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首页> 外文期刊>Biochemistry >Probing the catalytic mechanism of sulfite reductase by X-ray crystallography: structures of the Escherichia coli hemoprotein in complex with substrates, inhibitors, intermediates, and products
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Probing the catalytic mechanism of sulfite reductase by X-ray crystallography: structures of the Escherichia coli hemoprotein in complex with substrates, inhibitors, intermediates, and products

机译：通过X射线晶体学探索亚硫酸还原酶的催化机理：与底物，抑制剂，中间体和产物复合的大肠杆菌血红蛋白的结构

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To further understand the six-electron reductions of sulfite and nitrite catalyzed by the Escherichia coli sulfite reductase hemoprotein (SiRHP), we have determined crystallographic structures of the enzyme in complex with the inhibitors phosphate, carbon monoxide, and cyanide, the substrates sulfite and nitrite, the intermediate nitric oxide, the product sulfide (or, most likely, an oxidized derivative thereof), and an oxidized nitrogen species (probably nitrate). A hydrogen-bonded cage of ligand-binding arginine and lysine side chains, ordered water molecules, and siroheme carboxylates provides preferred locations for recognizing the common functional groups of these ligands and accommodates their varied sizes, shapes, and charged without requiring substantial structural changes. The coordination geometries presented here suggest that the successively deoxygenated sulfur and nitrogen species produced during catalysis need not alter their orientation in the active site to adopt new stable coordination states. Strong pi-acid ligands decrease the bond length between the siroheme and the proximal cysteine thiolate shared with the iron-sulfur cluster, emphasizing the ability of the coupled cofactors to promote electron tranfer into substrate. On binding the siroheme, the substrate sulfite provides an oxygen atom in a unique location of the binding site compared to all other ligands studied, induces a spin transition in the siroheme iron, flips an active-site arginine, and orders surrounding active-center loops. The loop that coalesces over the active center shields the positively charged ligand-coordinating residues from solvent, enhancing their ability to polarize the substrate. Hydrogen bonds supplied by active-site arginine and lysine residues facilitate charge transfer into the substrate from the electron-rich cofactors, activate S-O bonds for reductive cleavage, and provide potential proton sources for the formation of favorable aquo leaving groups on the substrate. Strong interactions between sulfite and ordered water molecules also implicate solvent as a source of protons for generating product water. From the structures reported here, we propose a series of key structural states of ligated SiRHP in the catalytic reduction of sulfite to sulfide.
机译：为了进一步了解大肠杆菌亚硫酸还原酶血蛋白（SiRHP）催化的亚硫酸盐和亚硝酸盐的六电子还原，我们确定了该酶与磷酸盐，一氧化碳和氰化物，亚硫酸盐和亚硝酸盐底物的复合物的晶体结构。 ，中间一氧化氮，产物硫化物（或最有可能的是其氧化衍生物）和氧化的氮物质（可能是硝酸盐）。配体结合的精氨酸和赖氨酸侧链，有序的水分子和西罗血红素羧酸盐的氢键笼提供了识别这些配体的常见官能团的理想位置，并适应了它们的大小，形状和带电荷，而无需进行实质性的结构改变。此处给出的配位几何结构表明，在催化过程中连续脱氧的硫和氮物质无需改变其在活性位点的取向即可采用新的稳定配位态。强大的pi酸配体可减少与铁硫簇共享的Siroheme和近端半胱氨酸硫代半胱氨酸之间的键长，从而增强了偶联辅因子促进电子转移至底物中的能力。与其他已研究的配体相比，在结合西罗血红素时，底物亚硫酸盐在结合位点的唯一位置提供了一个氧原子，在西罗血红素铁中诱导自旋转变，翻转了活性位点的精氨酸，并围绕了活性位点环。结合在活性中心上方的环将带正电荷的配体配位残基与溶剂隔离开，从而增强了其使底物极化的能力。活性位点精氨酸和赖氨酸残基提供的氢键促进电荷从富电子辅因子转移到基质中，激活S-O键进行还原裂解，并提供潜在的质子源，以在基质上形成有利的水离去基团。亚硫酸盐与有序水分子之间的强相互作用也暗示溶剂是质子源，用于产生产物水。根据此处报道的结构，我们提出了连接的SiRHP在亚硫酸盐催化还原成硫化物中的一系列关键结构状态。

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