Broadwater J A, Achim C, Münck E, Fox B G
The Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison 53705, USA.
Biochemistry. 1999 Sep 21;38(38):12197-204. doi: 10.1021/bi9914199.
Stearoyl-ACP Delta(9)-desaturase (Delta 9D) is a diiron enzyme that catalyzes 18:0-ACP desaturation. Each subunit of homodimeric resting Delta 9D contains a diferric cluster, while chemical reduction by 4e(-) produces a diferrous cluster in each subunit. Reaction of 4e(-)-reduced Delta 9D with 18:0-ACP and O(2) yields a blue chromophore (lambda(max) approximately 700 nm) that exhibits a vibrational spectrum indicative of a micro-1,2-peroxo complex; this species has been designated peroxo Delta9D. In contrast to other enzymic peroxodiiron intermediates, peroxo Delta 9D is long-lived (t(1/2) approximately 30 min at 25 degrees C) and decays via an oxidase reaction without formation of either H(2)O(2) or product (18:1-ACP). In this work, optical, transient kinetic, and Mössbauer techniques have been used to further investigate the origin and nature of this unusual peroxodiiron complex. Rapid mixing of 4e(-) Delta 9D with O(2)-equilibrated 18:0-ACP produced peroxo Delta 9D as revealed by a temperature-dependent, pseudo-first-order absorption increase at 700 nm (k = 46 s(-)(1) at 6 degrees C). The Mössbauer spectrum of peroxo Delta 9D, accounting for 96% of the total iron, consists of two quadrupole doublets present in equal proportions: delta(1) = 0.68(1) mm/s, and Delta E(Q)(1) = 1.90(2) mm/s; delta(2) = 0.64(1) mm/s, and Delta E(Q)(2) = 1.06(2) mm/s. Decay of the 700 nm optical band (k = 0.004 min(-)(1) at 6 degrees C) correlates with the complete conversion of peroxo Delta 9D into a complex called peroxo-cycled Delta 9D, which exhibits two new doublets present in equal proportions: delta(1) = 0.57(2) mm/s, and Delta E(Q)(1) = 1. 91(3) mm/s; delta(2) = 0.52(2) mm/s, and Delta E(Q)(2) = 1.41(3) mm/s. Thus, peroxo Delta 9D contains two asymmetric diferric clusters and reacts to yield peroxo-cycled Delta 9D, also containing two asymmetric diferric clusters that most probably represent a substrate complex state. The clusters of both peroxo Delta 9D and peroxo-cycled Delta 9D have a diamagnetic ground state. Because peroxo Delta 9D and peroxo-cycled Delta 9D are observed only in the presence of 18:0-ACP, substrate binding appears to have introduced asymmetry into the Delta 9D diiron clusters. In situ photolysis of peroxo Delta 9D at 4.2 K in the Mössbauer cryostat caused the release of O(2) and the reappearance of a diferrous Delta 9D.18:0-ACP complex with slightly changed parameters, suggesting a constrained cluster configuration was produced by the photolysis event. Annealing the photolyzed sample for 30 min at 77 K quantitatively restored the Mössbauer spectrum of peroxo Delta 9D, showing that the released O(2) was effectively sequestered within the active site.
硬脂酰-ACP Δ(9)-去饱和酶（Δ9D）是一种双铁酶，催化18:0-ACP的去饱和反应。同二聚体静止态的Δ9D的每个亚基都含有一个双铁簇，而通过4e(-)进行化学还原会在每个亚基中产生一个双亚铁簇。4e(-)还原的Δ9D与18:0-ACP和O₂反应会产生一种蓝色发色团（λ(max)约为700 nm），其振动光谱表明存在一个μ-1,2-过氧配合物；该物种被命名为过氧Δ9D。与其他酶促过氧双铁中间体不同，过氧Δ9D寿命较长（25℃下t(1/2)约为30分钟），并通过氧化酶反应衰变，不形成H₂O₂或产物（18:1-ACP）。在这项工作中，利用光学、瞬态动力学和穆斯堡尔技术进一步研究了这种不寻常的过氧双铁配合物的起源和性质。4e(-) Δ9D与O₂平衡的18:0-ACP快速混合产生了过氧Δ9D，这通过700 nm处温度依赖性的准一级吸收增加得以揭示（6℃下k = 46 s⁻¹）。过氧Δ9D的穆斯堡尔光谱占总铁的96%，由两个等比例存在的四极双峰组成：δ(1) = 0.68(1) mm/s，ΔE(Q)(1) = 1.90(2) mm/s；δ(2) = 0.64(1) mm/s，ΔE(Q)(2) = 1.06(2) mm/s。700 nm光带的衰变（6℃下k = 0.004 min⁻¹）与过氧Δ9D完全转化为一种称为过氧循环Δ9D的配合物相关，该配合物呈现出两个等比例存在的新双峰：δ(1) = 0.57(2) mm/s，ΔE(Q)(1) = 1.91(3) mm/s；δ(2) = 0.52(2) mm/s，ΔE(Q)(2) = 1.41(3) mm/s。因此，过氧Δ9D包含两个不对称的双铁簇，并反应生成过氧循环Δ9D，后者也包含两个不对称的双铁簇，很可能代表一种底物配合物状态。过氧Δ9D和过氧循环Δ9D的簇都具有抗磁性基态。由于仅在18:0-ACP存在时才观察到过氧Δ9D和过氧循环Δ9D，底物结合似乎给Δ9D双铁簇引入了不对称性。在穆斯堡尔低温恒温器中于4.2 K对过氧Δ9D进行原位光解导致O₂释放，并重新出现参数略有变化的双亚铁Δ9D·18:0-ACP配合物，这表明光解事件产生了一种受限的簇构型。将光解后的样品在77 K下退火30分钟可定量恢复过氧Δ9D的穆斯堡尔光谱，表明释放的O₂有效地被隔离在活性位点内。
