Yan Fang, Fojtikova Veronika, Man Petr, Stranava Martin, Martínková Markéta, Du Yongming, Huang Dongyang, Shimizu Toru
Department of Cell Biology and Genetics and Key Laboratory of Molecular Biology in High Cancer Incidence Coastal Chaoshan Area of Guangdong Higher Education Institutes, Shantou University Medical College, Shantou, 515041, Guangdong, China.
Biometals. 2015 Aug;28(4):637-52. doi: 10.1007/s10534-015-9847-7. Epub 2015 Mar 25.
EcDOS is a heme-based O2-sensing phosphodiesterase in which O2 binding to the heme iron complex in the N-terminal domain substantially enhances catalysis toward cyclic-di-GMP, which occurs in the C-terminal domain. Here, we found that hydrogen sulfide enhances the catalytic activity of full-length EcDOS, possibly owing to the admixture of 6-coordinated heme Fe(III)-SH(-) and Fe(II)-O2 complexes generated during the reaction. Alanine substitution at Met95, the axial ligand for the heme Fe(II) complex, converted the heme Fe(III) complex into the heme Fe(III)-SH(-) complex, but the addition of Na2S did not further reduce it to the heme Fe(II) complex of the Met95Ala mutant, and no subsequent formation of the heme Fe(II)-O2 complex was observed. In contrast, a Met95His mutant formed a stable heme Fe(II)-O2 complex in response to the same treatment. An Arg97Glu mutant, containing a glutamate substitution at the amino acid that interacts with O2 in the heme Fe(II)-O2 complex, formed a stable heme Fe(II) complex in response to Na2S, but this complex failed to bind O2. Interestingly, the addition of Na2S promoted formation of verdoheme (oxygen-incorporated, modified protoporphyrin IX) in an Arg97Ile mutant. Catalytic enhancement by Na2S was similar for Met95 mutants and the wild type, but significantly lower for the Arg97 mutants. Thus, this study shows the first isolation of spectrometrically separated, stable heme Fe(III)-SH(-), heme Fe(II) and heme Fe(II)-O2 complexes of full-length EcDOS with Na2S, and confirms that external-ligand-bound, 6-coordinated heme Fe(III)-SH(-) or heme Fe(II)-O2 complexes critically contribute to the Na2S-induced catalytic enhancement of EcDOS.
EcDOS是一种基于血红素的氧气感应磷酸二酯酶，其中氧气与N端结构域中的血红素铁复合物结合，可显著增强对C端结构域中环状二鸟苷酸的催化作用。在此，我们发现硫化氢可增强全长EcDOS的催化活性，这可能是由于反应过程中生成了六配位的血红素Fe(III)-SH(-)和Fe(II)-O2复合物的混合物。血红素Fe(II)复合物的轴向配体Met95处的丙氨酸取代，将血红素Fe(III)复合物转化为血红素Fe(III)-SH(-)复合物，但添加Na2S并未将其进一步还原为Met95Ala突变体的血红素Fe(II)复合物，且未观察到随后血红素Fe(II)-O2复合物的形成。相比之下，Met95His突变体在相同处理下形成了稳定的血红素Fe(II)-O2复合物。Arg97Glu突变体在与血红素Fe(II)-O2复合物中的O2相互作用的氨基酸处含有谷氨酸取代，在Na2S作用下形成了稳定的血红素Fe(II)复合物，但该复合物无法结合O2。有趣的是，添加Na2S促进了Arg97Ile突变体中胆绿素（氧结合的修饰原卟啉IX）的形成。Na2S对Met95突变体和野生型的催化增强作用相似，但对Arg97突变体则显著降低。因此，本研究首次通过光谱分离得到了全长EcDOS与Na2S形成的稳定的血红素Fe(III)-SH(-)、血红素Fe(II)和血红素Fe(II)-O2复合物，并证实外部配体结合的六配位血红素Fe(III)-SH(-)或血红素Fe(II)-O2复合物对Na2S诱导的EcDOS催化增强起着关键作用。
