在黑暗无活性和光照活性形式之间的光诱导FTIR差谱在（1154 - 1148）/1126和（1040 - 1034）/1019 cm⁻¹处显示出两个突出信号，在均匀³⁴S标记的NHase中，它们分别下移至1141/1114和1026/1012 cm⁻¹。此外，915/908 cm⁻¹处的一个小信号在³⁴S标记后也有相当大的下移。这些³⁴S敏感信号在D₂O缓冲液中基本保持不变，只是略有偏移。分别以Cys - SOH和Cys - SO₂H的简单模型化合物甲亚磺酸（CH₃SOH）和甲亚磺酸（CH₃SO₂H）为例，使用DFT方法计算了它们在质子化和去质子化形式以及金属配合物中的振动频率。将计算频率和同位素位移与观测值进行比较，确定了1140和1030 cm⁻¹附近的两个主要信号分别归属于与硫键合的Cys - SO₂⁻配合物的不对称和对称SO₂伸缩振动，910 cm⁻¹附近的小信号最有可能归属于与硫键合的Cys - SO⁻配合物的SO伸缩振动。这些归属以及氘代后的小频率位移与以下观点一致：去质子化的αCys112 - SO₂⁻和αCys114 - SO⁻与来自βArg56和/或βArg141的质子形成氢键，在α和β亚基的界面处形成一个反应腔。进一步推测，这些基团中的任何一个都与反应物水分子形成氢键，增加其碱性以促进对结合在铁中心的腈底物的亲核攻击。
