†Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470 Mülheim an der Ruhr, Germany.
‡Department of Chemistry "Ugo Schiff", University of Florence, Via della Lastruccia 3-13, I-50019 Sesto Fiorentino(Fi), Italy.
J Am Chem Soc. 2015 Apr 1;137(12):4141-50. doi: 10.1021/ja512938u. Epub 2015 Mar 24.
Nitrite is an important metabolite in the physiological pathways of NO and other nitrogen oxides in both enzymatic and nonenzymatic reactions. The ferric heme b protein nitrophorin 4 (NP4) is capable of catalyzing nitrite disproportionation at neutral pH, producing NO. Here we attempt to resolve its disproportionation mechanism. Isothermal titration calorimetry of a gallium(III) derivative of NP4 demonstrates that the heme iron coordinates the first substrate nitrite. Contrary to previous low-temperature EPR measurements, which assigned the NP4-nitrite complex electronic configuration solely to a low-spin (S = 1/2) species, electronic absorption and resonance Raman spectroscopy presented here demonstrate that the NP4-NO2(-) cofactor exists in a high-spin/low-spin equilibrium of 7:3 which is in fast exchange in solution. Spin-state interchange is taken as evidence for dynamic NO2(-) coordination, with the high-spin configuration (S = 5/2) representing the reactive species. Subsequent kinetic measurements reveal that the dismutation reaction proceeds in two discrete steps and identify an {FeNO}(7) intermediate species. The first reaction step, generating the {FeNO}(7) intermediate, represents an oxygen atom transfer from the iron bound nitrite to a second nitrite molecule in the protein pocket. In the second step this intermediate reduces a third nitrite substrate yielding two NO molecules. A nearby aspartic acid residue side-chain transiently stores protons required for the reaction, which is crucial for NPs' function as nitrite dismutase.
亚硝酸盐是 NO 及其他氮氧化物在酶促和非酶促反应中生理途径的重要代谢物。亚铁血红素 b 蛋白硝普酚 4（NP4）能够在中性 pH 值下催化亚硝酸盐歧化反应，生成 NO。在此，我们试图解析其歧化机制。NP4 的镓(III)衍生物的等温滴定量热法表明，亚铁血红素与第一个底物亚硝酸盐配位。与先前低 温 EPR 测量结果相反，后者将 NP4-亚硝酸盐络合物的电子构型仅分配给低自旋（S = 1/2）物种，这里呈现的电子吸收和共振拉曼光谱表明 NP4-NO2(-)辅因子存在于高自旋/低自旋平衡中，比例为 7:3，在溶液中快速交换。自旋态交换被视为动态 NO2(-)配位的证据，其中高自旋构型（S = 5/2）代表反应性物种。随后的动力学测量表明歧化反应分两步进行，并鉴定出 {FeNO}(7)中间物种。第一步反应生成 {FeNO}(7)中间产物，代表铁结合的亚硝酸盐中的氧原子向蛋白质口袋中第二个亚硝酸盐分子的转移。在第二步中，该中间产物还原第三个亚硝酸盐底物，生成两个 NO 分子。附近的天冬氨酸残基侧链暂时储存反应所需的质子，这对于 NPs 作为亚硝酸盐歧化酶的功能至关重要。
