UNUSUAL COPPER COORDINATION IN C112D PSEUDOMONAS AERUGINOSA AZURINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在折叠多肽环境中精确调节活性部位还原电位的能力使自然界能够自由地访问无数的化学成分，尽管其结合的元素库有限。铜绿假单胞菌天青素的还原潜力长期以来一直被认为对内外1类铜配位球内的取代反应都很敏感。这种灵敏度使得有可能将铜(II/I)电势调节到比水氧化还原电偶高0.5V以上。为了进一步探索这种对铜(II/I)氧化还原对的控制，我们构建了天青素的突变体，其中类型1的铜中心通过去除赤道半胱氨酸配体转变为类型2中心。这些蛋白质通过替换121位的甲硫氨酸进一步改变。通过SAM介导的循环伏安法和方波伏安法，我们已经证明了天青中的第二类铜还原电位也可以通过残基121来调节。在对这些天青C112D/M121X蛋白的研究中，我们观察到在M121L的情况下，相对于其他轴向突变蛋白，X-带EPR中的轴向超精细信号明显变窄。在100x10-4厘米-1，这将金属中心放置在类型1分类的极端边缘。通过伏安测量，我们发现除了具有282 mV的还原电势外，电极与铜中心之间的电子转移速度比天青C112D增加了一个数量级以上。这些发现表明，尽管没有硫连接，铜蛋白可能具有类型1的特征。我们建议使用EXAFS来研究C112D/M121X天青的氧化和还原形式相对于C112D天青发生的变化。精确的铜(I)和铜(II)蛋白质的金属-配位键距离将有助于深入了解共价性和重组能的贡献。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The capacity for precise tuning of active site reduction potentials in folded polypeptide environments has afforded nature the freedom to access myriad chemistries despite its limited repertoire of incorporated elements. The reduction potential of Pseudomonas aeruginosa azurin has long been known to be sensitive to substitutions within both the inner and outer type 1 copper coordination sphere. This sensitivity makes it possible to tune the Cu(II/I) potential over 0.5 V higher than the aqueous redox couple. In order to further explore this control over the Cu(II/I) redox couple, we have constructed mutants of azurin in which the type 1 copper center has been converted to a type 2 center through removal of the equatorial cysteine ligand. These proteins have been further altered through substitutions to the axial methionine at position 121. Through SAM-mediated cyclic and square wave voltammetry, we have demonstrated that the type 2 copper reduction potential in azurin is also tuned via residue 121. In studying these azurin C112D/M121X proteins, we made the observation that in the case of M121L, the axial hyperfine signal in the X-band EPR is significantly narrowed relative to the other axial mutant proteins. At 100x10-4 cm-1, this places the metal center at the extreme edge of the type 1 classification. Through voltammetry measurements, we found that in addition to possessing a reduction potential of 282 mV versus NHE, the rate of electron transfer between the electrode and the copper center has increased over an order of magnitude relative to azurin C112D. These findings suggest that despite the absence of sulfur ligation, copper proteins may adopt type 1 character. We propose to use EXAFS to examine the changes that occur in the oxidized and the reduced forms of C112D/M121X azurins relative to C112D azurin. Precise metal-ligand bond distances for the Cu(I) and Cu(II) proteins will provide insight into the contributions of covalency and reorganization energy.