A novel small metal-binding protein (SmbP), with only 93 residues and no similarity to other known proteins, has been isolated from the periplasm of Nitrosomonas europaea. It is characterized by its high percentage (17%) of histidines, a motif of ten repeats of seven residues, a four α-helix bundle structure, and a high binding affinity to about six equivalents of Cu2+. The goal of this study is to investigate the Cu2+ binding sites in SmbP and to understand how Cu2+ stabilizes the protein. Preliminary folding experiments indicated that Cu2+ greatly stabilizes SmbP. In this study, protein folding data from circular dichroism (CD) spectroscopy was used to elucidate the role of Cu2+ in stabilizing SmbP structure against unfolding induced by decreased pH, increased temperature, and chemical denaturants. The significant stabilization effects of Cu2+ were demonstrated by the observation that Cu2+-SmbP remained fully folded under extreme environmental conditions, such as acidic pH, 96 °C, and 8 M urea. Also, it was shown that Cu2+ is able to induce the refolding of unfolded SmbP in acidic solutions. These findings imply that the coordination of Cu2+ to histidine residues is responsible for the stabilization effects. The crystal structure of SmbP without Cu2+ has been determined. However, attempts to crystallize Cu2+-SmbP have not been successful. In this study, multidimensional NMR experiments were conducted in order to gain additional information regarding the Cu2+-SmbP structure, in particular its metal binding sites. Unambiguous resonance assignments were successfully made. Cα secondary chemical shifts confirmed that SmbP has a four α-helical structure. A Cu2+-protein titration experiment monitored by NMR indicated a top-to-bottom, sequential metal binding pattern for SmbP. In addition, several bioinformatics tools were used to complement the experimental approach and identity of the ligands in Cu2+-binding sites in SmbP is proposed.

The metalloenzyme quercetin 2,3-dioxygenase (QueD) catalyzes the oxidative decomposition of the aromatic compound, quercetin. The most recently characterized example is a product of the bacterium Bacillus subtilis (BsQueD); all previous examples were fungal enzymes from the genus Aspergillus (AQueD). AQueD contains a single atom of Cu(II) per monomer. However, BsQueD, over expressed in Escherichia coli, contains Mn(II) and has two metal-binding sites, and therefore two possible active sites per monomer. To understand the contribution of each site to BsQueD's activity, the N-terminal and C-terminal metal-binding sites have been mutated individually in an effort to disrupt metal binding. In wild type BsQueD, each Mn(II) is ligated by three histidines (His) and one glutamate (Glu). All efforts to mutate His residues to non-ligating residues resulted in insoluble protein or completely inactive enzyme. A soluble mutant was expressed that replaced the Glu residue with a fourth His at the N-terminal domain. This mutant (E69H) has a specific activity of 0.00572 &mumol;/min/mg, which is nearly 3000-fold lower than the rate of wild type BsQueD (15.9 &mumol;/min/mg). Further analysis of E69H by inductively couple plasma mass spectrometry revealed that this mutant contains only 0.062 mol of Mn(II) per mol of enzyme. This is evidence that disabling metal-ligation at one domain influences metal-incorporation at the other. During the course of the mutagenic study, a second, faster purification method was developed. A hexahistidine tag and an enterokinase cleavage site were fused to the N-terminus of BsQueD (6xHis-BsQueD). Active enzyme was successfully expressed and purified with a nickel column in 3 hours. This is much faster than the previous multi-column purification, which took two full days to complete. However, the concentration of soluble, purified enzyme (1.8 mg/mL) was much lower than concentrations achieved with the traditional method (30 mg/mL). While the concentration of 6xHis-BsQueD is sufficient for some analyses, there are several characterization techniques that must be conducted at higher concentrations. Therefore, it will be advantageous to continue using both purification methods in the future.