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-CuBMb, 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-CuBMb 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-CuBMb, 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

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

A designed functional metalloenzyme that reduces O 2 to H 2O with over one thousand turnovers

No spare Tyr: Rational design of functional enzymes with a high number of turnovers is a challenge, especially those with a complex active site, such as respiratory oxidases. Introducing two His and one Tyr residues into myoglobin resulted in enzymes that reduce O 2 to H 2O with more than 1000 turnovers (red line, see scheme) and minimal release of reactive oxygen species. The positioning of the Tyr residue is critical for activity. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Systematic Tuning of Heme Redox Potentials and Its Effects on O₂ Reduction Rates in a Designed Oxidase in Myoglobin

Cytochrome <i>c</i> Oxidase (C<i>c</i>O) is known to catalyze the reduction of O₂ to H₂O efficiently with a much lower overpotential than most other O₂ reduction catalysts. However, methods by which the enzyme fine-tunes the reduction potential (<i>E</i>°) of its active site and the corresponding influence on the O₂ reduction activity are not well understood. In this work, we report systematic tuning of the heme <i>E</i>° in a functional model of C<i>c</i>O in myoglobin containing three histidines and one tyrosine in the distal pocket of heme. By removing hydrogen-bonding interactions between Ser92 and the proximal His ligand and a heme propionate, and increasing hydrophobicity of the heme pocket through Ser92Ala mutation, we have increased the heme <i>E</i>° from 95 ± 2 to 123 ± 3 mV. Additionally, replacing the native heme <i>b</i> in the C<i>c</i>O mimic with heme <i>a</i> analogs, diacetyl, monoformyl, and diformyl hemes, that posses electron-withdrawing groups, resulted in higher <i>E</i>° values of 175 ± 5, 210 ± 6, and 320 ± 10 mV, respectively. Furthermore, O₂ consumption studies on these C<i>c</i>O mimics revealed a strong enhancement in O₂ reduction rates with increasing heme <i>E</i>°. Such methods of tuning the heme <i>E</i>° through a combination of secondary sphere mutations and heme substitutions can be applied to tune <i>E</i>° of other heme proteins, allowing for comprehensive investigations of the relationship between <i>E</i>° and enzymatic activity

Structure and Dynamics of CO₂ on Rutile TiO₂(110)-1×1

Adsorption, binding, and diffusion of CO₂ molecules on model rutile TiO₂(110)-1×1 surfaces were investigated experimentally using scanning tunneling microscopy, infrared reflection adsorption spectroscopy (IRAS), molecular beam scattering, and temperature programmed desorption and theoretically via dispersion corrected density functional theory and ab initio molecular dynamics. In accord with previous studies, bridging oxygen (O_b) vacancies (V_O’s) are found to be the most stable binding sites. Additional CO₂ adsorbs on 5-coordinated Ti sites (Ti_5c) with the initial small fraction stabilized by CO₂ adsorbed on V_O sites. The Ti_5c-bound CO₂ is found to be highly mobile at 50 K at coverages of up to 1/2 monolayer (ML). Theoretical studies show that the CO₂ diffusion on Ti_5c rows proceeds via a rotation-tumbling mechanism with extremely low barrier of 0.06 eV. The Ti_5c-bound CO₂ molecules are found to bind preferentially to a single Ti_5c with the OCO axis tilted away from the surface normal. The binding energy of tilted CO₂ molecules changes only slightly with changes in the azimuth of the CO₂ tilt angle. At 2/3 ML, CO₂ diffusion is hindered and at 1 ML an ordered (2×2) overlayer with a zigzag arrangement of tilted CO₂ molecules develops along the Ti_5c rows. Out of phase arrangement of the zigzag chains is observed across the rows. An additional 0.5 ML of CO₂ can be adsorbed at O_b sites with a binding energy only slightly lower than that on Ti_5c sites presumably due to quadrupole–quadrupole interactions with the Ti_5c-bound CO₂ molecules

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