Zipse Hendrik, Artin Erin, Wnuk Stanislaw, Lohman Gregory J S, Martino Debora, Griffin Robert G, Kacprzak Sylwia, Kaupp Martin, Hoffman Brian, Bennati Marina, Stubbe Joanne, Lees Nicholas
Department of Chemistry and Biochemistry, Ludwig-Maximilians Universitaet Muenchen, 81377 Muenchen, Germany.
J Am Chem Soc. 2009 Jan 14;131(1):200-11. doi: 10.1021/ja806693s.
The Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: alpha and beta. Incubation of the E441Q-alpha mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4'-radical of a 3'-keto-2'-deoxycytidine 5'-diphosphate. To identify the structure of the nucleotide radical [1'-(2)H], [2'-(2)H], [4'-(2)H], [5'-(2)H], [U-(13)C, (15)N], [U-(15)N], and [5,6 -(2)H] CDP and [beta-(2)H] cysteine-alpha were synthesized and incubated with E441Q-alpha2beta2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of (2)H at C4' and C1' altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5'-diphosphate. The relationship of this radical to the disulfide radical anion is discussed.
大肠杆菌核糖核苷酸还原酶（RNR）催化核苷二磷酸转化为脱氧核苷酸，催化过程需要一个双铁 - 酪氨酰自由基辅因子。RNR由两个同型二聚体亚基（α和β）以1:1的复合物形式组成。将E441Q - α突变型RNR与底物CDP和变构效应物TTP一起孵育，会导致酪氨酰自由基的丧失，并在200毫秒至分钟的时间尺度上形成两个新的自由基。第一个自由基先前已通过停流紫外/可见光谱和脉冲高场电子顺磁共振光谱确定为二硫自由基阴离子。第二个自由基被认为是3'-酮-2'-脱氧胞苷5'-二磷酸的4'-自由基。为了确定核苷酸自由基的结构，合成了[1'-(2)H]、[2'-(2)H]、[4'-(2)H]、[5'-(2)H]、[U-(13)C, (15)N]、[U-(15)N]和[5,6 -(2)H] CDP以及[β-(2)H]半胱氨酸-α，并将它们与E441Q - α2β2和TTP一起孵育。通过9 GHz和140 GHz脉冲电子顺磁共振光谱以及35 GHz电子核双共振光谱对核苷酸自由基进行了检测。在C4'和C1'处取代(2)H改变了观察到的核苷酸自由基的超精细相互作用，并确定观察到的结构并非预测的结构。进行了密度泛函理论计算（B3LYP/IGLO - III/B3LYP/TZVP），以努力重现光谱观测结果，并得出与所有实验数据一致的新结构。结果出乎意料地表明，该自由基是胞苷5'-二磷酸的半醌核苷酸自由基。讨论了这个自由基与二硫自由基阴离子的关系。
