Electronic structural information from Q-band ENDOR on the type 1 and type 2 copper liganding environment in wild-type and mutant forms of copper-containing nitrite reductase.
Electronic structural information from Q-band ENDOR on the type 1 and type 2 copper liganding environment in wild-type and mutant forms of copper-containing nitrite reductase.
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来自 Q 带 ENDOR 的关于野生型和突变型含铜亚硝酸还原酶的 1 型和 2 型铜配体环境的电子结构信息。
DOI：
10.1021/bi971604r
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Scholes,CP
中科院分区：
文献类型：
--
作者：
Veselov,A;Olesen,K;Sienkiewicz,A;Shapleigh,JP;Scholes,CP
Q-Band Endor阐明了质子和氮的超精细特征，以提供亚硝酸盐还原酶中蓝绿色类型1和催化类型2铜中心的配体的自旋密度信息。亚硝酸还原酶的蓝绿色中心1具有氧化还原、电子转移的作用，与塑料花青素的蓝色中心相比，它具有以下结构差异： 缩短了铜−键长，长了铜−键长，改变了配体−铜−键角(Adman，E.T.，Godden，J.W.，and Turley，S.(1995)J.Biol)。Chem.270,27458−27474)。两个1型组氨酸(Nδ)配体的超精细偶联的电子自旋密度比以前报道的其他蓝色1型蛋白质的电子自旋密度有更大的百分比差异，而半胱氨酸β-质子超精细偶联(半胱氨酸π-质子超精细偶联是连接半胱氨酸硫的未配对pπ自旋密度的指标)显示出较小的电子自旋密度。1型中心的突变M182T使铜配体Met182转变为Thr182，导致中心回复到光学上的“蓝色”中心，使其氧化还原电位提高了∼100 mV，并导致活性丧失(先前论文)。令人惊讶的是，在M182T中，无论是在组氨酸或半胱氨酸超精细偶合中，还是在InValue和铜核超精细偶合中，都没有从天然的1型铜发生变化。结论是，改变的1型蛋氨酸连接引起的光学和氧化还原改变不必与HOMO的电子自旋离域改变同时发生，从其基本的半胱氨酸和组氨酸中报道。2型中心三个组氨酸(Nε)配体的氮偶联的详细图像表明，其中一个组氨酸氮与2型HOMO中的另外两个组氨酸氮存在实质性的(∼200%)电子超精细不等价，从而为2型中心的电子扭曲提供了证据。在亚硝酸盐底物的存在下，所有组氨酸的超精细偶联减少。我们认为，这种亚硝酸盐诱导的共价性降低可能与2型氧化还原电位的增加有关，以帮助电子转移到2型中心。偶极耦合、角度选择的可交换质子特征，在一系列g值范围内观察到，预测了配体−的水质子与铜的距离为2.8ä，这些水质子被亚硝酸盐消除。他的287不是第二类配体，但定位为扰乱第二类铜的轴向水或亚硝酸盐。在亚硝酸盐存在的情况下，突变体H287E没有表现出水质子的损失和配基组氨酸共价性的降低。H287E的活性大大降低(以前的论文)，Endor的信息是NO2-不与H287E的2型铜结合。总之，来自这项对本地和适当选择的突变体的电子信息提供了对类型1和类型2铜的最高占据分子轨道(HOMO)波函数的测试，并提供了对功能酶性质的深入电子洞察。
Q-band ENDOR elucidated proton and nitrogen hyperfine features to provide spin density information at ligands of blue-green Type 1 and catalytic Type 2 copper centers in nitrite reductase. The blue-green Type 1 center of nitrite reductase has a redox, electron-transfer role, and compared to the blue center of plastocyanin, it has the following structural differences: a shortened Cu−Smetbond length, a longer Cu−Scysbond length, and altered ligand−copper−ligand bond angles (Adman, E. T., Godden, J. W., and Turley, S. (1995)J. Biol. Chem.270, 27458−27474). The hyperfine couplings of the two Type 1 histidine (Nδ) ligands showed a larger percentage difference from each other in electron spin density than previously reported for other blue Type 1 proteins, while the cysteine β-proton hyperfine couplings, a measure of unpaired pπ spin density on the liganding cysteine sulfur, showed a smaller electron spin density. A mutation of the Type 1 center, M182T, having the copper-liganding Met182transformed to Thr182, caused the center to revert to an optically “blue” center, raised its redox potential by ∼100 mV, and led to the loss of activity (prior paper). Surprisingly, in M182T there wasno changefrom native Type 1 copper either in the histidine or cysteine hyperfine couplings or ingvalues and Cu nuclear hyperfine couplings. The conclusion is that the optical and redox alterations due to changed Type 1 methionine ligation need not be concurrent with electron spin delocalization changes in the HOMO as reported from its essential cysteine and histidines. A detailed picture of the nitrogen couplings from the three histidine (Nε) ligands of the Type 2 center indicated a substantial (∼200%) electronic hyperfine inequivalence of one of the histidine nitrogens from the other two within the Type 2 HOMO and thus provided evidence for electronic distortion of the Type 2 site. In the presence of the nitrite substrate, hyperfine couplings of all histidines diminished. We suggest that this nitrite-induced decreased covalency would correlate with an increased Type 2 redox potential to assist electron transfer to the Type 2 center. Dipole-coupled, angle-selected exchangeable proton features, observed over a range ofgvalues, predicted a ligand−water proton distance of 2.80 Å from copper, and these water protons were eliminated by nitrite. His287is not a Type 2 ligand but is positioned to perturb an axial water or a nitrite of Type 2 copper. In the presence of nitrite the mutant H287E showed no evidence for the loss of water protons and no diminished ligand histidine covalency. H287E has vastly diminished activity (prior paper), and the ENDOR information is that NO2-does not bind to Type 2 copper of H287E. In summary, the electronic information from this study of native and suitably chosen mutants provided a test of the highest occupied molecular orbital (HOMO) wave function at Type 1 and Type 2 coppers and an intimate electronic insight into functional enzymatic properties.