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首页> 外文期刊>The FEBS journal >Critical roles of Leu99 and Leu115 at the heme distal side in auto-oxidation and the redox potential of a heme-regulated phosphodiesterase from Escherichia coli
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Critical roles of Leu99 and Leu115 at the heme distal side in auto-oxidation and the redox potential of a heme-regulated phosphodiesterase from Escherichia coli

机译：血红素远端的Leu99和Leu115在自氧化和血红素调节的磷酸二酯酶的氧化还原电位中的关键作用

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摘要

The heme-regulated phosphodiesterase from Escherichia coli (Ec DOS), which is a heme redox-dependent enzyme, is active with a ferrous heme but inactive with a ferric heme. Global structural changes including axial ligand switching and a change in the rigidity of the FG loop accompanying the heme redox change may be related to the dependence of Ec DOS activity on the redox state. Axial ligands such as CO, NO, and O-2 act as inhibitors of Ec DOS because they interact with the ferrous heme complex. The X-ray crystal structure of the isolated heme-bound domain (Ec DosH) shows that Leu99, Phe113 and Leu115 indirectly and directly form a hydrophobic triad on the heme plane and that they should be located at or near the ligand access channel of the heme iron. We generated L99T, L99F, L115T, and L115F mutants of Ec DosH and examined their physicochemical characteristics, including auto-oxidation rates, O-2 and CO binding kinetics, and redox potentials. The Fe(III) complex of the L115F mutant was unstable and had a Soret absorption spectrum located 5 nm lower than those of the wild-type and other mutants. Auto-oxidation rates of the mutants (0.049-0.33 min(-1)) were much higher than that of the wild-type (0.0063 min(-1)). Furthermore, the redox potentials of the former three mutants (23.1-34.6 mV versus SHE) were also significantly lower than that of the wild-type (63.9 mV versus SHE). Interaction between O-2 and the L99F mutant was different from that in the wild-type, whereas CO binding rates of the mutants were similar to those of the wild-type. Thus, it appears that Leu99 and Leu115 are critical for determining the characteristics of heme iron. Finally, we discuss the roles of these amino-acid residues in the heme electronic states.
机译：来自大肠杆菌的血红素调节的磷酸二酯酶（Ec DOS）是一种依赖于血红素的氧化还原酶，对亚铁血红素有活性，但对铁血红素无活性。总体结构变化包括轴向配体转换以及伴随血红素氧化还原变化的FG环刚性的变化可能与Ec DOS活性对氧化还原状态的依赖性有关。轴向配体（例如CO，NO和O-2）可作为Ec DOS的抑制剂，因为它们与亚铁血红素复合物相互作用。分离的血红素结合结构域（Ec DosH）的X射线晶体结构表明Leu99，Phe113和Leu115在血红素平面上间接和直接形成疏水三联体，并且它们应位于或接近其配体通道血红素铁。我们生成了Ec DosH的L99T，L99F，L115T和L115F突变体，并检查了它们的理化特性，包括自氧化率，O-2和CO结合动力学以及氧化还原电势。 L115F突变体的Fe（III）配合物不稳定，Soret吸收光谱比野生型和其他突变体低5 nm。突变体的自氧化率（0.049-0.33 min（-1））远高于野生型（0.0063 min（-1））。此外，前三个突变体的氧化还原电位（23.1-34.6 mV对SHE）也显着低于野生型（63.9 mV对SHE）。 O-2和L99F突变体之间的相互作用不同于野生型，而突变体的CO结合率与野生型相似。因此，看来Leu99和Leu115对于确定血红素铁的特性至关重要。最后，我们讨论了这些氨基酸残基在血红素电子态中的作用。

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