Department of Chemistry, University of Puerto Rico , Mayagüez Campus, Mayagüez 00680, Puerto Rico.
Biochemistry. 2013 Oct 8;52(40):7007-21. doi: 10.1021/bi400745a. Epub 2013 Sep 27.
Hemoglobin HbI from the clam Lucina pectinata is involved in H2S transport, whereas homologous heme protein HbII/III is involved in O2 metabolism. Despite similar tertiary structures, HbI and HbII/III exhibit very different reactivity toward heme ligands H2S, O2, and NO. To investigate this reactivity at the heme level, we measured the dynamics of ligand interaction by time-resolved absorption spectroscopy in the picosecond to nanosecond time range. We demonstrated that H2S can be photodissociated from both ferric and ferrous HbI. H2S geminately rebinds to ferric and ferrous out-of-plane iron with time constants (τgem) of 12 and 165 ps, respectively, with very different proportions of photodissociated H2S exiting the protein (24% in ferric and 80% in ferrous HbI). The Gln(E7)His mutation considerably changes H2S dynamics in ferric HbI, indicating the role of Gln(E7) in controling H2S reactivity. In ferric HbI, the rate of diffusion of H2S from the solvent into the heme pocket (kentry) is 0.30 μM(-1) s(-1). For the HbII/III-O2 complex, we observed mainly a six-coordinate vibrationally excited heme-O2 complex with O2 still bound to the iron. This explains the low yield of O2 photodissociation and low koff from HbII/III, compared with those of HbI and Mb. Both isoforms behave very differently with regard to NO and O2 dynamics. Whereas the amplitude of geminate rebinding of O2 to HbI (38.5%) is similar to that of myoglobin (34.5%) in spite of different distal heme sites, it appears to be much larger for HbII/III (77%). The distal Tyr(B10) side chain present in HbII/III increases the energy barrier for ligand escape and participates in the stabilization of bound O2 and NO.
来自帘蛤科 Lucina pectinata 的血红蛋白 HbI 参与 H2S 转运，而同源血红素蛋白 HbII/III 则参与 O2 代谢。尽管具有相似的三级结构，但 HbI 和 HbII/III 对血红素配体 H2S、O2 和 NO 的反应性却非常不同。为了在血红素水平上研究这种反应性，我们在皮秒到纳秒的时间范围内通过时间分辨吸收光谱测量了配体相互作用的动力学。我们证明 H2S 可以从 ferric 和 ferrous HbI 中光解。H2S 以时间常数 (τgem) 分别为 12 和 165 ps 的速度再次结合到 ferric 和 ferrous 出平面铁，并且光解的 H2S 离开蛋白质的比例非常不同（在 ferric HbI 中为 24%，在 ferrous HbI 中为 80%）。Gln(E7)His 突变极大地改变了 ferric HbI 中的 H2S 动力学，表明 Gln(E7)在控制 H2S 反应性方面的作用。在 ferric HbI 中，H2S 从溶剂扩散到血红素口袋的速率 (kentry) 为 0.30 μM(-1) s(-1)。对于 HbII/III-O2 配合物，我们主要观察到六配位振动激发的血红素-O2 配合物，其中 O2 仍与铁结合。这解释了与 HbI 和 Mb 相比，O2 光解的产率和 koff 较低的原因。两种同工酶在 NO 和 O2 动力学方面的行为非常不同。尽管远端血红素位点不同，但 O2 再次结合到 HbI 的复合振幅 (38.5%) 与肌红蛋白 (34.5%) 相似，但对于 HbII/III 而言，复合振幅似乎要大得多 (77%)。存在于 HbII/III 中的远端 Tyr(B10)侧链增加了配体逃逸的能垒，并参与了结合 O2 和 NO 的稳定。
