Copper-cobalt superoxide dismutase

Bovine copper-cobalt superoxide dismutase, activity, 45 152-153... 

Copper, silver and gold - The paramagnetism of Cu11 has limited the use of NMR for the direct study of copper complexes, but information is readily obtained from studies of ligand nuclei. For example, the broadening of the NMR signal by copper in copper-zinc and copper-cobalt superoxide dismutases (SODs) has been used to determine the distance between the copper and the proton on bound formate (Sette et al., 1992). Also, broadening of the formate 13C NMR resonance reveals information about the orientation of the formate. 

Fig. 6.2. Schematic structure of the active site of copper-zinc superoxide dismutase [72]. The zinc ion can be substituted, among others, by copper(II), cobalt(II) and nickel(II) ions.

Previous sections focused on the enzymatic determination of NO, N02 using copper, zinc superoxide dismutase (SODl), and NOs using nitrate reductase (NaR)-modified Pt electrodes. This is a costly, time-consuming, and tedious procedure and the enzymes are not very stable. In order to resolve these drawbacks, the highly stable metaUoporphyrin-modified electrode would be the preeminent choice for the determination of NO metaboHtes. Different metalloporphyrins including nickel, iron, cobalt, manganese, and some other metal complexes were previously used for the... 

The above system is similar to that built into the protein superoxide dismutase (Fig. 6.2). The zinc can be replaced by several metal ions. Cobalt(II) and nickel(II) are pertinent here. The cobalt-copper system has J 17 cm-1 with S = 1 ground state [25]. The electronic relaxation times for the two ions are expected to be either equal or similar. The H NMR spectrum is shown in Fig. 6.8A. The assignment has been performed through several steps. 

Fig. 9.10. (A) 200 MHz H NMR spectrum of copper-depleted, cobalt-substituted Cu,Zn superoxide dismutase (B) difference between the difference NOE spectra obtained by irradiating signals D and C, respectively. The spectra demonstrate that the NOE on E arises from D and not from C. The off-resonance position was placed symmetrical with respect to E in both cases [41].

Sette, M., Paci, M., Desideri, A. and Rotillo, G. (1992) Formate as an NMR probe of anion binding to copper-zinc and copper-cobalt bovine erythrocyte superoxide dismutase. Biochemistry, 31, 2410-2415. 

Cobalt has recently been used as an ESR active substitute in zinc metalloenzymes. Whilst liquid helium temperatures may be needed and theoretical aspects of the spectra are not yet as well understood, cobalt has two important advantages over copper as a metal substitute, namely that many cobalt derivatives show some enzymic activity (e.g. cobalt in carbonic anhydrase, alkaline phosphatase and superoxide dismutase) and that g values and hyperfine splitting are more sensitive to ligand environment, particularly when low spin. ESR data have been reported for cobalt substituted thermolysin, carboxypeptidase A, procarboxypeptidase A and alkaline phosphatase [51]. These are all high spin complexes. Cobalt carbonic anhydrase has been prepared and reacted with cyanide [52]. In... 

It has been concluded from a study of the optical and e.p.r. spectra of Co —Cu bovine superoxide dismutase, in which zinc has been replaced by cobalt, that the cobalt site reactivity should be described in terms of reaction of the Co-imidazolate-Cu system as a whole the crystal structure reported last year indicated that the metals were linked by a common histidine residue. There is an exchange interaction between the cobalt and copper however, this is abolished when the linking imidazole is protonated. Further evidence for the close proximity and interactive dependence of the zinc and copper binding sites was obtained from a study of the 4 Cu protein a two-fold enhancement of the activity of 2 Cu dismutase was observed upon occupation of the zinc sites by the Cu ". On the basis of C1 n.m.r. studies, Fee and Ward have suggested that one co-ordination position of Cu in superoxide dismutase is normally occupied by water they further suggest that superoxide can displace the solvent to form a cupric peroxide complex. 

The kinetics of formation of the intermediate complex between catalase and HgOa have been re-examined, and reports of pulse radiolysis experiments with superoxide dismutase and its manganese-containing form have appeared. Bovine superoxide dismutase is known to contain two Cu and two Zn atoms per molecule and it has been shown that the copper site, which is directly involved in the catalytic activity, comprises three nitrogens and a water molecule bound to the metal in a field with less than axial symmetry. Less is known of the zinc site but it has recently been shown, by e.p.r. spectroscopy with the cobalt-copper form of the enzyme, that the... 

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