Davydov R M, Davydov A, Ingemarson R, Thelander L, Ehrenberg A, Gräslund A
Department of Biophysics, Stockholm University, Arrhenius Laboratory, Sweden.
Biochemistry. 1997 Jul 29;36(30):9093-100. doi: 10.1021/bi9700375.
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.
通过γ辐射产生的移动电子在77 K下于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，会产生处于混合价态Fe(II)/Fe(III)的EPR可检测铁位点。主要的EPR信号提供了有关铁位点二价铁形式的配体排列信息，而样品退火后观察到的次要信号则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，77 K捕获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，在高达110 K的温度下可观察到。这些光谱被归属于μ-氧桥联双核铁位点。在小鼠metR2中，主要的EPR光谱是两种成分的混合物。将R2样品退火至160 - 170 K会将主要的EPR信号转变为菱形光谱，其特征为gav < 1.8，且仅在25 K以下可观察到。这些光谱被归属于由氧桥质子化形成的具有μ-羟基桥的部分弛豫形式。在220 K进一步退火会产生新的菱形EPR光谱，与在室温下化学还原后观察到并发现稳定的光谱非常相似。具有酪氨酰自由基的活性小鼠R2蛋白中主要混合价铁位点的EPR信号也有两个成分。两者都与在metR2中观察到的不同。在1型单纯疱疹病毒蛋白R2中，met蛋白观察到一个主要的混合价成分。活性小鼠R2中混合价态的剂量 - 产率曲线呈S形，表明酪氨酰自由基在铁位点之前被移动电子还原。对不含和含自由基的小鼠R2用连二亚硫酸盐进行还原的动力学实验表明，不含自由基的蛋白质中铁位点的还原速率显著提高。结果表明，在活性小鼠R2中，只有与相邻自由基形成的完整二价铁位点会产生混合价光谱，并且这些位点可能以两种结构不同的形式存在。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著影响铁位点的结构，还影响其反应性。
