Optical spectroscopic investigation of the alkaline transition in umecyanin from horseradish root.
Optical spectroscopic investigation of the alkaline transition in umecyanin from horseradish root.
复制标题
辣根根中梅花青苷碱性转变的光学光谱研究。
作者：
I. Delfino;Katsuko Sato;M. Harrison;L. Andolfi;A. Bizzarri;C. Dennison;S. Cannistraro
来源于辣根根的鱼藤花青素（UMC）属于植物铜氧还蛋白家族--植物蓝素的星花青素亚类。该蛋白质具有典型的1型His（2）Cys赤道配体，位于其单核铜位点，但具有轴向Gln配体，取代了plantacyanins，uclacyanins和大多数其他铜氧还蛋白的通常弱配位Met。UMC表现出，像其他植物蓝素，改变可见光，EPR，顺磁（1）H NMR光谱在升高的pH值，也修改了还原电位。这种碱性转变在pK（α）约为10时发生[Dennison，C.，Lawler，A. T.（2001）Biochemistry 40，3158-3166]。在这项研究中，我们调查的碱性过渡互补光谱技术。当代使用的吸收，荧光，动态光散射，共振拉曼光谱，使我们能够证明，碱性过渡诱导重组的蛋白质和UMC的整体大小增加，但蛋白质聚集不会发生。过渡并没有显着的影响，UMC的活性位点的环境，但有微妙的改变，在铜网站的几何形状。的Cu-N（His）键的加强的直接证据，这是在协议的假设，其中一个His配体的N（ε 2）H部分的去质子化是碱性过渡的原因。Cu-S（Cys）键的弱化也被观察到，这沿着弱化的轴向相互作用，一定是由于增强的Cu-N（His）相互作用。
Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His(2)Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N(epsilon2)H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.