An Engineered Glutamate in Biosynthetic Models of Heme-Copper Oxidases Drives Complete Product Selectivity by Tuning the Hydrogen-Bonding Network.

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 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.

A designed heme-[4Fe-4S] metalloenzyme catalyzes sulfite reduction like the native enzyme.

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 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.

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 O to HO, 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 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.

Effect of circular permutation on the structure and function of type 1 blue copper center in azurin.

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 and 2 β strands or between 3 and 4 β strands.

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).

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme.

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.

Recent advances in biosynthetic modeling of nitric oxide reductases and insights gained from nuclear resonance vibrational and other spectroscopic studies.

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.

Systematic tuning of heme redox potentials and its effects on O2 reduction rates in a designed oxidase in myoglobin.

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.

Metalloenzyme design and engineering through strategic modifications of native protein scaffolds.

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.

Spectroscopic and computational study of a nonheme iron nitrosyl center in a biosynthetic model of nitric oxide reductase.

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.

A designed functional metalloenzyme that reduces O2 to H2O with over one thousand turnovers.

[Image: see text] Rational design of functional enzymes with high turnovers is a significant challenge, especially those with complex active site and difficult reactions, such as in respiratory oxidases. Introducing 2 His and 1 Tyr into myoglobin resulted in designed enzymes that reduce O to HO with > 1000 turnovers and minimal release of reactive oxygen species. This also showed that presence and positioning of Tyr, not Cu, are critical for activity.