Reiter Tiffany A, Rusnak Frank
Section of Hematology Research and the Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Biochemistry. 2004 Jan 27;43(3):782-90. doi: 10.1021/bi0356956.
Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large superfamily of metallophosphoesterases, including serine/threonine protein phosphatases, purple acid phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. Members of this family share several common characteristics, including a common phosphoesterase motif, secondary structural fold (betaalphabetaalphabeta), and metal ligand environment, and often accommodate a dinuclear metal center. The identity of the active site metals often differs between family members. Despite the extensive spectroscopic studies of several family members, only the standard redox potential of porcine purple acid phosphate (PAP) has been measured. In this report, we investigate the redox properties of another member of this protein family. The standard redox potentials of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of lambdaPP were determined from anaerobic redox titration experiments. Two different S = 5/2, mono-Fe3+ lambdaPP species were identified: the first with an E/D approximately 0.17, g = 8.9 and 4.8, and an Eo' approximately +130 mV; the second with E/D approximately 0.05, g = 6.7, 5.9, and 4.4, and an Eo' approximately +120 mV. The first and second mono-Fe3+ species are thought to represent Fe present in the M2 and M1 sites, respectively. The addition of Zn2+ to mono-Fe3+ lambdaPP results in a decrease in both mono-Fe3+ species and the appearance of a new S = 5/2, Fe(3+)-Zn2+ species (E/D approximately 0.02, g = 5.9, and an Eo' > +175 mV). The Fe-Fe lambdaPP titration revealed an S = 1/2, Fe(3+)-Fe2+ (g < 2) species with an Eo' > +128 mV. These results suggest that the active site of lambdaPP supports a high oxidation potential for both metal sites and may indicate an equally oxidizing active site for other member metallophosphoesterases.
噬菌体λ蛋白磷酸酶（λPP）是金属磷酸酯酶大家族的一员，该家族包括丝氨酸/苏氨酸蛋白磷酸酶、紫色酸性磷酸酶、5'-核苷酸酶以及诸如Mre11等DNA修复酶。这个家族的成员具有几个共同特征，包括一个共同的磷酸酯酶基序、二级结构折叠（β-α-β-α-β）以及金属配体环境，并且通常容纳一个双核金属中心。活性位点金属的身份在家族成员之间常常有所不同。尽管对几个家族成员进行了广泛的光谱研究，但仅测量了猪紫色酸性磷酸酶（PAP）的标准氧化还原电位。在本报告中，我们研究了这个蛋白质家族的另一个成员的氧化还原性质。通过厌氧氧化还原滴定实验确定了λPP的单铁、铁-锌和铁-铁金属异构体的标准氧化还原电位。鉴定出了两种不同的S = 5/2的单铁³⁺λPP物种：第一种E/D约为0.17，g = 8.9和4.8，Eo'约为+130 mV；第二种E/D约为0.05，g = 6.7、5.9和4.4，Eo'约为+120 mV。第一种和第二种单铁³⁺物种被认为分别代表存在于M2和M1位点的铁。向单铁³⁺λPP中添加Zn²⁺会导致两种单铁³⁺物种减少，并出现一种新的S = 5/2的铁（³⁺）-锌²⁺物种（E/D约为0.02，g = 5.9，Eo' > +175 mV）。铁-铁λPP滴定揭示了一种S = 1/2的铁（³⁺）-铁²⁺（g < 2）物种，其Eo' > +128 mV。这些结果表明，λPP的活性位点对两个金属位点都支持高氧化电位，并且可能表明其他成员金属磷酸酯酶的活性位点同样具有氧化性。
