Desideri A, Cocco D, Calabrese L, Rotilio G
Biochim Biophys Acta. 1984 Mar 29;785(3):111-7. doi: 10.1016/0167-4838(84)90134-1.
Co(II) derivatives of Cu,Zn-superoxide dismutase having cobalt substituted for the copper (Co,Zn-superoxide dismutase and Co,Co-superoxide dismutase) were studied by optical and EPR spectroscopy. EPR and electronic absorption spectra of Co,Zn-superoxide dismutase are sensitive to solvent perturbation, and in particular to the presence of phosphate. This behaviour suggests that cobalt in Co,Zn-superoxide dismutase is open to solvent access, at variance with the Co(II) of the Cu,Co-superoxide dismutase, which is substituted for the Zn. Phosphate binding as monitored by optical titration is dependent on pH with an apparent pKa = 8.2. The absorption spectrum of Co,Zn-superoxide dismutase in water has three weak bands in the visible region (epsilon = 75 M-1 X cm-1 at 456 nm; epsilon = 90 M-1 X cm-1 at 520 nm; epsilon = 70 M-1 X cm-1 at 600 nm) and three bands in the near infrared region, at 790 nm (epsilon = 18 M-1 X cm-1), 916 nm (epsilon = 27 M-1 X cm-1) and 1045 nm (epsilon = 25 M-1 X cm-1). This spectrum is indicative of five-coordinate geometry. In the presence of phosphate, three bands are still present in the visible region but they have higher intensity (epsilon = 225 M-1 X cm-1 at 544 nm; epsilon = 315 M-1 X cm-1 at 575 nm; epsilon = 330 M-1 X cm-1 at 603 nm), whilst the lowest wavelength band in the near infrared region is at much lower energy, 1060 nm (epsilon = 44 M-1 X cm-1). The latter property suggests a tetrahedral coordination around the Co(II) centre. Addition of 1 equivalent of CN- gives rise to a stable Co(II) low-spin intermediate, which is characterized by an EPR spectrum with a highly rhombic line shape. Formation of this CN- complex was found to require more cyanide equivalents in the case of the phosphate adduct, suggesting that binding of phosphate may inhibit binding of other anions. Titration of the Co,Co-derivative with CN- provided evidence for magnetic interaction between the two metal centres. These results substantiate the contention that Co(II) can replace the copper of Cu,Zn-superoxide dismutase in a way that reproduces the properties of the native copper-binding site.
对用钴取代铜的铜锌超氧化物歧化酶的钴（II）衍生物（钴锌超氧化物歧化酶和钴钴超氧化物歧化酶）进行了光学光谱和电子顺磁共振光谱研究。钴锌超氧化物歧化酶的电子顺磁共振光谱和电子吸收光谱对溶剂扰动敏感，特别是对磷酸盐的存在敏感。这种行为表明，钴锌超氧化物歧化酶中的钴易于与溶剂接触，这与取代锌的铜钴超氧化物歧化酶中的钴（II）不同。通过光学滴定监测的磷酸盐结合取决于pH值，表观pKa = 8.2。钴锌超氧化物歧化酶在水中的吸收光谱在可见光区域有三个弱带（456 nm处ε = 75 M-1·cm-1；520 nm处ε = 90 M-1·cm-1；600 nm处ε = 70 M-1·cm-1），在近红外区域有三个带，分别在790 nm（ε = 18 M-1·cm-1）、916 nm（ε = 27 M-1·cm-1）和1045 nm（ε = 25 M-1·cm-1）。该光谱表明其为五配位几何结构。在存在磷酸盐的情况下，可见光区域仍有三个带，但强度更高（544 nm处ε = 225 M-1·cm-1；575 nm处ε = 315 M-1·cm-1；603 nm处ε = 330 M-1·cm-1），而近红外区域最低波长的带能量低得多，为1060 nm（ε = 44 M-1·cm-1）。后一特性表明钴（II）中心周围为四面体配位。加入1当量的CN-会产生一个稳定的钴（II）低自旋中间体，其特征是具有高度菱形线形的电子顺磁共振光谱。发现在磷酸盐加合物的情况下，形成这种CN-配合物需要更多当量的氰化物，这表明磷酸盐的结合可能会抑制其他阴离子的结合。用CN-滴定钴钴衍生物为两个金属中心之间的磁相互作用提供了证据。这些结果证实了这样的论点，即钴（II）可以以再现天然铜结合位点特性的方式取代铜锌超氧化物歧化酶中的铜。
