An engineered two-iron superoxide reductase lacking the [Fe(SCys)(4)] site retains its catalytic properties in vitro and in vivo

Superoxide reductases (SORs) contain a characteristic square-pyramidal [Fe(NHis)(4)(SCys)] active site that catalyzes reduction of superoxide to hydrogen peroxide in several anaerobic bacteria and archaea. Some SORs, referred to as two-iron SORs (2Fe-SORs), also contain a lower-potential [Fe(SCys)(4)] site that is presumed to have an electron transfer function. However, the intra- and inter-subunit distances between [Fe(SCys)(4)] and [Fe(NHis)(4)(SCys)] iron centers within the 2Fe-SOR homodimer seem too long for efficient electron transfer between these sites. The possible role of the [Fe(SCys)(4)] site in 2Fe-SORs was addressed in this work by examination of an engineered Desulfovibrio vulgaris 2Fe-SOR variant, C13S, in which one ligand residue of the [Fe(SCys)(4)] site, cysteine 13, was changed to serine. This single amino acid residue change destroyed the native [Fe(SCys)(4)] site with complete loss of its iron, but left the [Fe(NHis)(4)(SCys)] site and the protein homodimer intact. The spectroscopic, redox and superoxide reactivity properties of the [Fe(NHis)(4)(SCys)] site in the C13S variant were nearly indistinguishable from those of the wild-type 2Fe-SOR. Aerobic growth complementation of a superoxide dismutase (SOD)-deficient Escherichia coli strain showed that the presence of the [Fe(NHis)(4)(SCys)] site in C13S 2Fe-SOR was apparently sufficient to catalyze reduction of the intracellular superoxide to nonlethal levels. As is the case for the wild-type protein, C13S 2Fe-SOR did not show any detectable SOD activity, i.e., destruction of the [Fe(SCys)(4)] site did not unmask latent SOD activity of the [Fe(NHis)(4)(SCys)] site. Possible alternative roles for the [Fe(SCys)(4)] site in 2Fe-SORs are considered

p28, A first in class peptide inhibitor of cop1 binding to p53

BACKGROUND: A 28 amino-acid (aa) cell-penetrating peptide (p28) derived from azurin, a redox protein secreted from the opportunistic pathogen Pseudomonas aeruginosa, produces a post-translational increase in p53 in cancer cells by inhibiting its ubiquitination. METHODS: In silico computational simulations were used to predict motifs within the p53 DNA-binding domain (DBD) as potential sites for p28 binding. In vitro direct and competitive pull-down studies as well as western blot and RT-PCR analyses were used to validate predictions. RESULTS: The L1 loop (aa 112–124), a region within the S7–S8 loop (aa 214–236) and T140, P142, Q144, W146, R282 and L289 of the p53DBD were identified as potential sites for p28 binding. p28 decreased the level of the E3 ligase COP1 >80%, in p53(wt) and p53(mut) cells with no decrease in COP1 in p53dom/neg or p53null cells. Brief increases in the expression of the E3 ligases, TOPORS, Pirh2 and HDM2 (human double minute 2) in p53(wt) and p53(mut) cells were in response to sustained increases in p53. CONCLUSION: These data identify the specific motifs within the DBD of p53 that bind p28 and suggest that p28 inhibition of COP1 binding results in the sustained, post-translational increase in p53 levels and subsequent inhibition of cancer cell growth independent of an HDM2 pathway