H2S HEMOGLOBIN I STRUCTURE--SITE DIRECTED MUTAGENESIS STUDY

果胶Lucina pectinata的血红蛋白I可能是唯一在硫化氢结合铁态和二氧结合铁态下发挥作用的血红素蛋白。一种被提出的机制表明，Hbl与H2S反应形成血红素铁硫低自旋配合物，将H2S运输到细菌共生体内。硫化氢被认为是通过一种未知还原剂的电子转移，在铁蛋白形成时释放到细菌中。此外，血红素- fe /+ 3sh2片段和GInE7之间存在氢键，可以稳定HbISH2中心。同样，PheB10和PheE11残基分别与HbICO、HbIO2和HBICN中心之间的极性相互作用。该假说认为Hbl可能具有两种不同的配体稳定机制：一种是Hbl- sh2中心，另一种是HbICO、HBIO2和HbICN部分。本文提出的工作旨在揭示附近氨基酸与HbISH2、HbIO2、HbICO和HbICN基团之间的相互作用。将采用定点诱变方法。选择Hbl在E7、B10和E11位点的定向变异，以及配体的同位素标记与共振和FTIR振动光谱耦合，将阐明Hbl-配体的双重稳定机制和结构。我们计划解决控制Hbl对H2S的结合、运输和递送的因素。目前，关于血红素蛋白中观察到的配体亲和差异是由静电效应、空间约束还是氢键差异引起的，有激烈的争论和讨论。从广义上讲，Hbl为解决这些问题提供了真正的优势，因为在这里，铁蛋白和铁蛋白在发挥其生物功能时是重要的、独特的和可逆的。该项目的长期目标是阐明好氧果绿Lucina pectinata与未知厌氧菌之间的共生相互作用和交流。

Hemoglobin I from Lucina pectinata is probably the only heme protein that carries out its function in the hydrogen-sulfide bound ferric state and in the dioxygen bound ferrous state. A proposed mechanism suggests that Hbl reacts with H2S to form a heme ferric sulfide low spin complex to transport the H2S to a bacteria symbion. Hydrogen sulfide is believed to be released to the bacteria upon formation of the ferrous protein by electron transfer from a still unknown reductant. Furthermore, the presence of H-bonding between the heme-Fe/+3 SH2 moiety and GInE7 is postulated to stabilize the HbISH2 center. Likewise, polar interactions between the PheB10 and PheE11 residues and the HbICO, HbIO2, and HBICN centers, respectively. This hypothesis is that Hbl may possesses two distinct ligand stabilization mechanism: one for the Hbl-SH2 center and the other for the HbICO, HBIO2, and HbICN moieties. The work proposed here is intended to unravel the interactions between the nearby amino acids and the HbISH2, HbIO2, HbICO, and HbICN moieties. A site-directed mutagenesis approach will be used. Choices of Hbl site-directed variants in the E7, B10, and E11 positions, and isotope labeling of ligands coupled to resonance and FTIR vibrational spectroscopies will clarify the Hbl-ligand dual stabilization mechanism and structure. We plan to address the factors that controls the binding, transport, and delivery of H2S by Hbl. Right now, there are intense arguments and discussions going on about whether the ligand affinity differences that are observed in heme proteins arise from electrostatic effects, from steric constraints, or from hydrogen-bonding differences. In the broad sense, Hbl offers a real advantage to address these issues as here the ferric, as well as the ferrous, protein is significant, unique and reversible when it carries its biological function. A long term goal of this project is to clarify the symbiotic interaction and communication between the aerobic Lucina pectinata and the unknown anaerobic bacteria.