Han Wen-Ge, Noodleman Louis
Department of Molecular Biology TPC15, The Scripps Research Institute, 10550 North Torrey Pines Road La Jolla, California 92037, USA.
Dalton Trans. 2009 Aug 14(30):6045-57. doi: 10.1039/b903847g. Epub 2009 Jun 23.
Two models (I and II) for the active site structure of class-I ribonucleotide reductase (RNR) intermediate X in subunit R2 have been studied in this paper, using broken-symmetry density functional theory (DFT) incorporated with the conductor like screening (COSMO) solvation model and with the finite-difference Poisson-Boltzmann self-consistent reaction field (PB-SCRF) calculations. Only one of the bridging groups between the two iron centers is different between model-I and model-II. Model-I contains two mu-oxo bridges, while model-II has one bridging oxo and one bridging hydroxo. These are large active site models including up to the fourth coordination shell H-bonding residues. Mössbauer and ENDOR hyperfine property calculations show that model-I is more likely to represent the active site structure of RNR-X. However, energetically our pK(a) calculations at first highly favored the bridging oxo and hydroxo (in model-II) structure of the diiron center rather than having the di-oxo bridge (in model-I). Since the Arg236 and the nearby Lys42, which are very close to the diiron center, are on the protein surface of RNR-R2, it is highly feasible that one or two anion groups in solution would interact with the positively charged side chains of Arg236 and Lys42. The anion group(s) can be a reductant, phosphate, sulfate, nitrate, and other negatively charged groups existing in biological environments or in the buffer of the experiment. Since sulfate ions certainly exist in the buffer of the ENDOR experiment, we have examined the effect of the sulfate (SO(4)(2-), surrounded by explicit water molecules) H-bonding to the side chain of Arg236. We find that when sulfate interacts with Arg236, the carboxylate group of Asp237 tends to be protonated, and once Asp237 is protonated, the Fe(iii)Fe(iv) center in X favors the di-oxo bridge (model-I). This would explain that the ENDOR observed RNR-X active site structure is likely to be represented by model-I rather than model-II.
本文利用结合导体类屏蔽（COSMO）溶剂化模型及有限差分泊松-玻尔兹曼自洽反应场（PB-SCRF）计算的破缺对称性密度泛函理论（DFT），研究了R2亚基中I类核糖核苷酸还原酶（RNR）中间体X活性位点结构的两种模型（模型I和模型II）。模型I和模型II之间，两个铁中心之间只有一个桥连基团不同。模型I包含两个μ-氧桥，而模型II有一个桥连氧和一个桥连羟基。这些都是较大的活性位点模型，包括直至第四配位层的氢键残基。穆斯堡尔谱和电子核双共振超精细性质计算表明，模型I更有可能代表RNR-X的活性位点结构。然而，从能量角度看，我们最初的pK(a)计算强烈支持二铁中心的桥连氧和羟基（模型II）结构，而非双氧桥（模型I）结构。由于非常靠近二铁中心的精氨酸236和附近的赖氨酸42位于RNR-R2的蛋白质表面，溶液中的一个或两个阴离子基团与精氨酸236和赖氨酸42带正电的侧链相互作用是非常可行的。阴离子基团可以是生物环境或实验缓冲液中存在的还原剂、磷酸盐、硫酸盐、硝酸盐及其他带负电的基团。由于电子核双共振实验的缓冲液中肯定存在硫酸根离子，我们研究了硫酸根（SO(4)(2-)，被明确的水分子包围）与精氨酸236侧链形成氢键的影响。我们发现，当硫酸根与精氨酸236相互作用时，天冬氨酸237的羧基倾向于质子化，一旦天冬氨酸237质子化，X中的Fe(iii)Fe(iv)中心就有利于双氧桥（模型I）。这就解释了电子核双共振观察到的RNR-X活性位点结构可能由模型I而非模型II表示。
