Metal Cofactor Research Articles

Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation.

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII(η6‐arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non‐biological ligand. Tandem mass spectrometry and 19F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein‐derived ligands. By introduction of ruthenium cofactors into a 4‐helical bundle, transfer hydrogenation catalysts were generated that displayed a 35‐fold rate increase when compared to the respective small molecule reaction in solution.

Mechanical coupling in the nitrogenase complex.

The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. We report a graph theory analysis of the mechanical coupling in the nitrogenase complex as a key step to understanding the dynamics of allosteric regulation of nitrogen reduction. This analysis shows that regions near the active sites undergo large-scale, large-amplitude correlated motions that enable communications within each half and between the two halves of the complex. Computational predictions of mechanically regions were validated against an analysis of the solution phase dynamics of the nitrogenase complex via hydrogen-deuterium exchange. These regions include the P-loops and the switch regions in the Fe proteins, the loop containing the residue β-188Ser adjacent to the P-cluster in the MoFe protein, and the residues near the protein-protein interface. In particular, it is found that: (i) within each Fe protein, the switch regions I and II are coupled to the [4Fe-4S] cluster; (ii) within each half of the complex, the switch regions I and II are coupled to the loop containing β-188Ser; (iii) between the two halves of the complex, the regions near the nucleotide binding pockets of the two Fe proteins (in particular the P-loops, located over 130 Å apart) are also mechanically coupled. Notably, we found that residues next to the P-cluster (in particular the loop containing β-188Ser) are important for communication between the two halves.

Co-delivery of imidazolium Zn(II)salen and Origanum Syriacum essential oil by shrimp chitosan nanoparticles for antimicrobial applications

This study reports preparation and physicochemical characterization of natural antimicrobials (Origanum Syriacum essential oil (OSEO), shrimp chitosan nanoparticles (CSNPs)) and new imidazolium ionic liquid-supported Zn(II)Salen. These antimicrobials were separately or co-encapsulated by CSNPs to fabricate novel antimicrobial nanoplatforms "NPFs" (OSEO-loaded CSNPs (NPF-1), Zn(II)Salen-loaded CSNPs (NPF-2), and Zn(II)Salen@OSEO-loaded CSNPs (NPF-3)). The finding of loading, encapsulation, and antimicrobial release studies confirm the suitability of CSNPs for nanoencapsulation of Zn(II)Salen and OSEO. All NPFs can significantly suppress the growth of microbial species with performances dependent upon the microbial strain and nanoplatform concentration. The susceptibility of microbes toward new antimicrobials was as follows; Gram-positive bacteria > Gram-negative bacteria > fungi. The amazing physicochemical features of new nanoplatforms and their bioactive ingredients (Zn(II)Salen, OSEO, and CSNPs) signify the importance of our designs for developing a new generation of nanopharmaceuticals supported both natural products and biogenic ionic metal cofactors, targeting the multidrug resistant (MDR) pathogens.

Proximity proteomics in a marine diatom reveals a putative cell surface-to-chloroplast iron trafficking pathway.

Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed; however, proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.

Design and engineering of artificial metalloproteins: from de novo metal coordination to catalysis

Abstract Metalloproteins are essential to sustain life. Natural evolution optimized them for intricate structural, regulatory and catalytic functions that cannot be fulfilled by either a protein or a metal ion alone. In order to understand this synergy and the complex design principles behind the natural systems, simpler mimics were engineered from the bottom up by installing de novo metal sites in either natural or fully designed, artificial protein scaffolds. This review focuses on key challenges associated with this approach. We discuss how proteins can be equipped with binding sites that provide an optimal coordination environment for a metal cofactor of choice, which can be a single metal ion or a complex multinuclear cluster. Furthermore, we highlight recent studies in which artificial metalloproteins were engineered towards new functions, including electron transfer and catalysis. In this context, the powerful combination of de novo protein design and directed evolution is emphasized for metalloenzyme development.

Geological Factors Impacted Cadmium Availability and use as an Alternative Cofactor for Zinc in the Carbon Fixation Pathways of Marine Diatoms

AbstractTransition metal cofactors are crucial for many biological processes. Despite being primarily considered to be toxic, the transition metal cadmium (Cd) was discovered to be a substitute cofactor for zinc (Zn) in photosynthetic carbon fixation pathways of marine diatoms. However, it is not known how conditions in the geosphere impacted Cd availability and its incorporation as an alternative metal cofactor for phytoplankton. We employed mineral chemistry network analysis to investigate which geochemical factors may have influenced the availability of Cd and Zn during the putative time period that the alternative Cd‐based pathway evolved. Our results show that Zn minerals are more chemically diverse than are Cd minerals, but Zn‐ and Cd‐containing minerals have similar network centrality values when specifically considering sulfur (S)‐containing species. Cadmium and Zn sulfides are the most common Cd‐ and Zn‐containing mineral species over the past 500 million years. In particular, the Cd and Zn sulfides, respectively greenockite and sphalerite, were highly abundant during this time period. Furthermore, S‐containing Cd and Zn minerals are commonly co‐located in geologic time, allowing them to be weathered and transported to the ocean in tandem, rather than from separate sources. We suggest that the simultaneous weathering of Cd and Zn sulfides allowed for Cd to be a bioavailable direct substitute for Zn in protein complexes during periods of Zn depletion. The biogeochemical cycles of Zn and Cd exemplify the importance of the coevolution of the geosphere and biosphere in shaping primary production in the modern ocean.

