Structure-function relations in Rieske-type proteins

铁硫蛋白在自然界中普遍存在，存在于大多数代谢途径中，了解决定其物理化学性质的参数是生物化学的中心问题。其中最有趣的是2Fe-2S团簇，它们的共同特征是两个半胱氨酸和两个组氨酸将铁原子连接起来。其中包括Bc1(及相关)复合体的Rieske铁硫蛋白(Isp)，以及几种Rieske类型的细菌蛋白，包括Sulfolobus sp.的古生菌硫氧还蛋白(SDX)。7号菌株和Rieske型铁氧还蛋白(ARF)。这些蛋白质的一个显著特征是2Fe-2S簇表现出广泛的氧化还原电位变化，从-100 mV到300 mV，尽管它们有相似的连接。最近的晶体结构显示，在蛋白质的簇结合结构域中，配体在这一范围的两端都有类似的取向。这意味着每个特定簇的氧化还原电位和蛋白分解性质受蛋白质环境的其他独特特征控制。为了了解蛋白质的功能，影响这些参数的因素必须在每个蛋白质的原子水平上确定。我们建议用先进的磁共振技术研究上述三个Rieske型团簇的蛋白质环境，重点是二维ESEEM，以探索顺磁中心与邻近核自旋之间的相互作用。我们将分析数据以提供结构信息，并将其与isp的晶体结构和萘-1，2-双加氧酶的晶体结构进行比较。光谱结果将为精确确定团簇环境提供约束，包括组氨酸和半胱氨酸配体的配位、氢键和非配位氮的存在以及溶剂的可及性。通过对分子工程产生的突变菌株进行类似的实验，我们预计我们将能够确定控制簇的氧化还原和蛋白分解属性的蛋白质环境的局部特征，它们在反应机制中的作用，以及在不同菌株中产生功能修饰的变化。这些结果将回答有关结构因素控制Rieske型蛋白氧化还原电位的一些基本问题，并为其他氧化还原蛋白的类似问题提供借鉴。

Iron-sulfur proteins are ubiquitous in nature and occur in most metabolic pathways, and an understanding of the parameters determining their physico-chemical properties is a central problem of biochemistry. Among the most interesting are the 2Fe-2S clusters that have as a common feature the liganding of the irons by two cysteines and two histidines. These include the Rieske iron-sulfur protein (ISP) of the bc1 (and related) complexes, and several Rieske-type bacterial proteins, including the archaeal sulredoxin (SDX) of Sulfolobus sp. Strain 7 and Rieske-type ferredoxin (ARF) from S. solfaricus to be investigated in this project. A remarkable feature of these proteins is the wide variation in redox potentials, from -100 to 300 mV, exhibited by the 2Fe-2S clusters, despite their similar ligation. Recent crystallographic structures show a similar orientation of ligands in the cluster binding domain in proteins at both extremes of this range. This implies that the redox potentials and protolytic properties of each particular cluster are controlled by other unique features of the protein environment. To understand how the proteins function, the factors influencing these parameters must be determined at the atomic level for each protein. We propose to investigate the protein environment of the three Rieske-type clusters above by using advanced magnetic resonance techniques, with an emphasis on 2-D ESEEM to explore the interaction between the paramagnetic centers and nuclear spins in the neighborhood. We will analyze the data so as to provide structural information, and compared this with the crystallographic structured of ISP, and of naphthalene- 1,2-dioxygenase. The spectroscopic results will provide constraints to allow precise determination of the cluster environment including coordination of histidine and cysteine ligands, presence of hydrogen bonds and non-coordinated nitrogens, and accessibility of solvent. By performing similar experiments with mutant strains generated by molecular engineering, we anticipate that we will be able to identify the local features of the protein environment that control the redox and protolytic properties of the clusters, their role in reaction mechanisms, and the changes that produce functional modification in different strains. The results will answer some fundamental questions about structural factors controlling the redox potentials of Rieske-type proteins, and provide insights to similar questions in other redox proteins.