Whittaker J W
Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, OR 97006, U.S.A.
Biochem Soc Trans. 2003 Dec;31(Pt 6):1318-21. doi: 10.1042/bst0311318.
The manganese and iron SODs (superoxide dismutases) form a superfamily of closely related antioxidant defence metalloenzymes. MnSOD requires Mn (not Fe) for activity. However, when MnSOD is expressed in Escherichia coli grown in medium supplemented with ferrous salts, Fe substitutes for Mn in the active site, reflecting relatively indiscriminate uptake of either Mn or Fe and a surprisingly low selectivity for the identity of the bound metal ion. X-ray crystallographic studies on Fe-substituted MnSOD show that the substrate access channel is blocked by solvent (hydroxide), providing a structural explanation for the observed metal specificity of the catalytic activity. The mechanism of metal binding has been investigated in vitro using recombinant thermophilic SODs. The thermophilic Thermus thermophilus MnSOD expressed in E. coli was isolated as the metal-free apoprotein when heat treatment was eliminated from the purification procedure. While incubation of the purified MnSOD apoprotein with metal salts at ambient temperatures did not restore SOD activity, re-activation could be achieved by heating the protein with Mn salts at elevated temperatures. This in vitro thermally triggered metal uptake is non-specific for the metal ion; both Mn and Fe bind, but only Mn restores catalytic activity. Formation of the metal complex is essentially irreversible under these conditions. The metallation process is strongly temperature-dependent, suggesting that there are substantial activation barriers to metal uptake at ambient temperatures that are overcome by a transition in the apoprotein structure under physiological conditions. Two mechanisms may be proposed for SOD metallation: one involving subunit dissociation and another involving domain separation. Thermally triggered metal binding by thermophilic SODs is providing new insight into the metallation mechanism of the SOD apoprotein, which is likely to be conserved over this family of enzymes.
锰超氧化物歧化酶（SOD）和铁超氧化物歧化酶构成了一个由密切相关的抗氧化防御金属酶组成的超家族。锰超氧化物歧化酶的活性需要锰（而非铁）。然而，当锰超氧化物歧化酶在添加了亚铁盐的培养基中生长的大肠杆菌中表达时，铁会在活性位点取代锰，这反映出对锰或铁的摄取相对不加区分，并且对结合金属离子的种类选择性出奇地低。对铁取代的锰超氧化物歧化酶的X射线晶体学研究表明，底物进入通道被溶剂（氢氧化物）阻断，这为观察到的催化活性的金属特异性提供了结构解释。已使用重组嗜热超氧化物歧化酶在体外研究了金属结合机制。当从纯化过程中去除热处理步骤时，在大肠杆菌中表达的嗜热栖热菌锰超氧化物歧化酶被分离为无金属的脱辅基蛋白。虽然在室温下将纯化的锰超氧化物歧化酶脱辅基蛋白与金属盐一起孵育不能恢复超氧化物歧化酶活性，但通过在高温下将蛋白质与锰盐一起加热可以实现重新激活。这种体外热触发的金属摄取对金属离子不具有特异性；锰和铁都能结合，但只有锰能恢复催化活性。在这些条件下，金属复合物的形成基本上是不可逆的。金属化过程强烈依赖温度，这表明在室温下存在大量阻碍金属摄取的活化能垒，而在生理条件下脱辅基蛋白结构的转变可以克服这些能垒。对于超氧化物歧化酶的金属化可以提出两种机制：一种涉及亚基解离，另一种涉及结构域分离。嗜热超氧化物歧化酶的热触发金属结合为超氧化物歧化酶脱辅基蛋白的金属化机制提供了新的见解，这一机制可能在该酶家族中是保守的。
