Spectroscopic and Crystallographic Evidence for the Role of a Water-Containing H‑Bond Network in Oxidase Activity of an Engineered Myoglobin

Heme-copper oxidases (HCOs) catalyze efficient reduction of oxygen to water in biological respiration. Despite progress in studying native enzymes and their models, the roles of non-covalent interactions in promoting this activity are still not well understood. Here we report EPR spectroscopic studies of cryo­reduced oxy-F33Y-Cu_BMb, a functional model of HCOs engineered in myoglobin (Mb). We find that cryo­reduction at 77 K of the O₂-bound form, trapped in the conformation of the parent oxy­ferrous form, displays a ferric-hydro­peroxo EPR signal, in contrast to the cryo­reduced oxy-wild-type (WT) Mb, which is unable to deliver a proton and shows a signal from the peroxo-ferric state. Crystallography of oxy-F33Y-Cu_BMb reveals an extensive H-bond network involving H₂O molecules, which is absent from oxy-WTMb. This H-bonding proton-delivery network is the key structural feature that transforms the reversible oxygen-binding protein, WTMb, into F33Y-Cu_BMb, an oxygen-activating enzyme that reduces O₂ to H₂O. These results provide direct evidence of the importance of H-bond networks involving H₂O in conferring enzymatic activity to a designed protein. Incorporating such extended H-bond networks in designing other metallo­enzymes may allow us to confer and fine-tune their enzymatic activities

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

23 mars 19041904/03/23 (A5,N1258)

Manganese and Cobalt in the Nonheme-Metal-Binding Site of a Biosynthetic Model of Heme-Copper Oxidase Superfamily Confer Oxidase Activity through Redox-Inactive Mechanism

The presence of a nonheme metal, such as copper and iron, in the heme-copper oxidase (HCO) superfamily is critical to the enzymatic activity of reducing O₂ to H₂O, but the exact mechanism the nonheme metal ion uses to confer and fine-tune the activity remains to be understood. We herein report that manganese and cobalt can bind to the same nonheme site and confer HCO activity in a heme–nonheme biosynthetic model in myoglobin. While the initial rates of O₂ reduction by the Mn, Fe, and Co derivatives are similar, the percentages of reactive oxygen species (ROS) formation are 7%, 4%, and 1% and the total turnovers are 5.1 ± 1.1, 13.4 ± 0.7, and 82.5 ± 2.5, respectively. These results correlate with the trends of nonheme-metal-binding dissociation constants (35, 22, and 9 μM) closely, suggesting that tighter metal binding can prevent ROS release from the active site, lessen damage to the protein, and produce higher total turnover numbers. Detailed spectroscopic, electrochemical, and computational studies found no evidence of redox cycling of manganese or cobalt in the enzymatic reactions and suggest that structural and electronic effects related to the presence of different nonheme metals lead to the observed differences in reactivity. This study of the roles of nonheme metal ions beyond the Cu and Fe found in native enzymes has provided deeper insights into nature’s choice of metal ion and reaction mechanism and allows for finer control of the enzymatic activity, which is a basis for the design of efficient catalysts for the oxygen reduction reaction in fuel cells