Centre for Metals in Biology, School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD, 4072, Australia.
J Biol Inorg Chem. 2010 May;15(4):505-14. doi: 10.1007/s00775-009-0619-0. Epub 2010 Jan 19.
Continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy have been used to characterize two variants of bacterial sulfite dehydrogenase (SDH) from Starkeya novella in which the conserved active-site arginine residue (R55) is replaced by a neutral amino acid residue. Substitution by the hydrophobic methionine residue (SDH(R55M)) has essentially no effect on the pH dependence of the EPR properties of the Mo(V) center, even though the X-ray structure of this variant shows that the methionine residue is rotated away from the Mo center and a sulfate anion is present in the active-site pocket (Bailey et al. in J Biol Chem 284:2053-2063, 2009). For SDH(R55M) only the high-pH form is observed, and samples prepared in H(2)(17)O-enriched buffer show essentially the same (17)O hyperfine interaction and nuclear quadrupole interaction parameters as SDH(WT) enzyme. However, the pH dependence of the EPR spectra of SDH(R55Q), in which the positively charged arginine is replaced by the neutral hydrophilic glutamine, differs significantly from that of SDH(WT). For SDH(R55Q) the blocked form with bound sulfate is generated at low pH, as verified by (33)S couplings observed upon reduction with (33)S-labeled sulfite. This observation of bound sulfate for SDH(R55Q) supports our previous hypothesis that sulfite-oxidizing enzymes can exhibit multiple pathways for electron transfer and product release (Emesh et al. in Biochemistry 48:2156-2163, 2009). At pH > or = 8 the high-pH form dominates for SDH(R55Q).
连续波和脉冲电子顺磁共振（EPR）光谱已被用于表征来自新星鞘氨醇单胞菌的两种变体的细菌亚硫酸盐脱氢酶（SDH），其中保守的活性位点精氨酸残基（R55）被中性氨基酸残基取代。用疏水性甲硫氨酸残基（SDH（R55M））取代几乎不会影响 Mo（V）中心的 EPR 特性的 pH 依赖性，尽管该变体的 X 射线结构表明甲硫氨酸残基从 Mo 中心旋转并存在硫酸根阴离子在活性位点口袋中（Bailey 等人，在 J Biol Chem 284:2053-2063, 2009）。对于 SDH（R55M），仅观察到高 pH 形式，并且在用 H（2）（17）O 富集缓冲液制备的样品中，（17）O 超精细相互作用和核四极相互作用参数与 SDH（WT）酶基本相同。然而，SDH（R55Q）的 EPR 光谱的 pH 依赖性与 SDH（WT）的显著不同，其中带正电荷的精氨酸被中性亲水性谷氨酰胺取代。对于 SDH（R55Q），在低 pH 下生成带结合硫酸根的受阻形式，这可以通过用（33）S 标记的亚硫酸盐还原时观察到的（33）S 偶合来验证。对于 SDH（R55Q）观察到结合硫酸根，这支持了我们之前的假设，即亚硫酸盐氧化酶可以表现出多种电子转移和产物释放途径（Emesh 等人，在 Biochemistry 48:2156-2163, 2009）。在 pH > 或 = 8 时，SDH（R55Q）的高 pH 形式占主导地位。
