Proton NMR of Escherichia coli sulfite reductase: studies of the heme protein subunit with added ligands.
Proton NMR of Escherichia coli sulfite reductase: studies of the heme protein subunit with added ligands.
复制标题
大肠杆菌亚硫酸盐还原酶的质子核磁共振：添加配体的血红素蛋白亚基的研究。
DOI：
10.1021/bi00085a008
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Spicer,LD
中科院分区：
文献类型：
--
作者：
Kaufman,J;Siegel,LM;Spicer,LD
1993年6月1日收到的修订手稿摘要：分离自大肠杆菌的亚硫酸盐还原酶的血红素蛋白亚基（SiR-HP; MT 64 000）含有与2+氧化态[4 Fe-4S]簇交换偶联的异菌氯环西罗血红素。SiR-HP在合适的电子供体存在下可以催化亚硫酸盐还原为硫化物和亚硝酸盐还原为氨。利用顺磁NMR研究了SiR-HP与外源抑制剂氰化物或底物亚硫酸盐和亚硝酸盐结合形成的低自旋复合物。作为模型，还制备了纯化的siroheme的氰化物络合物。分离的铁低自旋siroheme-CN的NMR光谱与自旋密度转移到α 2 η分子轨道中一致，这是卟啉中禁止的相互作用。分离的铁低自旋siroheme-CN的质子NMR位移的模式是非常相似的tothose获得的蛋白质氰化物复合物。SiR-HP的氰化物络合物的NMR光谱在所有三个可访问的氧化还原状态下获得。观察到的超精细位移的模式为one-electronand two-electron还原氰化物配合物是典型的[4Fe-4S]集群中的2+和1+氧化态，分别。从簇半胱氨酸的（3-CH 2）质子产生的共振已被分配给所有的配合物研究利用氘取代。氰化物，亚硫酸盐，亚硝酸盐连接状态具有几乎相同的移位高场簇半胱氨酸共振，其存在表明，在溶液中siroheme和簇之间存在共价耦合。还提供了存在第二阴离子结合位点的数据，其占据扰乱了氧化的SiR-HP NMR谱，其中结合以比配体与血红素结合的速率快得多的速率发生。大肠杆菌亚硫酸盐还原酶血红素蛋白亚基（SiR-HP）1的活性位点由与西罗血红素辅基交换偶联的[4Fe-4S]簇组成（Christner等人，1981; Janick & Siegel，1982）。已经提出了活性位点的模型（Janick & Siegel，1982），其中一个簇Fe通过半胱氨酸簇配体的硫共价桥连到sirohemeFe。X射线晶体学数据（McRee等人，1986）与该模型一致，尽管它缺乏足够的分辨率来识别桥接配体。最近，我们研究了未连接SiR-HP的质子NMR，并提供了与半胱氨酸作为桥连配体一致的数据（考夫曼等人，1993 b）。在siroheme的远端侧，与[4Fe-4S]簇相对，氧化SiR-HP的X射线晶体结构没有显示出显著的电子密度（McRee等人，1986年）。因此，这可能是许多外源配体结合的位点，包括底物亚硫酸盐和亚硝酸盐以及抑制剂如氰化物和CO。在迄今为止研究的所有复合物中，配体结合似乎涉及血红素
Revised Manuscript Received June 1, 1993 abstract: The heme protein subunit of sulfite reductase (SiR-HP; MT 64 000) from Escherichia coli as isolated contains the isobacteriochlorin siroheme exchange-coupled to a [4Fe-4S] cluster in the 2+ oxidation state. SiR-HP in the presence of a suitable electron donor can catalyze the six-electron reductions of sulfite to sulfide and nitrite to ammonia. Paramagnetic NMR was used to study the low-spin complexes of SiR-HP formed by binding the exogenous inhibitor cyanide or the substrates sulfite and nitrite. As a model, the cyanide complex of purified siroheme was also prepared. The NMR spectrum of isolated ferric low-spin siroheme-CN is consistent with spin density being transferred into the a2 „molecular orbital, an interaction which is symmetry-forbidden in porphyrins. The pattern of proton NMR shifts observed for isolated ferric low-spin siroheme-CN is very similar tothose obtained for the protein-cyanide complex. NMR spectra of the cyanide complex of SiR-HP were obtained in all three accessible redox states. The pattern of hyperfine shifts observed for the one-electronand two-electron reduced cyanide complexes is typical of those seen for [4Fe-4S] clusters in the 2+ and 1+ oxidation states, respectively. Resonances arising from the (3-CH2 protons of cluster cysteines have been assigned for all complexes studied utilizing deuterium substitution. The cyanide-, sulfite-, and nitrite-ligated states possessed an almost identically shifted upfield cluster cysteine resonance whose presence indicates that covalent coupling exists between siroheme and cluster in solution. Data are also presented for the existence of a secondary anion binding site, the occupancy of which perturbs the oxidized SiR-HP NMR spectrum, where binding occurs at a rate much faster than thatof ligand binding to heme.The active site of Escherichia coli sulfite reductaseheme protein subunit (SiR-HP) 1 consists of a [4Fe-4S] cluster exchange-coupled to a siroheme prosthetic group (Christner et al., 1981; Janick & Siegel, 1982). A model of the active site has been proposed (Janick & Siegel, 1982) in which one cluster Fe is covalently bridged to the sirohemeFe by the sulfur of a cysteinal cluster ligand. X-ray crystallographic data (McRee et al., 1986) are consistent with thismodel, although it lacks sufficient resolution to identify the bridging ligand. Recently, we have examined theproton NMR of unligated SiR-HP andhave presented data consistent with cysteine acting as the bridging ligand (Kaufman et al, 1993b). On the distal side of the siroheme, opposite the [4Fe-4S] cluster, the X-ray crystalstructure of oxidized SiR-HP does not show significant electron density (McRee et al., 1986). This may therefore be the site of binding for a number of exogenous ligands, including the substrates sulfite and nitrite and inhibitory agents such as cyanide and CO. In all complexes studied to date, ligand binding appears to involve the heme