Vega J M, Kamin H
J Biol Chem. 1977 Feb 10;252(3):896-909.
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 The high spin EPR signals disappear (J. M. Vega, H. Kamin, N. R. Orme-Johnson, and W. H. Orme-Johnson, unpublished observations).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.
菠菜中的铁氧化还原蛋白-亚硝酸盐还原酶（EC 1.7.7.1.）已被纯化至同质，比活性为110单位/毫克蛋白质。该酶的分子量为61,000，含有3个铁原子（其中1个在西罗血红素中）和2个不稳定硫化物，即每分子1个（Fe2-S2），在276、386（Soret）、573（α）和690纳米处有吸收最大值，E386为3.97×10⁴ M⁻¹·cm⁻¹，A276/A386吸光率比为1.8。厌氧条件下加入连二亚硫酸盐会导致690纳米峰的消失以及573纳米吸收带分裂为545和585纳米处的两个宽峰。连二亚硫酸盐的还原作用会因氰化物而增强（图7），每摩尔酶大约需要3个电子当量。对于亚硝酸盐或羟胺（该酶的底物）、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纳米吸收带会消失，Soret和α区域的吸光度会改变。高自旋EPR信号消失（J.M.维加、H.卡明、N.R.奥姆-约翰逊和W.H.奥姆-约翰逊，未发表的观察结果）。滴定法可计算出每摩尔酶结合1摩尔亚硝酸盐，解离常数Kdiss为3.2×10⁻⁶ M。连二亚硫酸盐还原的酶也会与添加的亚硝酸盐、羟胺或氰化物形成复合物，其特征是573（α）吸收带发生明显变化。因此，底物或竞争性抑制剂可以与氧化态或还原态的酶形式结合。一氧化碳抑制亚硝酸盐还原酶并与还原态的酶形成复合物（在395、543和585纳米处有最大吸收峰）。分光光度法可检测到的一氧化碳复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。在用连二亚硫酸盐和亚硝酸盐进行稳态周转期间，该酶会与添加的亚硝酸盐形成复合物，相对于还原态的酶，在445、538和580纳米处有吸收差异最大值。当几乎所有底物耗尽时，会出现一种新物种的光谱，表明亚硝酸盐还原酶可能会与多种氧化态的含氮化合物形成复合物。亚硝酸盐按化学计量比还原为氨，未检测到中间还原态的游离含氮化合物。对氯汞苯甲酸（pCMB）抑制亚硝酸盐还原酶活性，亚硝酸盐可部分保护该酶免受这种抑制。用汞制剂滴定天然酶表明，每摩尔亚硝酸盐还原酶可结合6摩尔pCMB。天然亚硝酸盐还原酶的Soret吸收带在pCMB处理的酶中会发生改变并部分褪色，573（α）带消失。
