Hirasawa M, Shaw R W, Palmer G, Knaff D B
J Biol Chem. 1987 Sep 15;262(26):12428-33.
Chemical analysis of the ferredoxin-dependent native form (Mr = 85,000) of spinach nitrite reductase has demonstrated a siroheme content that approaches 2 mol of siroheme/mol of enzyme. A widely studied modified (Mr = 61,000) form of nitrite reductase, that has lost much of the native enzyme's ability to use ferredoxin as an electron donor, contains approximately 1 mol of siroheme/mol of enzyme. Quantitation of the high spin ferri-siroheme EPR signals and of nitrite-binding sites of the two preparations confirmed that the native enzyme's siroheme content is approximately twice that of the modified enzyme. Plots of nitrite and cyanide binding to the native enzyme versus ligand concentration are sigmoidal, with Hill coefficients of 1.6-1.8 and 2.3-2.8, respectively. Plots of enzyme activity versus nitrite concentration for the native enzyme are sigmoidal with a Hill coefficient of 2.4. Cyanide inhibition of enzymatic activity was shown to be not competitive. Addition of cyanide to the native enzyme resulted in a diminution of the high spin ferri-siroheme EPR signal and produced EPR signals with g values of 2.71, 2.33, and 1.49 due to low spin ferri-siroheme.
对菠菜亚硝酸还原酶的铁氧化还原蛋白依赖性天然形式（Mr = 85,000）进行化学分析表明，其西罗血红素含量接近2摩尔西罗血红素/摩尔酶。一种经过广泛研究的亚硝酸还原酶修饰形式（Mr = 61,000），已丧失了天然酶将铁氧化还原蛋白用作电子供体的大部分能力，其西罗血红素含量约为1摩尔西罗血红素/摩尔酶。对这两种制剂的高自旋铁-西罗血红素EPR信号和亚硝酸盐结合位点进行定量分析，证实天然酶的西罗血红素含量约为修饰酶的两倍。天然酶与亚硝酸盐和氰化物的结合曲线与配体浓度的关系呈S形，希尔系数分别为1.6 - 1.8和2.3 - 2.8。天然酶的酶活性与亚硝酸盐浓度的关系曲线呈S形，希尔系数为2.4。氰化物对酶活性的抑制作用是非竞争性的。向天然酶中添加氰化物会导致高自旋铁-西罗血红素EPR信号减弱，并产生g值为2.71、2.33和1.49的低自旋铁-西罗血红素EPR信号。
