Dose M M, Hirasawa M, Kleis-SanFrancisco S, Lew E L, Knaff D B
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409-1061, USA.
Plant Physiol. 1997 Jul;114(3):1047-53. doi: 10.1104/pp.114.3.1047.
Spinach (Spinacea oleracea) leaf ferredoxin (Fd)-dependent nitrite reductase was treated with either the arginine-modifying reagent phenyl-glyoxal or the lysine-modifying reagent pyridoxal-5'-phosphate under conditions where only the Fd-binding affinity of the enzyme was affected and where complex formation between Fd and the enzyme prevented the inhibition by either reagent. Modification with [14C]phenylglyoxal allowed the identification of two nitrite reductase arginines, R375 and R556, that are protected by Fd against labeling. Modification of nitrite reductase with pyridoxal-5'-phosphate, followed by reduction with NaBH4, allowed the identification of a lysine, K436, that is protected by Fd against labeling. Positive charges are present at these positions in all of the Fd-dependent nitrite reductase for which sequences are available, suggesting that these amino acids are directly involved in electrostatic binding of Fd to the enzyme.
菠菜（Spinacea oleracea）叶片铁氧还蛋白（Fd）依赖性亚硝酸还原酶在仅影响该酶与Fd结合亲和力的条件下，用精氨酸修饰剂苯乙二醛或赖氨酸修饰剂磷酸吡哆醛 - 5'-磷酸处理，并且Fd与该酶之间的复合物形成可防止这两种试剂的抑制作用。用[14C]苯乙二醛进行修饰可鉴定出两个亚硝酸还原酶精氨酸，即R375和R556，它们受Fd保护不被标记。用磷酸吡哆醛 - 5'-磷酸修饰亚硝酸还原酶，然后用NaBH4还原，可鉴定出一个赖氨酸，即K436，它受Fd保护不被标记。在所有已知序列的Fd依赖性亚硝酸还原酶中，这些位置都存在正电荷，这表明这些氨基酸直接参与Fd与该酶的静电结合。
