Sulfur K-edge X-ray absorption Spectroscopy and density functional theory calculations on superoxide reductase: Role of the axial thiolate in reactivity
Sulfur K-edge X-ray absorption Spectroscopy and density functional theory calculations on superoxide reductase: Role of the axial thiolate in reactivity
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DOI：
10.1021/ja064167p
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发表时间：
2007-10-17
影响因子：
15
通讯作者：
Solomon, Edward I.
中科院分区：
文献类型：
--
作者：
Dey, Abhishek;Jenney, Francis E., Jr.;Solomon, Edward I.
超氧化物还原酶（SOR）是一种非血红素铁酶，以扩散控制的速率将超氧化物还原为过氧化物。硫K边X射线吸收光谱（XAS）用于研究来自激烈火球菌的I Fe SOR的静止高自旋和CN结合低自旋Fe-III形式的基态电子结构。一个计算模型与约束咪唑环（必要的复制自旋状态），氢键相互作用的硫醇（必要的复制Fe-S键共价的高自旋和低自旋形式），和氢键的可交换的轴向配体（必要的复制基态的低自旋形式）开发，然后用于研究酶促反应机制。的休息亚铁网站与超氧化物和质子化导致高自旋Fe-III-OOH物种和随后的质子化导致过氧化氢释放的反应计算是最积极有利的反应途径。我们的研究结果表明，硫醇盐作为一个共价阴离子配体。用中性非共价配体取代硫醇盐使质子化非常吸热，并大大提高了还原电位。硫醇盐的共价性质削弱了Fe-III键与该氢过氧物种的近端氧的键合，这使其pK（a）相对于主要阴离子配体的pKa增加了额外的5个对数单位，促进了其质子化。与细胞色素P450的比较表明，较强的赤道配位场从卟啉的结果在低自旋Fe-III-OOH的物种，这将是不能够有效的H2 O2释放由于自旋交叉势垒与高自旋5C Fe-III产品的形成。此外，细胞色素P450中双阴离子卟啉π环的存在允许O-O异裂解，形成Fe-IV-氧代卟啉自由基物质，这被计算为对非血红素SOR配体环境极其不利。最后，计算出O-2（-）还原循环结束时产物释放产生的5C Fe-III位点能够与第二个O-2反应，产生超氧化物歧化酶（SOD）活性。然而，与FeSOD相反，SOR的5C Fe-III位点，其带更多正电荷，被计算为具有结合第六阴离子配体的高亲和力，这将抑制其SOD活性。
Superoxide reductase (SOR) is a non-heme iron enzyme that reduces superoxide to peroxide at a diffusion-controlled rate. Sulfur K-edge X-ray absorption spectroscopy (XAS) is used to investigate the ground-state electronic structure of the resting high-spin and CN- bound low-spin Fe-III forms of the I Fe SOR from Pyrococcus furiosus. A computational model with constrained imidazole rings (necessary for reproducing spin states), H-bonding interaction to the thiolate (necessary for reproducing Fe-S bond covalency of the high-spin and low-spin forms), and H-bonding to the exchangeable axial ligand (necessary to reproduce the ground state of the low-spin form) was developed and then used to investigate the enzymatic reaction mechanism. Reaction of the resting ferrous site with superoxide and protonation leading to a high-spin Fe-III-OOH species and its subsequent protonation resulting in H2O2 release is calculated to be the most energetically favorable reaction pathway. Our results suggest that the thiolate acts as a covalent anionic ligand. Replacing the thiolate with a neutral noncovalent ligand makes protonation very endothermic and greatly raises the reduction potential. The covalent nature of the thiolate weakens the Fe-III bond to the proximal oxygen of this hydroperoxo species, which raises its pK(a), by an additional 5 log units relative to the pKa of a primarily anionic ligand, facilitating its protonation. A comparison with cytochrome P450 indicates that the stronger equatorial ligand field from the porphyrin results in a low-spin Fe-III-OOH species that would not be capable of efficient H2O2 release due to a spin-crossing barrier associated with formation of a high-spin 5C Fe-III product. Additionally, the presence of the dianionic porphyrin pi ring in cytochrome P450 allows O-O heterolysis, forming an Fe-IV-oxo porphyrin radical species, which is calculated to be extremely unfavorable for the non-heme SOR ligand environment. Finally, the 5C Fe-III site that results from the product release at the end of the O-2(-) reduction cycle is calculated to be capable of reacting with a second O-2, resulting in superoxide dismutase (SOD) activity. However, in contrast to FeSOD, the 5C Fe-III site of SOR, which is more positively charged, is calculated to have a high affinity for binding a sixth anionic ligand, which would inhibit its SOD activity.