Machine Learning Guided Rational Design of a Non-Heme Iron-Based Lysine Dioxygenase Improves its Total Turnover Number.

Highly selective C-H functionalization remains an ongoing challenge in organic synthetic methodologies. Biocatalysts are robust tools for achieving these difficult chemical transformations. Biocatalyst engineering has often required directed evolution or structure-based rational design campaigns to improve their activities.

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Determination of substrate binding affinity (K) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the K of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis coupled with High Performance Liquid Chromatography (HPLC) for subsequent detection.

Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells.

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O) concentrations. Here, we have reprogrammed cellular hypoxia (low O) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O to PHD2's catalytic core.

Machine learning guided rational design of a non-heme iron-based lysine dioxygenase improves its total turnover number.

Highly selective C-H functionalization remains an ongoing challenge in organic synthetic methodologies. Biocatalysts are robust tools for achieving these difficult chemical transformations. Biocatalyst engineering has often required directed evolution or structure-based rational design campaigns to improve their activities.

Controllable multi-halogenation of a non-native substrate by SyrB2 iron halogenase.

Geminal, multi-halogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multi-halogenation of unactivated C-H bonds. Biocatalysts are powerful tools for late-stage C-H functionalization, as they operate with high degrees of regio-, chemo-, and stereoselectivity. 2-oxoglutarate (2OG)-dependent non-heme iron halogenases chlorinate and brominate aliphatic C-H bonds offering a solution for achieving these challenging transformations.

Oxygen affinities of DosT and DosS sensor kinases with implications for hypoxia adaptation in Mycobacterium tuberculosis.

DosT and DosS are heme-based kinases involved in sensing and signaling O tension in the microenvironment of Mycobacterium tuberculosis (Mtb). Under conditions of low O, they activate >50 dormancy-related genes and play a pivotal role in the induction of dormancy and associated drug resistance during tuberculosis infection. In this work, we reexamine the O binding affinities of DosT and DosS to show that their equilibrium dissociation constants are 3.

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Determination of substrate binding affinity () is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis with subsequent detection with High Performance Liquid Chromatography (HPLC).

Oxygen affinities of DosT and DosS sensor kinases with implications for hypoxia adaptation in .

DosT and DosS are heme-based kinases involved in sensing and signaling O tension in the microenvironment of (). Under conditions of low O, they activate >50 dormancy-related genes and play a pivotal role in the induction of dormancy and associated drug resistance during tuberculosis infection. In this work, we reexamine the O binding affinities of DosT and DosS to show that their equilibrium dissociation constants are 3.

Understanding ATP Binding to DosS Catalytic Domain with a Short ATP-Lid.

DosS is a heme-containing histidine kinase that triggers dormancy transformation in. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases reveals a short ATP-lid. This feature has been thought to block binding of ATP to DosS's CA domain in the absence of interactions with DosS's dimerization and histidine phospho-transfer (DHp) domain.

Gas tunnel engineering of prolyl hydroxylase reprograms hypoxia signaling in cells.

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O) concentrations. Here, we have reprogrammed cellular hypoxia (low O) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O to PHD2's catalytic core.

Understanding ATP binding to DosS catalytic domain with a short ATP-lid.

DosS is a heme-sensor histidine kinase that responds to redox-active stimuli in mycobacterial environments by triggering dormancy transformation. Sequence comparison of the catalytic ATP-binding (CA) domain of DosS to other well-studied histidine kinases suggests that it possesses a rather short ATP-lid. This feature has been thought to inhibit DosS kinase activity by blocking ATP binding in the absence of interdomain interactions with the dimerization and histidine phospho-transfer (DHp) domain of full-length DosS.

Metalloprotein enabled redox signal transduction in microbes.

Microbes utilize numerous metal cofactor-containing proteins to recognize and respond to constantly fluctuating redox stresses in their environment. Gaining an understanding of how these metalloproteins sense redox events, and how they communicate such information downstream to DNA to modulate microbial metabolism, is a topic of great interest to both chemists and biologists. In this article, we review recently characterized examples of metalloprotein sensors, focusing on the coordination and oxidation state of the metals involved, how these metals are able to recognize redox stimuli, and how the signal is transmitted beyond the metal center.

Electrostatically regulated active site assembly governs reactivity in non-heme iron halogenases.

Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride/bromide ion at an unactivated carbon position using a high-valent haloferryl intermediate. Despite more than a decade of structural and mechanistic characterization, how NHFe-Hals preferentially bind specific anions and substrates for C-H functionalization remains unknown. Herein, using lysine halogenating BesD and HalB enzymes as model systems, we demonstrate strong positive cooperativity between anion and substrate binding to the catalytic pocket.

Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins.

Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (K) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of K of heme-containing oxygen sensors.

An enzymatic method for precise oxygen affinity measurements over nanomolar-to-millimolar concentration regime.

Oxygen affinity is an important property of metalloproteins that helps elucidate their reactivity profile and mechanism. Heretofore, oxygen affinity values were determined either using flash photolysis and polarography techniques that require expensive instrumentation, or using oxygen titration methods which are erroneous at low nanomolar and at high millimolar oxygen concentrations. Here, we describe an inexpensive, easy-to-setup, and a one-pot method for oxygen affinity measurements that uses the enzyme chlorite dismutase (Cld) as a precise in situ oxygen source.

