Raitsimring Arnold M, Astashkin Andrei V, Feng Changjian, Wilson Heather L, Rajagopalan K V, Enemark John H
Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA.
Inorganica Chim Acta. 2008 Mar 3;361(4):941-946. doi: 10.1016/j.ica.2007.05.023.
The Mo(V) forms of the Tyr343Phe (Y343F) mutant of human sulfite oxidase (SO) have been investigated by continuous wave (CW) and variable frequency pulsed EPR spectroscopies as a function of pH. The CW EPR spectrum recorded at low pH (∼6.9) has g-values similar to those known for the low-pH form of the native vertebrate SO (original lpH form); however, unlike the spectrum of original lpH SO, it does not show any hyperfine splittings from a nearby exchangeable proton. The detailed electron spin echo (ESE) envelope modulation (ESEEM) and pulsed electron-nuclear double resonance (ENDOR) experiments also did not reveal any nearby protons that could belong to an exchangeable ligand at the molybdenum center. These results suggest that under low-pH conditions the active site of Y343F SO is in the "blocked" form, with the Mo(V) center coordinated by sulfate. With increasing pH the EPR signal from the "blocked" form decreases, while a signal similar to that of the original lpH form appears and becomes the dominant signal at pH>9. In addition, both the CW EPR and ESE-detected field sweep spectra reveal a considerable contribution from a signal similar to that usually detected for the high-pH form of native vertebrate SO (original hpH form). The nearby exchangeable protons in both of the component forms observed at high pH were studied by the ESEEM spectroscopy. These results indicate that the Y343F mutation increases the apparent pK(a) of the transition from the lpH to hpH forms by ∼2 pH units.
已通过连续波（CW）和变频脉冲电子顺磁共振光谱研究了人亚硫酸盐氧化酶（SO）的Tyr343Phe（Y343F）突变体的Mo（V）形式随pH的变化。在低pH（约6.9）下记录的CW EPR光谱的g值与天然脊椎动物SO的低pH形式（原始低pH形式）已知的g值相似；然而，与原始低pH SO的光谱不同，它没有显示出来自附近可交换质子的任何超精细分裂。详细的电子自旋回波（ESE）包络调制（ESEEM）和脉冲电子 - 核双共振（ENDOR）实验也未揭示任何可能属于钼中心可交换配体的附近质子。这些结果表明，在低pH条件下，Y343F SO的活性位点处于“封闭”形式，Mo（V）中心由硫酸盐配位。随着pH升高，来自“封闭”形式的EPR信号减弱，而类似于原始低pH形式的信号出现并在pH>9时成为主要信号。此外，CW EPR和ESE检测的场扫描光谱均显示出来自类似于天然脊椎动物SO高pH形式（原始高pH形式）通常检测到的信号的相当大的贡献。通过ESEEM光谱研究了在高pH下观察到的两种组分形式中附近的可交换质子。这些结果表明，Y343F突变使从低pH形式到高pH形式转变的表观pK（a）增加了约2个pH单位。
