How can a single second sphere amino acid substitution cause reduction midpoint potential changes of hundreds of millivolts?
How can a single second sphere amino acid substitution cause reduction midpoint potential changes of hundreds of millivolts?
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DOI：
10.1021/ja069224t
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发表时间：
2007-08-15
影响因子：
15
通讯作者：
Miller, Anne-Frances
中科院分区：
文献类型：
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作者：
Yikilmaz, Emine;Porta, Jason;Miller, Anne-Frances
超氧化物歧化酶(SOD)的活性中心金属离子被还原和再氧化，因为它不成比例地将超氧化物转化为二氧化碳和过氧化氢。因此，还原中点电位(E-m)是催化活性的关键决定因素。在大肠杆菌含铁超氧化物歧化酶(FeSOD)中，Fe3+的还原伴随着一个配位的OH-质子化，生成与H2O配位的Fe2+。配位溶剂与活性部位以外的蛋白质的唯一接触是保守的谷氨酰胺残基。将该Gln突变为His或Glu导致E-m分别升高220 mV和660 mV以上，尽管整体蛋白质结构保持不变，但His是Gln的化学保守替代品，中性Glu与Gln是等结构和等电子的。因此，我们研究了E-m升高的几个可能的基础，包括改变的Fe电子结构，改变的活性位静电，改变的氢键和改变的氧化还原偶联质子转移。利用EPR、MCD和核磁共振光谱，我们发现两个突变体的活性中心电子结构都与WT酶的相似，Q69E-FeSOD在氧化态明显偏离WT-like Fe3+配位，这可以用对小阴离子的亲和力增加来解释。外源阴离子的自发配位只能稳定氧化的Q69E-Fe3+SOD，因此不能解释Q69E FeSOD的E-m增加。WT类阴离子结合亲和力和活性中心pK表明，Q69H-FeSOD的His69在两个氧化态都是中性的，就像WT-FeSOD的Gln69一样，而Glu69在氧化态是中性的，但在Q69E-FeSOD的还原态是电离的。Q69E-Fe2+SOD的1.1A分辨晶体结构表明，Glu69在还原状态下接受配位溶剂提供的强氢键，而WT-FeSOD中Gln69提供氢键。这些数据和密度泛函理论认为，Q69E-FeSOD的E-m升高主要可以由以下因素解释：(1)还原状态下强制氢键捐赠的缓解，(2)Glu69‘S为质子耦合的Fe3+还原提供质子的能力，以及(3)还原状态下强烈的氢键接受，这稳定了配位H2O。因此，我们的结果支持这样的假设，即蛋白质基质可以通过影响氧化还原耦合的质子转移和与之相关的能量来应用显着的氧化还原调节。
The active site metal ion of superoxide dismutase (SOD) is reduced and reoxidized as it disproportionates superoxide to dioxygen and hydrogen peroxide. Thus, the reduction midpoint potential (E-m) is a critical determinant of catalytic activity. In E. coli Fe-containing SOD (FeSOD), reduction of Fe3+ is accompanied by protonation of a coordinated OH-, to produce Fe2+ coordinated by H2O. The coordinated solvent's only contact with the protein beyond the active site is a conserved Gln residue. Mutation of this Gln to His or Glu resulted in elevation of the E-m by 220 mV and more than 660 mV, respectively [Yikilmaz et al., Biochemistry 2006, 45, 1151-1161], despite the fact that overall protein structure was preserved, His is a chemically conservative replacement for Gln, and neutral Glu is isostructural and isoelectronic with Gln. Therefore, we have investigated several possible bases for the elevated E-m's, including altered Fe electronic structure, altered active site electrostatics, altered H-bonding and altered redox-coupled proton transfer. Using EPR, MCD, and NMR spectroscopies, we find that the active site electronic structures of the two mutants resemble that of the WT enzyme, for both oxidation states, and Q69E-FeSOD's apparent deviation from WT-like Fe3+ coordination in the oxidized state can be explained by increased affinity for a small anion. Spontaneous coordination of an exogenous anion can only stabilize oxidized Q69E-Fe3+SOD and, therefore, cannot account for the increased E-m of Q69E FeSOD. WT-like anion binding affinities and active site pK's indicate that His69 of Q69H-FeSOD is neutral in both oxidation states, like Gln69 of WT-FeSOD, whereas Glu69 appears to be neutral in the oxidized state but ionized in the reduced state of Q69E-FeSOD. A 1.1 A resolution crystal structure of Q69E-Fe2+SOD indicates that Glu69 accepts a strong H-bond from coordinated solvent in the reduced state, in contrast to the case in WT-FeSOD where Gln69 donates an H-bond. These data and DFT calculations lead to the proposal that the elevated E-m of Q69E-FeSOD can be substantially explained by (1) relief from enforced H-bond donation in the reduced state, (2) Glu69's capacity to provide a proton for proton-coupled Fe3+ reduction, and (3) strong hydrogen bond acceptance in the reduced state, which stabilizes coordinated H2O. Our results thus support the hypothesis that the protein matrix can apply significant redox tuning via its influence over redox-coupled proton transfer and the energy associated with it.