An engineered metalloprotein as a functional and structural bioinorganic model system.

Despite the rapid advances which have occurred in structural biology and computational chemistry, the rational design of functional protein structures continues to be one of the most challenging objectives in the field of protein engineering involving synthetic model compounds and de novo peptides. The engineering of active sites is more complicated for metalloenzymes than for non-metalloenzymes because individual metal coordination geometries and oxidation states must be taken into account to ensure the desired function. Although elegant de novo designs of heme and non-heme proteins with symmetrical coordination centers have been reported recently, metalloenzymes with asymmetrical coordination centers have rarely been mimicked, even through the use of model compounds. Lu and co-workers recently described the rational design of a model metalloprotein containing a sophisticated metal center with the structural and functional properties of a native metalloenzyme. The use of a metalloprotein as a molecular template for mechanistic investigations of reactions catalyzed by other metalloenzymes is expected to be a powerful approach in the field of bioinorganic chemistry. The target protein of this research was nitric oxide reductase (NOR), which promotes the two-electron reduction of NO to N2O in a bacterial denitrification system. [6] Despite the lack of a three-dimensional structure, spectroscopic and genetic investigations of native NORs have shown that, structurally, NOR is most closely related to heme–copper oxidases (HCOs). One exception is that NOR contains a unique Fe(His)3 non-heme iron active site instead of the common Cu(His)3 center of the HCOs. Previous genetic investigations of NOR indicated the existence at the active center of an invariant glutamate residue, which is not conserved in the HCO active centers. This invariant glutamate residue is crucial for enzymatic activity. Model complexes of NOR have been synthesized; however, the role of the glutamate has not yet been proven, as a more complicated design is needed for the construction of the asymmetric coordination sphere. Thus, the modeling of NOR is much more challenging than the preparation of active-site models of typical heme and nonheme metal active centers. To address the problem, Lu and co-workers used myoglobin (Mb), a dioxygen-carrier heme protein, in a semisynthetic approach. The distal histidine residue (His64) of Mb plays a role in the capture of a dioxygen molecule by providing a hydrogenbonding interaction. His64 was identified as a candidate for a ligand in a model of a non-heme iron center. A comparison of the structures of Mb and cytochrome c oxidase (an HOC) suggested that two residues adjacent to His64 (Leu29 and Phe43) could be replaced with histidine residues to construct a Fe(His)3 center (Figure 1). On the basis of this minimized structure, a glutamate residue was introduced at position 68. The triple mutant L29H/F43H/V68E Mb (FeBMb) was prepared as an NORmodel scaffold. Iron(II)-titration experiments and the high-resolution crystal structure of FeBMb containing an Fe ion (Fe-FeBMb) indicated the likelihood that the conserved glutamate residue helps to stabilize the binding of iron at the active center through monodentate ligation (Figure 2a). Interestingly, the EPR-spectral and electrochemical properties of Fe-FeBMb are almost identical to those of native NOR. The activity of Fe-FeBMb for the reduction of NO was confirmed by gas chromatography/mass spectrometry, although the amount of N2O formed was very low. The enzymatic mechanism was investigated in detail by comparison with other Mb composites, such as wild-type and Glu-deleted FeBMb. Finally, Lu and co-workers concluded that the glutamate residue and the three histidine residues are essential both for iron binding and for NO-reduction activity.