An exposed tyrosine on the surface of trimethylamine dehydrogenase facilitates electron transfer to electron transferring flavoprotein: Kinetics of transfer in wild-type and mutant complexes
An exposed tyrosine on the surface of trimethylamine dehydrogenase facilitates electron transfer to electron transferring flavoprotein: Kinetics of transfer in wild-type and mutant complexes
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DOI：
10.1021/bi961224q
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发表时间：
1997-01-07
期刊：
影响因子：
2.9
通讯作者：
Scrutton, NS
中科院分区：
文献类型：
--
作者：
Wilson, EK;Huang, LX;Scrutton, NS
在野生型三甲基胺脱氢酶中，酪氨酸-442位于酶表面凹区的中心，该凹区被提议形成生理氧化还原受体电子转移黄素蛋白的对接位点。用停流光谱法研究了电子转移黄素蛋白对连二亚硫酸盐还原的野生型三甲胺脱氢酶（苯肼修饰）的再氧化过程中电子转移的本征速率常数。通过电子转移理论分析反应速率的温度依赖性，得到重组能为1.4eV，电子耦合矩阵元为0.82cm-1。通过分离三种突变形式的酶来研究残基Tyr-442在促进TMADH还原ETF中所起的作用，其中Tyr-442被苯丙氨酸、亮氨酸或甘氨酸残基。通过停流光谱法研究了从TMADH到ETF的这些突变体的电子转移速率。在25 ℃时，观察到Y 442 F的速率略有降低（1.4倍）和Y 442 L（2.2倍）突变体复合物，但速率大幅下降对于Y 442 G突变酶，观察到复合物的解离常数升高（30.5倍），对野生型TMADH的晶体结构的检查揭示了Tyr-442沿着酶表面上的小空腔的沿着定位：位于该空腔底部的瓦尔344是最接近TMADH的4Fe-4S中心的表面残基，并且可能位于到ETF的主要电子转移途径上。突变体复合物中电子转移速率的降低可能是由于复合物内电子转移供体和受体之间的电子偶联降低而引起的，直接或间接地由于两种蛋白质相对于彼此的取向的不利变化。
In wild-type trimethylamine dehydrogenase, tyrosine-442 is located at the center of a concave region on the surface of the enzyme that is proposed to form the docking site for the physiological redox acceptor, electron transferring flavoprotein. The intrinsic rate constant for electron transfer in the reoxidation of one-electron dithionite-reduced wild-type trimethylamine dehydrogenase (modified with phenylhydrazine) by electron transferring flavoprotein was investigated by stopped-flow spectroscopy. Analysis of the temperature dependence of the reaction rate by electron transfer theory yielded values for the reorganizational energy of 1.4 eV and the electronic coupling matrix element of 0.82 cm-l The role played by residue Tyr-442 in facilitating reduction of ETF by TMADH was investigated by isolating three mutant forms of the enzyme in which Tyr-442 was exchanged for a phenylalanine, leucine, or glycine residue. Rates of electron transfer from these mutants of TMADH to ETF were investigated by stopped-flow spectroscopy. At 25 degrees C, modest reductions in rate were observed for the Y442F (1.4-fold) and Y442L (2.2-fold) mutant complexes, but a substantial decrease in rate (30.5-fold) and an elevated dissociation constant for the complex were seen for the Y442G mutant enzyme, Inspection of the crystal structure of wild-type TMADH reveals that Tyr-442 is positioned along one side of a small cavity on the surface of the enzyme: Val 344, located at the bottom of this cavity, is the closest surface residue to the 4Fe-4S center of TMADH and is likely to be postioned on a major electron transfer pathway to ETF. The reduced electron transfer rates in the mutant complexes are probably brought about by decreases in electronic coupling between the electron transfer donor and acceptor within the complex, either directly or indirectly due to unfavorable change in the orientation of the two proteins with respect to one another.