Direct detection and measurement of electron relays in a multicentered enzyme: Voltammetry of electrode-surface films of E-coli fumarate reductase, an iron-sulfur flavoprotein
Direct detection and measurement of electron relays in a multicentered enzyme: Voltammetry of electrode-surface films of E-coli fumarate reductase, an iron-sulfur flavoprotein
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DOI：
10.1021/ja9723242
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发表时间：
1997-12-03
影响因子：
15
通讯作者：
Armstrong, FA
中科院分区：
文献类型：
--
作者：
Heering, HA;Weiner, JH;Armstrong, FA
蛋白质薄膜伏安法揭示了多中心酶中的分子内电子传递。E.大肠杆菌延胡索酸还原酶（FrdAB）吸附到电活性单层覆盖在旋转热解石墨边缘电极，给出特征伏安信号，解决和分配到氧化还原活性位点。在pH 7.3（2 ° C）下，归因于中心1（[2Fe-2S]）和3（[3Fe-4S]）和FAD的信号在约-50 mV处被包封在一起，而中心2（[4Fe-4S]）在-305 mV处表现为较弱的信号。在pH 9.5时，观察到类似的伏安法，主要区别在于FAD组分向荧光体的负边缘移动。双电子FAD信号的突出使得活性位点氧化还原转化能够在一系列条件下被跟踪和检查。在不存在富马酸盐的情况下，以高达20 V s（-1）的速率扫描，电子被传递到FAD，最明显的是中心1和3。在加入富马酸盐后，信号发生转变，使特定中心参与催化电子传递。在FAD包络区域中起源的S形波与接近中心2的电势的第二波结合。这在优化酶催化控制（与质量传递控制相反）的条件下尤其明显，即高富马酸盐水平、高旋转速率和pH 9.0，此时酶的活性低于pH 7.0。分子内电子传递根据催化需求和FAD作为电子受体的熟练程度在不同的中继系统之间划分。在高pH下，从中心1和3的电子转移的不利驱动力对中心2造成更大的负担。催化伏安图显示滞后的存在下，乙酸乙酯，和抑制剂结合优先氧化FAD。还原活化是缓慢的，但在中心2的电位以下急剧加速，表明该簇在减少与通道结合的活性位点方面比其他簇有效得多。结果表明，伏安法可以用来量化复杂酶中多个位点之间的分子内电子转移。
Intramolecular electron relays operating in a multicentered enzyme are revealed by protein film voltammetry. The membrane-extrinsic catalytic domain of E. coli fumarate reductase (FrdAB) adsorbs to electroactive monolayer coverage at a rotating pyrolytic graphite edge electrode, giving characteristic voltammetric signals that are resolved and assigned to redox-active sites. At pH 7.3 (2 degrees C) signals attributable to Centers 1 ([2Fe-2S]) and 3 ([3Fe-4S]) and FAD are enveloped together around -50 mV, while Center 2 ([4Fe-4S]) appears as a weaker signal at -305 mV. At pH 9.5, similar voltammetry is observed, the main difference being that the FAD component shifts to the negative edge of the enveloper. The prominence of the two-electron FAD signal enables active-site redox transformations to be tracked and examined over a range of conditions. Scans at rates up to 20 V s(-1) in the absence of fumarate shaw that electrons are relayed to the FAD, most obviously by Centers 1 and 3. Upon adding fumarate, the signals undergo transformations ss specific centers engage in catalytic electron transport. A sigmoidal wave originating in the FAD envelope region is joined by a second wave close to the potential of Center 2. This is particularly evident under conditions optimizing enzyme catalytic control (as opposed to mass-transport control), i.e. high fumarate levels, high rotation rate, and pH 9.0 at which the enzyme is less active than at pH 7.0. Intramolecular electron transport is partitioned between different relay systems depending on catalytic demand and proficiency of the FAD as electron acceptor. At high pH, the less favorable driving force for electron transfer from Centers 1 and 3 places a greater burden on Center 2. Catalytic voltammograms show hysteresis in the presence of oxalacetate, and inhibitor binding preferentially to oxidized FAD. Reductive activation is slow but accelerates sharply below the potential of Center 2, showing that this cluster is much more effective than the others in reducing the inhibitor-bound active site. The results demonstrate how voltammetry can be used to quantify intramolecular electron transfer among multiple sites in complex enzymes.