Mutational Analysis of Sulfite Reductase Hemoprotein Reveals the Mechanism for Coordinated Electron and Proton Transfer
Mutational Analysis of Sulfite Reductase Hemoprotein Reveals the Mechanism for Coordinated Electron and Proton Transfer
复制标题
DOI：
10.1021/bi300947a
复制
发表时间：
2012-12-11
期刊：
影响因子：
2.9
通讯作者：
Stroupe, M. Elizabeth
中科院分区：
文献类型：
--
作者：
Smith, Kyle W.;Stroupe, M. Elizabeth
亚硫酸盐还原酶催化亚硫酸盐的六电子还原为硫化物。在血红素蛋白亚基（SiRHP）中发现的活性位点位于带负电荷的卟啉类（称为siroheme）的远侧表面上，其中心铁原子与近侧Fe4S4簇耦合。四个带正电荷的氨基酸位于活性位点空腔周围。这两个赖氨酸（R83和R153）和两个赖氨酸（K215和K217）一起减轻了西罗血红素大环上的负电荷。它们还充当远端结合阴离子周围的笼，当底物结合和活性位点环夹紧时，所述笼收紧。天然SiRHP的结构指出这些氨基酸是重要的，但它们的具体作用是不明确的。在这里，我们已经改变了这四个活性位点的氨基酸和一个氨基酸的灵活的环（N149），以探测它们在SiRHP活性的作用。这些带正电荷的残基都不是电子转移所必需的，但只有R83S和N149W变体可以产生完全还原的产物。通过测量每单位释放的还原硫所使用的电子，我们发现K215，R153和K217负责中间和晚期质子转移，而N149和R153在控制阴离子结合和释放的柔性环结构中发挥作用。R83主要负责siroheme结合。总之，这些变体的活性和结构揭示了各自在阴离子结合和促进电子转移的偶联质子转移中的特定作用。
Sulfite reductase catalyzes the six-electron reduction of sulfite to sulfide. The active site, found in the hemoprotein subunit (SiRHP), sits on the distal face of a negatively charged porphyrinoid called siroheme whose central iron atom is coupled to a proximal Fe4S4 cluster. Four positively charged amino acids are positioned around the active site cavity. Together, these two arginines (R83 and R153) and two lysines (K215 and K217) mitigate the negative charge on the siroheme macrocycle. They also serve as a cage around the distally bound anion that tightens when substrate binds and an active site loop clamps down. Structures of native SiRHP point to these amino acids as being important, but their specific roles are ill-defined. Here, we have altered those four active site amino acids and one amino acid on the flexible loop (N149) to probe their roles in SiRHP activity. None of these positively charged residues is required for electron transfer, but only R83S and N149W variants can produce a fully reduced product. By measuring the electrons used per unit of reduced sulfur released, we show that K215, R153, and K217 are responsible for intermediate and late proton transfers, whereas N149 and R153 play a role in the structure of the flexible loop that controls anion binding and release. R83 is primarily responsible for siroheme binding. Together, the activities and structures of these variants reveal specific roles for each in anion binding and in coupled proton transfer that facilitates electron transfer.