The crucial importance of chemistry in the structure-function link: manipulating hydrogen bonding in iron-containing superoxide dismutase.
The crucial importance of chemistry in the structure-function link: manipulating hydrogen bonding in iron-containing superoxide dismutase.
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化学在结构-功能链接中的至关重要性：操纵含铁超氧化物歧化酶中的氢键。
DOI：
10.1021/bi051495d
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Miller,Anne-Frances
中科院分区：
文献类型：
--
作者：
Yikilmaz,Emine;Rodgers,DavidW;Miller,Anne-Frances
含铁超氧化物歧化酶的活性位点Fe由溶剂分子配位，溶剂分子的质子化状态与Fe的氧化态偶联。因此，我们已经提出，谷氨酰胺69和这种溶剂分子之间的氢键可以强烈地影响超氧化物歧化酶（SOD）中的Fe的氧化还原活性。我们在这里表明，这种谷氨酰胺突变为他微妙地改变了活性位点的结构，但保留了30%的活性。相反，突变为Glu则保留了活性位点结构，但使酶失活。因此，在这种情况下，酶的功能与原子位置无关，而与残基身份（化学）有关。我们观察到Q69 E-FeSOD氧化态相对于氧化态Q69 H-FeSOD的还原态和中间不稳定的强烈不稳定。事实上，氧化还原滴定表明，Gln 69突变为His使还原电位增加240 mV，而突变为Glu似乎使还原电位增加660 mV以上。我们发现这足以解释突变体的活性丧失，尽管其他因素也可能起作用。Q69 E-FeSOD的还原电位的强烈升高可能反映了活性位点H-键合网络的重组，包括残基69和配位溶剂之间的关键H-键的极性的可能逆转。
Fe-containing superoxide dismutase's active site Fe is coordinated by a solvent molecule, whose protonation state is coupled to the Fe oxidation state. Thus, we have proposed that H-bonding between glutamine 69 and this solvent molecule can strongly influence the redox activity of the Fe in superoxide dismutase (SOD). We show here that mutation of this Gln to His subtly alters the active site structure but preserves 30% activity. In contrast, mutation to Glu otherwise preserves the active site structure but inactivates the enzyme. Thus, enzyme function correlates not with atom positions but with residue identity (chemistry), in this case. We observe strong destabilization of the Q69E-FeSOD oxidized state relative to the reduced state and intermediate destabilization of oxidized Q69H-FeSOD. Indeed, redox titrations indicate that mutation of Gln69 to His increases the reduction potential by 240 mV, whereas mutation to Glu appears to increase it by more than 660 mV. We find that this suffices to explain the mutants' loss of activity, although additional factors may also contribute. The strongly elevated reduction potential of Q69E-FeSOD may reflect reorganization of the active site H-bonding network, including possible reversal of the polarity of the key H-bond between residue 69 and coordinated solvent.