AN ENDOR STUDY OF THE TYROSYL FREE-RADICAL IN RIBONUCLEOTIDE REDUCTASE FROM ESCHERICHIA-COLI
AN ENDOR STUDY OF THE TYROSYL FREE-RADICAL IN RIBONUCLEOTIDE REDUCTASE FROM ESCHERICHIA-COLI
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DOI：
10.1021/ja00203a002
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发表时间：
1989-10-11
影响因子：
15
通讯作者：
SJOBERG, BM
中科院分区：
文献类型：
--
作者：
BENDER, CJ;SAHLIN, M;SJOBERG, BM
酪氨酰自由基已被确定为几种蛋白质的组成部分，其功能是氧化还原化学。核糖核苷酸还原酶是这类酶的代表，因为其B2亚基含有酪氨酸残基，该残基是活性所必需的自由基。然而，固定化酶的EPR谱变宽，并且从其线形分析中只能提取有限的信息。在这种情况下，ENDOR光谱是一种分辨率更高的技术，我们用它来详细表征酶结合自由基。核糖核苷酸还原酶中的ENDOR增强仅在低于110 K的温度下观察到，这是由于通过μ-Δ Δ的自由基的温度依赖性弛豫增强。氧桥高自旋铁离子对。低于这个温度，获得良好的光谱和特定的蛋白质中的酪氨酸残基的氘化被用来分配测量的超精细张量的各种质子的自由基。邻位质子（Ax =-26.9MHz，Ay =-7.8MHz，Az =-19.7MHz）和强耦合β-质子的主张量分量的分析亚甲基质子确定该自由基具有七元奇数交替物种的特征，自旋密度分布为0.16（酚氧）、0.26（邻位）、-0.07（Meta）、-0.03（携带酚氧的环碳）和0.49（帕拉）。这些计算也提供了一个确定的麦康奈尔X值环质子在这类自由基。的β 质子以30 °的二面角定位。90度。相对于芳环的碳-1上的PZ轨道。结果表明，核糖核苷酸还原酶的酪氨酰自由基是不带电荷的，并且在蛋白质内的其环境中不与供体氢键合。
Tyrosyl radicals have been identified as components of several proteins whose function is redox chemistry. Ribonucleotide reductase is representative of this class of enzymes as its B2 subunit contains a tyrosine residue that is necessarily a radical for activity. The EPR spectrum of the immobilized enzyme is broadened, however, and only limited information can be extracted from an analysis of its line shape. In this situation, ENDOR spectroscopy is a higher resolution technique, and we used it to characterize the enzyme-bound radical in detail. ENDOR enhancement in ribonucleotide reductase is observed only at temperatures below 110 K due to a temperature-dependent relaxation enhancement of the radical by a .mu.-oxo-bridged pair of high-spin ferric ions. Below this temperature, excellent spectra are obtained and specific deuteration of the tyrosine residues in the protein was used to assign measured hyperfine tensors to the various protons in the radical. An analysis of the principal tensor components of the ortho protons (Ax = -26.9 MHz, Ay = -7.8 MHz, Az = -19.7 MHz) and the strongly coupled .beta.-methylene protons establishes that the radical had characteristics of a seven-member odd-alternate species with a spin density distribution of 0.16 (phenol oxygen), 0.26 (ortho), -0.07 (meta), -0.03 (ring carbon carrying phenol oxygen), and 0.49 (para). These calculations also provide a determination of the McConnell X value for ring protons in this class of radical. The .beta. protons are situated with dihedral angles of 30.degree. and 90.degree. with respect to the pz orbital on carbon-1 of the aromatic ring. The results indicate that the tyrosyl radical of ribonucleotide reductase is uncharged and not hydrogen bonded to donors in its environment within the protein.