UNUSUAL COPPER COORDINATION IN C112D PSEUDOMONAS AERUGINOSA AZURINS

C112D 铜绿假单胞菌天青蛋白中异常的铜配位

• 批准号: 7954476
• 负责人: KYLE M LANCASTER
• 金额: $ 0.1万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954476
• 关键词: Active Sites; Adopted; Azurin; Chemistry; Classification; Computer Retrieval of Information on Scientific Projects Database; Copper; Cysteine; Electrodes; Electron Transport; Elements; Environment; Excision; Freedom; Funding; Grant; Institution; Ligands; Ligation; Measurement; Mediating; Metals; Methionine; Nature; Oxidation-Reduction; Positioning Attribute; Proteins; Pseudomonas aeruginosa; Relative (related person); Research; Research Personnel; Resources; Signal Transduction; Source; Sulfur; United States National Institutes of Health; aqueous; insight; mutant; polypeptide; structural biology; synchrotron radiation

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在折叠多肽环境中精确调节活性位点还原电位的能力已经为自然界提供了获得无数化学物质的自由，尽管其有限的掺入元件库。 铜绿假单胞菌天青蛋白的还原电位长期以来一直被认为对内部和外部1型铜配位球内的取代敏感。这种灵敏度使得可以将Cu（II/I）电位调节到比水性氧化还原对高0.5V以上。为了进一步探索这种控制的Cu（II/I）氧化还原对，我们已经构建了天青蛋白的突变体，其中1型铜中心已被转换为2型中心，通过去除赤道半胱氨酸配体。 这些蛋白质已经通过在位置121处替换为轴向甲硫氨酸而进一步改变。 通过SAM介导的循环和方波伏安法，我们已经证明，2型铜还原电位天青蛋白也通过残基121调整。在研究这些天青蛋白C112 D/M121 X蛋白时，我们观察到，在M121 L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显著变窄。 在100 x10 -4 cm-1处，这将金属中心置于1型分类的最边缘。通过伏安法测量，我们发现，除了拥有282 mV的还原电位相对于NHE，电极和铜中心之间的电子转移速率增加了一个数量级相对于天青C112 D。 这些发现表明，尽管没有硫连接，铜蛋白可能采用1型字符。我们建议使用EXAFS检查发生在氧化和还原形式的C112 D/M121 X天青蛋白相对于C112 D天青蛋白的变化。 精确的金属配体键的Cu（I）和Cu（II）蛋白质的距离将提供深入了解共价和重组能的贡献。