Spectroscopic study of cobalt II substituted in some copper and zinc enzymes.

The substitution of the cobalt atom into the active site of zinc metalloenzymes is becoming a useful and common practice. The zinc atom, in fact, is magnetically and optically silent, whillst the cobalt has a characteristic optical and E.P.R. spectrum, so that it can be followed spectroscopically to obtain structural and functional information on the enzyme. In this thesis a spectroscopic study of the cobalt substituted into two metalloenzymes, namely copper-zinc-Superoxide Dismutase and Zinc- Carbonic Anhydrase, and of some low molecular weight cobalt complexes has been performed. In Superoxide Dismutase the cobalt has been selectively substituted either into the zinc or into the copper site, so that all the possible derivatives [Co-Cu], [Co-...], [Co-Co] and [Zn-Co], have been investigated. The [Co-..]SOD derivative is characterized by an axial E.P.R. spectrum and by an absorption spectrum in the visible region of intermediate intensity e530=315, e560=425, e588= 450 and a near infrared band at 1000 nm (e=90), indicating a tetrahedral coordination with a quite strong crystal field around the Co(II) centre. The [Co-Cu]SOD derivative does not show any E.P.R. signal and a magnetic susceptibility study, carried out between 30-210 K indicates that this is due to the strong antiferromagnetic coupling (2J ? 600 cm-1) occurring between the two metal centres. The [Zn-Co]SOD derivative shows an interesting phosphate buffer spectral dependence. In particular, the electronic spectrum, carried out in the presence of phosphate buffer, has three quite intense bands in the visible region (e540=225, e580=330, e605=330) and a band in the near infrared at 1050 nm (e=40). When the same spectrum is carried out in the absence of phosphate buffer the bands in the visible region are much less intense and the near infrared band is shifted toward lower wavelengths. This behaviour indicates a change in symmetry around the Co(II) centre, from tetrahedral to pentacoordinate in the presence and in the absence of phosphate respectively. The E.P.R. spectra also support this hypothesis. The reaction of cyanide and H2O2 with the [Zn-Co] and [Co-Co] SOD derivatives have also been investigated. The binding of CN- to the cobalt is temperature or freezing dependent. The E.P.R. spectra carried out at 77 K shows, in fact, that the CN- easily binds to the cobalt which is transformed into a low spin form, whilst the electronic room temperature spectra show that a very large CN-:Co ratio is needed to decrease the d-d bands of the unreacted high spin cobalt. The E.P.R. spectrum is rhombic with gz=2.027 and Az=115 x 10-4cm-1, suggesting a distorted pentacoordinate structure around the metal. The reaction with H2O2 shows a decrease of the electronic absorption spectrum of the cobalt and the appearance of a radical at g~2 in the E.P.R. spectrum. Oxidation of the Co (II) to Co (III) can probably be excluded because addition of sodium dithionite does not restore back the original spectrum. A comparative study of the K-absorption edge of several cobalt derivatives has allowed several structural correlations to be done. In particular, the [Co(II)-...] and the [Co(II)-Cu(I)]SOD derivatives have a completely identical K-edge spectrum, which is slightly different from that of the [Co(II)-Cu(II)] derivative, indicating that a conformational change and/or a different charge on the imidazole bridging the two metal sites occurs coincidentally with the change of copper valence. An E.P.R. study on low molecular weight model compounds suggests that the zero field splitting value ? between the two Kramers doublets in a high spin cobalt (II) ion can be used diagnostically to assign the geometry around the metal centre. The zero field splitting value is, in fact, greater in the pentacoordinate case than in the tetrahedral one. By comparison with the model compounds a pentacoordinate structure is assigned to the high pH and the iodide form of the cobalt carbonic anhydrase derivative. The study of the reaction of the native and the copper and cobalt substituted Carbonic Anhydrase shows that Cu(II) is easily extracted from the enzyme, Co(II) with some difficulty and Zn(II) is unaffected in any condition. Before the depletion, a stable pentacoordinate species and two stable and different intermediates in the case of cobalt and copper respectively are observed. A ?-irradiation investigation of the native Carbonic Anhydrase and Superoxide Dismutase has allowed detection of the E.P.R. signal of the Zn+ ion with a configuration 3d10 4s1. In both cases, the electron seems to be in an approximately sp hybrid orbital so explaining the lack of magnetic interaction in the native SOD. In fact, the O hybrid orbital of the zinc is in a wrong symmetry to couple with the ? electron system of the imidazole and with the ground state of the copper.