Understanding and Modulating Metalloenzymes with Unnatural Amino Acids, Non-Native Metal Ions, and Non-Native Metallocofactors.

Metalloproteins set the gold standard for performing important functions, including catalyzing demanding reactions under mild conditions. Designing artificial metalloenzymes (ArMs) to catalyze abiological reactions has been a major endeavor for many years, but most ArM activities are far below those of native enzymes, making them unsuitable for most pratical applications. A critical step to advance the field is to fundamentally understand what it takes to not only confer but also fine-tune ArM activities so they match those of native enzymes.

The Periodic Table's Impact on Bioinorganic Chemistry and Biology's Selective Use of Metal Ions.

Despite the availability of a vast variety of metal ions in the periodic table, biology uses only a selective few metal ions. Most of the redox active metals used belong to the first row of transition metals in the periodic table and include Fe, Co, Ni, Mn and Cu. On the other hand, Ca, Zn and Mg are the most commonly used redox inactive metals in biology.

O Reduction by Biosynthetic Models of Cytochrome Oxidase: Insights into Role of Proton Transfer Residues from Perturbed Active Sites Models of CcO.

Myoglobin based biosynthetic models of perturbed cytochrome oxidase (CcO) active site are reconstituted, in situ, on electrodes where glutamate residues are systematically introduced in the distal site of the heme/Cu active site instead of a tyrosine residue. These biochemical electrodes show efficient 4e/4H reduction with turnover rates and numbers more than 10 M s and 10, respectively. The HO/DO isotope effects of these series of crystallographically characterized mutants bearing zero, one, and two glutamate residues near the heme Cu active site of these perturbed CcO mimics are 16, 4, and 2, respectively.

Heme redox potentials hold the key to reactivity differences between nitric oxide reductase and heme-copper oxidase.

Despite high structural homology between NO reductases (NORs) and heme-copper oxidases (HCOs), factors governing their reaction specificity remain to be understood. Using a myoglobin-based model of NOR (FeMb) and tuning its heme redox potentials (E°') to cover the native NOR range, through manipulating hydrogen bonding to the proximal histidine ligand and replacing heme with monoformyl (MF-) or diformyl (DF-) hemes, we herein demonstrate that the E°' holds the key to reactivity differences between NOR and HCO. Detailed electrochemical, kinetic, and vibrational spectroscopic studies, in tandem with density functional theory calculations, demonstrate a strong influence of heme E°' on NO reduction.

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.

Insights Into How Heme Reduction Potentials Modulate Enzymatic Activities of a Myoglobin-based Functional Oxidase.

Heme-copper oxidase (HCO) is a class of respiratory enzymes that use a heme-copper center to catalyze O reduction to H O. While heme reduction potential (E°') of different HCO types has been found to vary >500 mV, its impact on HCO activity remains poorly understood. Here, we use a set of myoglobin-based functional HCO models to investigate the mechanism by which heme E°' modulates oxidase activity.

Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases.

Haem-copper oxidase (HCO) catalyses the natural reduction of oxygen to water using a haem-copper centre. Despite decades of research on HCOs, the role of non-haem metal and the reason for nature's choice of copper over other metals such as iron remains unclear. Here, we use a biosynthetic model of HCO in myoglobin that selectively binds different non-haem metals to demonstrate 30-fold and 11-fold enhancements in the oxidase activity of Cu- and Fe-bound HCO mimics, respectively, as compared with Zn-bound mimics.

Using Biosynthetic Models of Heme-Copper Oxidase and Nitric Oxide Reductase in Myoglobin to Elucidate Structural Features Responsible for Enzymatic Activities.

In biology, a heme-Cu center in heme-copper oxidases (HCOs) is used to catalyze the four-electron reduction of oxygen to water, while a heme-nonheme diiron center in nitric oxide reductases (NORs) is employed to catalyze the two-electron reduction of nitric oxide to nitrous oxide. Although much progress has been made in biochemical and biophysical studies of HCOs and NORs, structural features responsible for similarities and differences within the two enzymatic systems remain to be understood. Here, we discuss the progress made in the design and characterization of myoglobin-based enzyme models of HCOs and NORs.

Design and engineering of artificial oxygen-activating metalloenzymes.

Many efforts are being made in the design and engineering of metalloenzymes with catalytic properties fulfilling the needs of practical applications. Progress in this field has recently been accelerated by advances in computational, molecular and structural biology. This review article focuses on the recent examples of oxygen-activating metalloenzymes, developed through the strategies of de novo design, miniaturization processes and protein redesign.

A biosynthetic model of cytochrome c oxidase as an electrocatalyst for oxygen reduction.

Creating an artificial functional mimic of the mitochondrial enzyme cytochrome c oxidase (CcO) has been a long-term goal of the scientific community as such a mimic will not only add to our fundamental understanding of how CcO works but may also pave the way for efficient electrocatalysts for oxygen reduction in hydrogen/oxygen fuel cells. Here we develop an electrocatalyst for reducing oxygen to water under ambient conditions. We use site-directed mutants of myoglobin, where both the distal Cu and the redox-active tyrosine residue present in CcO are modelled.