CNRS URA 2096. CEA, iBiTec-S, Laboratoire des Hyperfréquences, Métalloprotéines et Systèmes de Spin, Gif-sur-Yvette, France.
Biochemistry. 2010 Oct 19;49(41):8857-72. doi: 10.1021/bi100499a.
We have identified a novel enzymatic reaction for nitrophorin 2 (NP2), a heme protein previously characterized as a nitric oxide carrier in the saliva of the Rhodnius prolixus insect. NP2 exhibited levels of peroxidase activity comparable to those of the bifunctional peroxidases (KatGs), despite their heme pocket structural differences (heme ruffling, Tyr38 and Tyr85 in hydrogen bonding interactions with the propionates in NP2). The intermediates of the peroxidase-like reaction of NP2 were identified by Electron Paramagnetic Resonance (EPR) and electronic absorption spectroscopies. The EPR spectrum consistent with an [Fe(IV)=O Por•]+ species was detected at pH <7. At pH ≥ 7, the change from a strong to a weak antiferromagnetic coupling interaction for the [Fe(IV)=O Por•]+ species was accompanied by the subsequent formation of an Fe(IV)=O Por intermediate. Tyr38 was shown to be the unique naturally occurring radical site in NP2. The Y38F mutant stabilized the radical on the tyrosine in hydrogen-bonding interaction with the other heme propionate (Tyr85). Kinetic studies using stopped-flow electronic absorption spectrophotometry revealed that the [Fe(IV)=O Por•]+ species reacts with histamine and norepinephrine in a peroxidase-like manner. Our findings demonstrate that NP2 has pH-dependent dual function: at the acidic pH of the insect saliva the protein behaves as a NO carrier, and, if exposed to the higher pH of the tissues and capillaries of the host, NP2 is able to bind histamine or it can efficiently inactivate norepinephrine through a peroxidase-like reaction, in the presence of hydrogen peroxide. Accordingly, the unprecedented peroxidase-like activity of NP2 is concluded to be a key biological function.
我们发现了一种新型的硝磷蛋白 2（NP2）酶促反应，NP2 是一种血红素蛋白，先前被鉴定为 Rhodnius prolixus 昆虫唾液中的一氧化氮载体。尽管 NP2 的血红素口袋结构存在差异（血红素起皱，Tyr38 和 Tyr85 与 NP2 中的丙酸盐发生氢键相互作用），但其过氧化物酶活性水平可与双功能过氧化物酶（KatGs）相媲美。通过电子顺磁共振（EPR）和电子吸收光谱鉴定了 NP2 类过氧化物酶反应的中间体。在 pH <7 时，检测到与 [Fe(IV)=O Por•]+ 物种一致的 EPR 谱。在 pH ≥ 7 时，[Fe(IV)=O Por•]+ 物种的强至弱反铁磁耦合相互作用的变化伴随着随后形成 Fe(IV)=O Por中间体。结果表明，Tyr38 是 NP2 中唯一的天然自由基位点。Y38F 突变体稳定了与另一个血红素丙酸盐（Tyr85）发生氢键相互作用的酪氨酸上的自由基。使用停流电子吸收分光光度法进行的动力学研究表明，[Fe(IV)=O Por•]+ 物种以类过氧化物酶的方式与组氨酸和去甲肾上腺素反应。我们的研究结果表明，NP2 具有 pH 依赖性的双重功能：在昆虫唾液的酸性 pH 下，该蛋白作为 NO 载体发挥作用，并且，如果暴露于宿主组织和毛细血管的较高 pH 下，NP2 能够结合组氨酸，或者能够通过类过氧化物酶反应有效灭活去甲肾上腺素，前提是存在过氧化氢。因此，NP2 的前所未有的类过氧化物酶活性被认为是一个关键的生物学功能。
