Topham R, Tesh S, Cole G, Mercatante D, Westcott A, Bonaventura C
Department of Chemistry, University of Richmond, Richmond, Virginia 23173, USA.
Arch Biochem Biophys. 1998 Apr 1;352(1):103-13. doi: 10.1006/abbi.1998.0574.
The crystal structure analysis of Subunit II of Limulus hemocyanin has shown that its polypeptide chain is folded into three distinct structural domains. The oxygen-binding, dinuclear copper center is located deep in the core of Domain 2. Two disulfide bonds are located in a bridging domain, Domain 3. These disulfide bonds are remote from the oxygen-binding site, but are positioned so that they could affect its stability. When the disulfide bonds are broken by dithiothreitol or other disulfide-bond reductants, the 340-nm absorption band, associated with oxygen binding, is lost. Disulfide-bond reductants also cause the loss of the oxygen-binding capacity of all seven of the other subunits of Limulus hemocyanin. Thus, disulfide bonding is a general feature of the Limulus hemocyanin subunits that is important to the maintenance of the physiologically effective geometry of the oxygen-binding site. The rate of loss of oxygen-binding capacity, however, is highly dependent on subunit type, aggregation state, and protein conformation. Evidence that protein conformation markedly affects the rate of disruption of the oxygen-binding site comes from the finding that the addition of dithiothreitol to fully oxygenated samples results in a slow initial loss of oxygen-binding capacity followed by an appreciably faster reaction rate. In contrast, in the deoxygenated conformation, the reaction rate is monophasic and never attains the faster rates observed for oxygenated samples. When the disulfide bonds are broken and oxygen-binding capacity is lost, there is subunit-specific variability in the extent of polypeptide-chain unfolding, subunit aggregation, and loss of active-site copper ions. When the disulfide-bond reductant is removed by dialysis so that disulfide bonds can re-form, there is also subunit-specific variability in the extent of restoration of oxygen-binding capacity. Complete restoration of structure and function as the disulfide bonds re-form occurs only for the 48-subunit native molecule, whose architecture is stabilized by bound Ca2+ and extensive intersubunit contacts. We have found a similar loss of oxygen-binding capacity upon breaking disulfide bonds in a number of other arthropod and mollusc hemocyanins, suggesting that the active site of Limulus hemocyanin is not unique in its dependence upon intact disulfides. The results presented in this paper suggest that disulfide-bond reduction may provide a simple, but powerful, chemical tool with which to probe internal and environmental factors that govern physiologically important structure-function relationships in the hemocyanins.
鲎血蓝蛋白亚基II的晶体结构分析表明，其多肽链折叠成三个不同的结构域。氧结合双核铜中心位于结构域2的核心深处。两个二硫键位于一个桥接结构域，即结构域3。这些二硫键远离氧结合位点，但其位置可能会影响其稳定性。当二硫键被二硫苏糖醇或其他二硫键还原剂破坏时，与氧结合相关的340纳米吸收带消失。二硫键还原剂还会导致鲎血蓝蛋白其他七个亚基的氧结合能力丧失。因此，二硫键结合是鲎血蓝蛋白亚基的一个普遍特征，对维持氧结合位点的生理有效几何结构很重要。然而，氧结合能力丧失的速率高度依赖于亚基类型、聚集状态和蛋白质构象。蛋白质构象显著影响氧结合位点破坏速率的证据来自以下发现：向完全氧合的样品中加入二硫苏糖醇会导致氧结合能力最初缓慢丧失，随后反应速率明显加快。相比之下，在脱氧构象中，反应速率是单相的，从未达到氧合样品中观察到 的较快速率。当二硫键断裂且氧结合能力丧失时，多肽链展开程度、亚基聚集和活性位点铜离子丧失的程度存在亚基特异性差异。当通过透析去除二硫键还原剂以使二硫键能够重新形成时，氧结合能力恢复的程度也存在亚基特异性差异。只有对于48亚基的天然分子，随着二硫键重新形成，结构和功能才能完全恢复，其结构通过结合的Ca2+和广泛的亚基间接触得以稳定。我们发现在许多其他节肢动物和软体动物血蓝蛋白中，二硫键断裂时也会出现类似的氧结合能力丧失，这表明鲎血蓝蛋白的活性位点在依赖完整二硫键方面并非独一无二。本文给出的结果表明，二硫键还原可能提供一种简单但强大的化学工具，用于探究控制血蓝蛋白中生理重要结构-功能关系的内部和环境因素。
