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首页> 外文期刊>Biochemistry >Intramolecular Electron Transfer in Sulfite-Oxidizing Enzymes: Elucidating the Role of a Conserved Active Site Arginine
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Intramolecular Electron Transfer in Sulfite-Oxidizing Enzymes: Elucidating the Role of a Conserved Active Site Arginine

机译：亚硫酸氧化酶中的分子内电子转移：阐明保守的活性位点精氨酸的作用

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All reported sulfite-oxidizing enzymes have a conserved arginine in their active site whichnhydrogen bonds to the equatorial oxygen ligand on the Mo atom. Previous studies on the pathogenicnR160Q mutant of human sulfite oxidase (HSO) have shown that Mo-heme intramolecular electron transfern(IET) is dramatically slowed when positive charge is lost at this position. To improve our understandingnof the function that this conserved positively charged residue plays in IET, we have studied the equivalentnuncharged substitutions R55Q and R55M as well as the positively charged substitution R55K in bacterialnsulfite dehydrogenase (SDH). The heme and molybdenum cofactor (Moco) subunits are tightly associatednin SDH, which makes it an ideal system for improving our understanding of residue function in IETnwithout the added complexity of the interdomain movement that occurs in HSO. Unexpectedly, thenuncharged SDH variants (R55Q and R55M) exhibited increased IET rate constants relative to that of thenwild type (3-4-fold) when studied by laser flash photolysis. The gain in function observed in SDHR55Qnand SDHR55M suggests that the reduction in the level of IET seen in HSOR160Q is not due to a required rolenof this residue in the IET pathway itself, but to the fact that it plays an important role in heme orientationnduring the interdomain movement necessary for IET in HSO (as seen in viscosity experiments). The pHnprofiles of SDHWT, SDHR55M, and SDHR55Q show that the arginine substitution also alters the behavior ofnthe Mo-heme IET equilibrium (Keq) and rate constants (ket) of both variants with respect to the SDHWTnenzyme. SDHWT has a ket that is independent of pH and a Keq that increases as pH decreases; on the othernhand, both SDHR55M and SDHR55Q have a ket that increases as pH decreases, and SDHR55M has a Keq thatnis pH-independent. IET in the SDHR55Q variant is inhibited by sulfate in laser flash photolysis experiments,na behavior that differs from that of SDHWT, but which also occurs in HSO. IET in SDHR55K is slower thannin SDHWT. A new analysis of the possible mechanistic pathways for sulfite-oxidizing enzymes is presentednand related to available kinetic and EPR results for these enzymes.
机译：所有报道的亚硫酸盐氧化酶在其活性位点均具有保守的精氨酸，其氢键合至Mo原子上的赤道氧配体。先前对人类亚硫酸氧化酶（HSO）的致病性nR160Q突变体的研究表明，当该位置失去正电荷时，Mo-血红素分子内电子转移（IET）会显着​​减慢。为了增进我们对这个保守的带正电残基在IET中发挥功能的了解，我们研究了亚硫酸细菌脱氢酶（SDH）中的等价不带电取代R55Q和R55M以及带正电的取代R55K。血红素和钼辅因子（Moco）亚基与SDH紧密相关，这使其成为提高我们对IETn中残基功能的理解的理想系统，而不会增加HSO中发生的域间运动的复杂性。出乎意料的是，当通过激光闪光光解法研究时，未充电的SDH变体（R55Q和R55M）相对于野生型（3-4倍）显示出更高的IET速率常数。 SDHR55Qn和SDHR55M中观察到的功能增强表明，在HSOR160Q中观察到的IET水平降低不是由于该残基在IET途径本身中的必需作用，而是由于它在血红素定向过程中起着重要作用HSO中IET必需的运动（如粘度实验所示）。 SDHWT，SDHR55M和SDHR55Q的pHn谱显示，精氨酸取代还改变了相对于SDHWTn酶的两个变体的血红素IET平衡（Keq）和速率常数（ket）的行为。 SDHWT具有与pH无关的ket和随pH降低而增加的Keq。另一方面，SDHR55M和SDHR55Q的ket随着pH的降低而增加，而SDHR55M的Keq thatnis与pH无关。 SDHR55Q变体中的IET在激光闪光光解实验中受到硫酸盐的抑制，其行为不同于SDHWT，但也发生在HSO中。 SDHR55K中的IET比SDHWT中的IET慢。提出了对亚硫酸盐氧化酶可能的机理途径的新分析，并与这些酶的可用动力学和EPR结果相关。

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