Reduction of ribonucleotide reductase (EC 1.17.4.1) R2 proteins in a frozen glycerol-buffer solution at 77 K by mobile electrons generated by gamma-irradiation produces EPR-detectable iron sites in mixed-valent Fe(II)/Fe(III) states. The primary EPR signals give information about the ligand arrangement of the diferric form of the iron site, whereas secondary signals observed after annealing of the sample show the effects of structural relaxation. In recombinant metR2 proteins (without free radical) from mouse and herpes virus type 1, the mixed-valent sites trapped at 77 K give rise to axial S = 1/2 EPR spectra with g values in the range 1.79-1.94, observable at temperatures up to 110 K. The spectra are assigned to mu-oxo-bridged dinuclear iron sites. In mouse metR2, the primary EPR spectrum is a mixture of two components. Annealing the R2 samples to 160-170 K transforms the primary EPR signals into rhombic spectra, characterized by gav < 1.8, and observable only below 25 K. These spectra are assigned to partially relaxed forms with a mu-hydroxo bridge, formed by protonation of the oxo bridge. Further annealing at 220 K produces new rhombic EPR spectra, which are closely similar with those observed and found to be stable after chemical reduction at room temperature. The EPR signal of the primary mixed-valent iron site in active mouse R2 protein with a tyrosyl radical also has two components. Both are different from those observed in metR2. In herpes simplex virus type 1 protein R2, one primary mixed-valent component was observed for the met protein. The dose-yield curve for the mixed-valent state in active mouse R2 is sigmoidal in shape, indicating that the tyrosyl radical is reduced by mobile electrons before the iron site. Kinetic experiments on the reduction by dithionite on mouse R2 without and with radical show a significantly enhanced rate for reduction of the iron site in the protein without radical. The results suggest that in active mouse R2 only complete diferric sites with neighboring radicals give rise to the mixed-valent spectra, and that these sites may exist in two structurally distinct forms. The results on the mouse R2 proteins confirm and extend previous results obtained on the Escherichia coli protein R2 showing that the presence of the tyrosyl radical significantly affects not only the structure but also the reactivity of the iron site.

译文

通过伽马辐照产生的移动电子在77 K的冷冻甘油缓冲溶液中还原核糖核苷酸还原酶 (EC 1.17.4.1) R2蛋白,在混合价的Fe(II)/Fe(III) 状态下产生可检测到EPR的铁位点。初级EPR信号提供有关铁位点二铁形式的配体排列的信息,而样品退火后观察到的次级信号显示了结构弛豫的影响。在来自小鼠和1型疱疹病毒的重组metR2蛋白 (无自由基) 中,捕获在77 k的混合价位点产生轴向S = 1/2 EPR光谱,g值在1.79-1.94范围内,可在高达110 K的温度下观察到。光谱分配给mu-氧桥双核铁位点。在小鼠metR2中,主要EPR光谱是两种成分的混合物。将R2样品退火至160-170 K将初级EPR信号转换为菱形光谱,其特征在于gav <1.8,并且仅在25 k以下可观察到。这些光谱被分配为具有 μ-羟基桥的部分松弛形式,该形式是由氧桥的质子化形成的。在220 K下进一步退火产生新的菱形EPR光谱,该光谱与观察到的光谱非常相似,并且在室温下化学还原后被发现是稳定的。具有酪氨酰自由基的活性小鼠R2蛋白中初级混合价铁位点的EPR信号也有两个成分。两者都不同于在metr2中观察到的。在1型单纯疱疹病毒蛋白R2中,观察到met蛋白的一种主要混合价成分。活性小鼠R2中混合价态的剂量-产量曲线呈s形,表明酪氨酰自由基在铁位点之前被移动电子还原。在没有自由基的情况下,连二亚硫酸盐在小鼠R2上还原的动力学实验表明,没有自由基的蛋白质中铁位的还原速率显着提高。结果表明,在活性小鼠R2中,只有具有相邻自由基的完整二价位点会产生混合价光谱,并且这些位点可能以两种结构上不同的形式存在。小鼠R2蛋白的结果证实并扩展了先前在大肠杆菌蛋白R2上获得的结果,表明酪氨酰自由基的存在不仅显着影响铁位点的结构,而且还影响铁位点的反应性。

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