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Efficiently carrying out the oxygen reduction reaction (ORR) is critical for many applications in biology and chemistry, such as bioenergetics and fuel cells, respectively. In biology, this reaction is carried out by large, transmembrane oxidases such as heme-copper oxidases (HCOs) and cytochrome bd oxidases. Common to these oxidases is the presence of a glutamate residue next to the active site, but its precise role in regulating the oxidase activity remains unclear. To gain insight into its role, we herein report that incorporation of glutamate next to a designed heme-copper center in two biosynthetic models of HCOs improves O 2 binding affinity, facilitates protonation of reaction intermediates, and eliminates release of reactive oxygen species. High-resolution crystal structures of the models revealed extended, water-mediated hydrogen-bonding networks involving the glutamate. Electron paramagnetic resonance of the cryoreduced oxy-ferrous centers at cryogenic temperature followed by thermal annealing allowed observation of the key hydroperoxo intermediate that can be attributed to the hydrogen-bonding network. By demonstrating these important roles of glutamate in oxygen reduction biochemistry, this work offers deeper insights into its role in native oxidases, which may guide the design of more efficient artificial ORR enzymes or catalysts for applications such as fuel cells.

Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. We report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved-through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites-to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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 O 2 to H 2 O, 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 O 2 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.

Type 1 copper (T1Cu) proteins are electron transfer (ET) proteins involved in many important biological processes. While the effects of changing primary and secondary coordination spheres in the T1Cu ET function have been extensively studied, few report has explored the effect of the overall protein structural perturbation on active site configuration or reduction potential of the protein, even though the protein scaffold has been proposed to play a critical role in enforcing the entatic or "rack-induced" state for ET functions. We herein report circular permutation of azurin by linking the N- and C-termini and creating new termini in the loops between 1 st and 2 nd β strands or between 3 rd and 4 th β strands. Characterization by electronic absorption, electron paramagnetic spectroscopies, as well as crystallography and cyclic voltammetry revealed that, while the overall structure and the primary coordination sphere of the circular permutated azurins remain the same as those of native azurin, their reduction potentials increased by 18 and 124 mV over that of WTAz. Such increases in reduction potentials can be attributed to subtle differences in the hydrogen-bonding network in secondary coordination sphere around the T1Cu center., (© 2016 The Protein Society.)

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.

Heteronuclear metalloenzymes catalyze some of the most fundamentally interesting and practically useful reactions in nature. However, the presence of two or more metal ions in close proximity in these enzymes makes them more difficult to prepare and study than homonuclear metalloenzymes. To meet these challenges, heteronuclear metal centers have been designed into small and stable proteins with rigid scaffolds to understand how these heteronuclear centers are constructed and the mechanism of their function. This chapter describes methods for designing heterobinuclear metal centers in a protein scaffold by giving specific examples of a few heme-nonheme bimetallic centers engineered in myoglobin and cytochrome c peroxidase. We provide step-by-step procedures on how to choose the protein scaffold, design a heterobinuclear metal center in the protein scaffold computationally, incorporate metal ions into the protein, and characterize the resulting metalloproteins, both structurally and functionally. Finally, we discuss how an initial design can be further improved by rationally tuning its secondary coordination sphere, electron/proton transfer rates, and the substrate affinity., (© 2016 Elsevier Inc. All rights reserved.)

This Forum Article focuses on recent advances in structural and spectroscopic studies of biosynthetic models of nitric oxide reductases (NORs). NORs are complex metalloenzymes found in the denitrification pathway of Earth's nitrogen cycle where they catalyze the proton-dependent two-electron reduction of nitric oxide (NO) to nitrous oxide (N2O). While much progress has been made in biochemical and biophysical studies of native NORs and their variants, a clear mechanistic understanding of this important metalloenzyme related to its function is still elusive. We report herein UV-vis and nuclear resonance vibrational spectroscopy (NRVS) studies of mononitrosylated intermediates of the NOR reaction of a biosynthetic model. The ability to selectively substitute metals at either heme or nonheme metal sites allows the introduction of independent (57)Fe probe atoms at either site, as well as allowing the preparation of analogues of stable reaction intermediates by replacing either metal with a redox inactive metal. Together with previous structural and spectroscopic results, we summarize insights gained from studying these biosynthetic models toward understanding structural features responsible for the NOR activity and its mechanism. The outlook on NOR modeling is also discussed, with an emphasis on the design of models capable of catalytic turnovers designed based on close mimics of the secondary coordination sphere of native NORs.

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s(-1)) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s(-1)) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.

Cytochrome c Oxidase (CcO) 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 (E°) 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 E° in a functional model of CcO 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 E° from 95 ± 2 to 123 ± 3 mV. Additionally, replacing the native heme b in the CcO mimic with heme a analogs, diacetyl, monoformyl, and diformyl hemes, that posses electron-withdrawing groups, resulted in higher E° values of 175 ± 5, 210 ± 6, and 320 ± 10 mV, respectively. Furthermore, O2 consumption studies on these CcO mimics revealed a strong enhancement in O2 reduction rates with increasing heme E°. Such methods of tuning the heme E° through a combination of secondary sphere mutations and heme substitutions can be applied to tune E° of other heme proteins, allowing for comprehensive investigations of the relationship between E° and enzymatic activity.

Metalloenzymes are among the major targets of protein design and engineering efforts aimed at attaining novel and efficient catalysis for biochemical transformation and biomedical applications, due to the diversity of functions imparted by the metallo-cofactors along with the versatility of the protein environment. Naturally evolved protein scaffolds can often serve as robust foundations for sustaining artificial active sites constructed by rational design, directed evolution, or a combination of the two strategies. Accumulated knowledge of structure-function relationship and advancement of tools such as computational algorithms and unnatural amino acids incorporation all contribute to the design of better metalloenzymes with catalytic properties approaching the needs of practical applications., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A major barrier to understanding the mechanism of nitric oxide reductases (NORs) is the lack of a selective probe of NO binding to the nonheme FeB center. By replacing the heme in a biosynthetic model of NORs, which structurally and functionally mimics NORs, with isostructural ZnPP, the electronic structure and functional properties of the FeB nitrosyl complex was probed. This approach allowed observation of the first S=3/2 nonheme {FeNO}(7) complex in a protein-based model system of NOR. Detailed spectroscopic and computational studies show that the electronic state of the {FeNO}(7) complex is best described as a high spin ferrous iron (S=2) antiferromagnetically coupled to an NO radical (S=1/2) [Fe(2+)-NO(.)]. The radical nature of the FeB -bound NO would facilitate N-N bond formation by radical coupling with the heme-bound NO. This finding, therefore, supports the proposed trans mechanism of NO reduction by NORs., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)