Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.
Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.
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一氧化氮对维氏固氮菌固氮酶各个成分的失活作用的动力学和光谱分析。
DOI：
10.1021/bi00126a015
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Mortenson,LE
中科院分区：
文献类型：
--
作者：
Hyman,MR;Seefeldt,LC;Morgan,TV;Arp,DJ;Mortenson,LE
摘要：用动力学和光谱学方法研究了一氧化氮（NO）对棕色固氮菌固氮酶各组分的影响。孵育的Fe蛋白（Av 2）为1小时的化学计量的4-和8-倍摩尔过量的NO Av 2二聚体导致在C2 H2-还原试验中的Av 2的活性完全丧失。失活的动力学表明，NO的Av 2所需的最低化学计量完全取代Av 2位于1和2之间。在MgATP和MgADP的存在下，NO对Av 2活性的失活速率可增加2倍，但连二亚硫酸钠的存在对Av 2活性的失活速率无影响。出乎意料的是，通过NO与Av 2的低比率使Av 2完全失活也导致其结合MgATP和MgADP的能力完全丧失。紫外-可见光谱表明，NO对Av 2的影响主要是通过氧化[4Fe-4S]中心来实现的。EPR光谱显示，在由NO失活Av 2期间的活性损失与S-1/2和S = 3* 5*/2信号的损失相关。经典的和强烈的铁-亚硝酰基信号（g= 2.03）的外观，只有当Av 2孵育大摩尔过量的NO和该信号的外观没有相关的Av 2活性的损失。NO对MoFe蛋白（Av 1）的影响比对Av 2的影响更为复杂。观察到Avl活性的时间依赖性失活（C2 H2还原），其需要比使Av 2活化所需的NO浓度高得多的NO浓度（高达10 kPa）。此外，连二亚硫酸钠的存在显着影响NO对Avl的影响。事实上，动力学证据表明，Avl催化的，NO依赖的消费连二亚硫酸盐发生之前Avl是由NO失活。紫外可见光和EPR光谱特征和NO失活的程度之间的相关性已经建立。由NO的固氮酶组分的失活不会导致聚集或溶解成它们的组成亚基。然而，NO inactivationdo导致的变化，因为没有NO处理的蛋白质抑制C2 H2还原活性在含有等摩尔浓度的未处理的蛋白质的测定。NO对两种固氮酶组分的影响可以用已知的NO与Fe-S中心的反应性来解释。棕色固氮菌固氮酶由两种蛋白质组分组成。Fe蛋白（Av 2）是一种同源二聚体蛋白（MT 64000），其含有一个连接两个亚基的[4Fe-4S]铁氧还蛋白样中心。铁蛋白催化电子从还原载体转移到第二种固氮酶组分，即钼铁蛋白。这种电子转移与MgATP的水解相结合。MoFe蛋白（Avl，Mr 250 000）是一种复杂的蛋白质（α 202），每个四聚体含有约30个Fe和2个Mo。MoFe蛋白中的金属存在于两组化学性质不同的簇中（Mortenson & Thorneley，1979）。其中之一，M簇（6-8 Fe，1 Mo，8 S '，1高柠檬酸盐），可以以FeMo辅因子的形式与蛋白质完整分离，并被认为是底物被还原的位点（Orme-Johnson，1985）。四聚体中剩余的Fe大部分存在于两个Fe-S簇中，每个Fe-S簇包括两个连接的[4Fe-4S]簇或两个各自为8 Fe的新簇
Revised Manuscript Received December 19, 1991 abstract: The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4-and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by thepresence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicatedthat the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S-1/2 and 5= 3* 5*/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g= 2.03) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Avl) were more complex thanfor Av2. A time-dependent inactivation of Avl activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa). In addition, the effects of NO on Avl were significantly affected by the presence of sodium dithionite. In fact, kinetic evidence suggests that an Avl-catalyzed, NO-dependent consumption of dithionite occurs before Avl is inactivated by NO. A correlation between UV-visible and EPR spectral features and the extent of NO inactivation has been established. The inactivation of either nitrogenase component by NO did not lead to aggregation or dissolution into their constitutive subunits. However, NO inactivationdid cause changes in both proteins since neither NO-treated protein inhibited C2H2-reducing activity in assays containing equimolar concentrations of untreated protein. The effects of NO on both nitrogenase components are interpreted in terms of the known reactivity of NO with Fe-S centers.^ íitrogenase from Azotobacter vinelandii is composed of two protein components. The Fe protein (Av2) is a homo-dimeric protein (MT 64000) which contains one [4Fe-4S] ferredoxin-like center which bridges the two subunits. The Fe protein catalyzes the transfer of an electron from reduced carriers to the second nitrogenase component, the molybde-num-iron (MoFe) protein. This electron transfer is coupled with hydrolysis of MgATP. The MoFe protein (Avl, Mr 250 000) is a complex protein (a202) which contains approximately 30 Fe and 2 Mo per tetramer. The metals in the MoFe protein are found in two sets of two chemically distinct clusters (Mortenson & Thorneley, 1979). One of these, the M cluster (6-8 Fe, 1 Mo, 8 S', 1 homocitrate), can be separated intact from the protein in the form of a FeMo cofactor and is re-garded as the site where substrate is reduced (Orme-Johnson, 1985). The majority of the remaining Fe in the tetramer is found in two Fe-S clusters, each of which comprises either two linked [4Fe-4S] clusters or two new clusters of 8 Fe each
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梭菌铁氧化还原蛋白化学性质的研究。
DOI：
10.1016/s0021-9258(18)51805-6
发表时间：
1963
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
W. Lovenberg;B. Buchanan;J. Rabinowitz
通讯作者：
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5-三磷酸腺苷镁对梭菌偶氮铁氧化还原蛋白（固氮酶的一种成分）的铁可及性的影响。
影响因子：
2.9
作者：
G. Walker;L. Mortenson
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影响因子：
2.9
作者：
Morgan,TV;McCracken,J;Orme-Johnson,WH;Mims,WB;Mortenson,LE;Peisach,J
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固氮酶的结构和功能。
影响因子：
16.6
作者：
L. Mortenson;R. Thorneley
通讯作者：
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N2 酶促还原机制：5-三磷酸腺苷和氰化物与 N2 还原系统的结合。
DOI：
10.1073/pnas.61.3.1021
发表时间：
1968
影响因子：
11.1
作者：
P. T. Bui;L. Mortenson
通讯作者：
L. Mortenson