Spinach nitrite reductase. Purification and properties of a siroheme-containing iron-sulfur enzyme.
Spinach nitrite reductase. Purification and properties of a siroheme-containing iron-sulfur enzyme.
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菠菜亚硝酸还原酶。
DOI：
10.1016/s0021-9258(19)75183-7
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发表时间：
1977
期刊：
影响因子：
--
通讯作者：
H. Kamin
中科院分区：
文献类型：
--
作者：
J. Vega;H. Kamin
铁氧还蛋白-亚硝酸盐还原酶(EC 1.7.7.1)从菠菜中提纯到均一，比活性为110个单位/毫克蛋白质。该酶具有3个铁原子(其中1个为铁血红素)和2个不稳定的硫化物，即每个分子1个(Fe2-S2)，其最大吸收峰位于276、386、573和690 nm，E386为3.97×10~(4)M~(-1)·cm~(-1)，A276/A386吸收比为1.8。厌氧加入亚硫酸盐后，690 nm吸收峰消失，573 nm吸收带分裂为545和585 nm两个宽峰。氰化物(图7)促进了二亚硫酸盐的还原，每摩尔酶需要大约3个电子当量。对于亚硝酸盐或羟胺(酶的底物)、氰化物(相对于亚硝酸盐的竞争性抑制剂)或亚硫酸盐，无底物酶的690 nm吸收带消失，Soret区和α区的吸光度发生变化。高自旋EPR信号消失了(J.M.Vega，H.Kamin，N.R.Orme-Johnson和W.H.Orme-Johnson，未发表的观测)。滴定允许计算1摩尔亚硝酸盐结合/摩尔酶，Kdiss为3.2×10(-6)M。二亚硫酸盐还原的酶也与添加的亚硝酸盐、羟胺或氰化物形成络合物，其特征是573(α)吸收带发生显著变化。因此，底物或竞争性抑制剂可以结合到氧化或还原的酶形式上。CO抑制亚硝酸还原酶，并与还原的酶形成络合物(最大吸收峰位于395、543和585 nm)。分光光度法可检测的CO络合物的形成或解离与亚硝酸盐还原催化的抑制或抑制-逆转相关。在与亚硝酸根和亚硝酸根的稳态转换过程中，酶与添加的亚硝酸根形成络合物，其最大吸收波长分别为445、538和580 nm。当几乎所有底物被耗尽时，一个新物种的光谱出现，这表明亚硝酸还原酶可能与不止一个氧化态的氮化合物形成络合物。亚硝酸盐按化学计量还原为氨，没有检测到中间还原状态的游离氮化合物。对氯汞苯甲酸盐(PCMB)抑制亚硝酸还原酶活性，亚硝酸盐部分拮抗这种抑制作用。用汞滴定天然酶，结果表明6摩尔的pCMB可与亚硝酸还原酶结合或结合。天然亚硝酸还原酶的Soret吸收带在pCMB处理的酶中发生改变并部分漂白，573(α)带消失。
Ferredoxin-nitrite reductase (EC 1.7.7.1.) from spinach has been purified to homogeneity with a specific activity of 110 units/mg of protein. The enzyme, Mr = 61,000 has 3 iron atoms (of which one is in siroheme) and 2 labile sulfides, i.e. 1 (Fe2-S2) per molecule, with absorption maxima at 276, 386 (Soret), 573 (alpha), and 690 nm, with an E386 of 3.97 X 10(4) M-1-cm-1, and A276/A386 absorptivity ratio of 1.8. Anaerobic addition of dithionite results in the loss of the 690 nm peak and the splitting of the 573 nm absorption band into two broad peaks at 545 and 585 nm. Reduction by dithionite is enhanced by cyanide (Fig. 7) and requires about 3 electron eq per mol of enzyme. With nitrite or hydroxylamine (substrates of the enzyme), cyanide (a competitive inhibitor with respect to nitrite), or sulfite, the 690 nm absorption band of substrate-free enzyme disappears and the absorbance in the Soret and alpha region are altered. The high spin EPR signals disappear (J. M. Vega, H. Kamin, N. R. Orme-Johnson, and W. H. Orme-Johnson, unpublished observations). Titration permits calculation of 1 mol of nitrite bound/mol of enzyme with a Kdiss of 3.2 X 10(-6) M. Dithionite-reduced enzyme also forms complexes with added nitrite, hydroxylamine, or cyanide, characterized by marked alterations in the 573 (alpha) absorption band. THus, substrates or competitive inhibitors can be bound to the oxidized or reduced enzyme forms. CO inhibits nitrite reductase and forms a complex with reduced enzyme (epsilonmax at 395, 543, and 585 nm). Formation or dissociation of the spectrophotometrically detectable CO complex correlates with inhibition or inhibition-reversal of nitrite reduction catalysis. During steady state turnover with dithionite and nitrite, the enzyme forms a complex with added nitrite with absorption difference maxima at 445, 538, and 580 nm with respect to reduced enzyme. When nearly all substrate is depleted the spectrum of a new species appears, indicating that nitrite reductase may form complexes with nitrogen compounds of more than one oxidation state. Nitrite is stoichiometrically reduced to ammonia without detectable free nitrogen compounds of intermediate reduction state. p-Chloromercuribenzoate (pCMB) inhibits nitrite reductase activity and nitrite partially protects against this inhibition. Titration of native enzyme with the mercurial shows that 6 mol of pCMB can be bound/mol or nitrite reductase. The Soret absorption band of the native nitrite reductase is altered and partially bleached in the pCMB-treated enzyme, and the 573 (alpha) band disappears.