Department of Cell Biology, Shantou University Medical College, 22 Xinling Road, Shantou, 515041, Guangdong, China.
Biometals. 2013 Oct;26(5):839-52. doi: 10.1007/s10534-013-9640-4. Epub 2013 Jun 5.
The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O2) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H2S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)-SH and heme Fe(II)-O2 complexes formed by H2S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O2 binding site in the heme Fe(II)-O2 complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H2S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H2S combined with H2O2 generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H2S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H2S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O2 binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H2S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.
大肠杆菌（Ec DOS）血红素基氧传感器磷酸二酯酶由一个 N 端血红素结合的氧感应结构域和一个 C 端催化结构域组成。氧（O2）与 Ec DOS 血红素 Fe(II)配合物结合可显著增强催化作用。向 Ec DOS 血红素 Fe(III)配合物中加入硫化氢（H2S）也能显著刺激催化作用，部分原因是 H2S 形成了血红素 Fe(III)-SH 和血红素 Fe(II)-O2 配合物。在这项研究中，我们研究了血红素远端氨基酸 M95（血红素 Fe(II)配合物的轴向配体）和 R97（血红素 Fe(II)-O2 配合物中的 O2 结合位点）在有氧条件下 Ec DOS 血红素结合结构域（Ec DOS-PAS）与 H2S 结合中的作用。有趣的是，R97A 和 R97I 突变蛋白在加入 H2S 后结合由反应产生的 H2O2，形成了氧合的改性血红素，verdoheme。时间依赖性质谱数据证实了这一发现。相比之下，H2S 没有与 M95H 和 R97E 突变体的血红素 Fe(III)配合物相互作用。因此，Ec DOS-PAS 中的血红素远端的 M95 和/或 R97 显著促进了 H2S 与 Fe(III)血红素配合物的相互作用，也促进了活性氧对血红素 Fe(III)配合物的修饰。重要的是，血红素蛋白的 O2 结合位点的突变将其功能从氧传感器转换为血红素加氧酶。本研究确立了 H2S 在修饰血红素铁配合物以形成与活性氧结合的 verdoheme 中的新作用。
