Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Aoba-ku, Sendai, Japan.
J Inorg Biochem. 2012 Mar;108:163-70. doi: 10.1016/j.jinorgbio.2011.09.019. Epub 2011 Sep 17.
YddV is a globin-coupled oxygen sensor enzyme in that O(2) binding to the Fe(II) heme in the sensor domain substantially enhances its diguanylate cyclase activity. The Fe(III) heme-bound enzyme is also the active form. Amino acid sequence comparisons indicate that Leu65 is well conserved in globin-coupled oxygen sensor enzymes. Absorption spectra of the Fe(III) heme complexes of L65G, L65M, L65Q and L65T mutants of the isolated heme domain of YddV (YddV-heme) were substantially different from that of the wild-type protein. Specifically, Soret bands of the 6-coordinated high-spin Fe(III) complexes of mutant proteins (with H(2)O and His98 as axial ligands) were located at around 403-406 nm, distinct from that (391 nm) of the 5-coordinated high-spin Fe(III) complex of wild-type protein with His98 as the axial ligand. The autooxidation rate constant (>0.10 min(-1)) of the Fe(II)-O(2) complex of L65G was substantially higher than that (0.011 min(-1)) of the wild-type protein. Affinities of O(2) for the Fe(II) complexes of L65G and L65T were markedly higher than that for the wild-type protein. Thus, we suggest that the well-conserved Leu65 located in the heme distal side is critical for restricting water access to the heme distal side to avoid rapid autooxidation of YddV, which needs a stable Fe(II)-O(2) complex with a low autooxidation rate.
YddV 是一种球蛋白偶联的氧传感器酶，因为 O(2)与传感器结构域中的 Fe(II)卟啉结合会显著增强其鸟苷酸环化酶活性。Fe(III)卟啉结合的酶也是活性形式。氨基酸序列比较表明，Leu65 在球蛋白偶联的氧传感器酶中高度保守。分离的 YddV 血红素结构域（YddV-heme）中 L65G、L65M、L65Q 和 L65T 突变体的 Fe(III)血红素复合物的吸收光谱与野生型蛋白的光谱有很大不同。具体而言，突变蛋白（以 H(2)O 和 His98 为轴向配体）的 6 配位高自旋 Fe(III)配合物的 Soret 带位于约 403-406nm，与轴向配体为 His98 的野生型蛋白的 5 配位高自旋 Fe(III)配合物的 391nm 不同。L65G 的 Fe(II)-O(2)复合物的自动氧化速率常数（>0.10 min(-1)) 显著高于野生型蛋白（0.011 min(-1))。L65G 和 L65T 的 O(2)对 Fe(II)配合物的亲和力明显高于野生型蛋白。因此，我们认为位于血红素远端的高度保守的 Leu65 对于限制水分子进入血红素远端以避免 YddV 的快速自动氧化至关重要，YddV 需要一个具有低自动氧化速率的稳定 Fe(II)-O(2)复合物。