Construction of a whole-cell biohybrid catalyst using a Cp*Rh(III)-dithiophosphate complex as a precursor of a metal cofactor

A whole-cell biohybrid catalyst where a (pentamethylcyclopentadienyl)rhodium(III) (Cp*Rh(III)) complex was covalently incorporated into the cavity of nitrobindin (NB), a β-barrel protein, was prepared on an E. coli cell surface to produce isoquinolines via C(sp2)–H bond activation. In this whole-cell biohybrid system, the Cp*Rh(III)-dithiophosphate complex with latent catalytic activity was utilized as a precursor of the metal cofactor. Strong chelation of the dithiophosphate ligands protects the rhodium complex from being deactivated by abundant nucleophiles in cellular environments during conjugation of the cofactor with the protein scaffold. The whole-cell biohybrid catalyst was then activated upon addition of Ag+ ion to dissociate the dithiophosphate ligands and promoted cycloaddition of acetophenone oxime with diphenylacetylene. Furthermore, the activity of the Cp*Rh(III)-linked whole-cell biohybrid catalyst was enhanced 2.1-fold by introducing glutamate residues at positions adjacent to the Cp*Rh(III) cofactor. These results indicate that the use of the Cp*Rh(III)-dithiophosphate complex with switchable activity from a “latent” form to an “active” form provides a new strategy for generating whole-cell biohybrid catalysts.

SUPEROXIDE DISMUTASE FAMILY GENES IN WATERMELON AND THEIR RESPONSES TO DIFFERENT ABIOTIC STRESSES

<List> <ListItem><ItemContent> • A total of 8 <i>SOD</i> genes from watermelon were identified and bioinformatically analyzed. </ItemContent></ListItem> <ListItem><ItemContent> • The SOD proteins from watermelon and other different plant species can be classified into five groups consistent with their metal cofactors. </ItemContent></ListItem> <ListItem><ItemContent> • <i>ClSOD</i> genes exhibited distinctive tissue-specific and abiotic stress responsive expression patterns. </ItemContent></ListItem></List> Superoxide dismutase (SOD) is an important enzyme in the antioxidant system of plants and plays a vital role in stress responses by maintaining the dynamic balance of reactive oxygen species (ROS) concentrations. Genome-wide analysis of the <i>SOD</i> gene family in various plant species has been conducted but little is known about this gene family in watermelon (<i>Citrullus lanatus</i>). Here, eight <i>SOD</i> genes were identified in the watermelon genome and are designated <i>ClCSD1-5</i>, <i>ClFSD1-2</i> and <i>ClMSD</i> according to their metal cofactors. Phylogenetic analysis shows that SOD proteins from various plant species can be classified into five groups and members in the same group possess the same metal cofactor and similar subcellular localizations. Expression analysis of the <i>ClSOD</i> genes indicates that they had tissue-specific expression patterns with high expression in different tissues including the leaves, flowers and fruit. In addition, the expression of <i>ClSOD</i> genes differed appreciably under salinity, drought and abscisic acid (ABA) treatments, indicating that they may be involved in ROS scavenging under different abiotic stresses via an ABA-dependent signaling pathway. These results lay the foundation for elucidating the function of <i>ClSOD</i> genes in stress tolerance and fruit development in watermelon.

A conserved allosteric element controls specificity and activity of functionally divergent PP2C phosphatases from Bacillus subtilis

Reversible phosphorylation relies on highly regulated kinases and phosphatases that target specific substrates to control diverse cellular processes. Here, we address how protein phosphatase activity is directed to the correct substrates under the correct conditions. The serine/threonine phosphatase SpoIIE from Bacillus subtilis, a member of the widespread protein phosphatase 2C (PP2C) family of phosphatases, is activated by movement of a conserved α-helical element in the phosphatase domain to create the binding site for the metal cofactor. We hypothesized that this conformational switch could provide a general mechanism for control of diverse members of the PP2C family of phosphatases. The B. subtilis phosphatase RsbU responds to different signals, acts on a different substrates, and produces a more graded response than SpoIIE. Using an unbiased genetic screen, we isolated mutants in the α-helical switch region of RsbU that are constitutively active, indicating conservation of the switch mechanism. Using phosphatase activity assays with phosphoprotein substrates, we found that both phosphatases integrate substrate recognition with activating signals to control metal-cofactor binding and substrate dephosphorylation. This integrated control provides a mechanism for PP2C family of phosphatases to produce specific responses by acting on the correct substrates, under the appropriate conditions.

Biochemistry of aerobic biological methane oxidation.

Methanotrophic bacteria represent a potential route to methane utilization and mitigation of methane emissions. In the first step of their metabolic pathway, aerobic methanotrophs use methane monooxygenases (MMOs) to activate methane, oxidizing it to methanol. There are two types of MMOs: a particulate, membrane-bound enzyme (pMMO) and a soluble, cytoplasmic enzyme (sMMO). The two MMOs are completely unrelated, with different architectures, metal cofactors, and mechanisms. The more prevalent of the two, pMMO, is copper-dependent, but the identity of its copper active site remains unclear. By contrast, sMMO uses a diiron active site, the catalytic cycle of which is well understood. Here we review the current state of knowledge for both MMOs, with an emphasis on recent developments and emerging hypotheses. In addition, we discuss obstacles to developing expression systems, which are needed to address outstanding questions and to facilitate future protein engineering efforts.

In situ self-doped biomass-derived porous carbon as an excellent oxygen reduction electrocatalyst for fuel cells and metal–air batteries

The concept of metal cofactor was utilized in fabricating superior ORR electrocatalysts for fuel cells and metal–air batteries.

Switch-1 instability at the active site decouples ATP hydrolysis from force generation in myosin II.

Myosin active site elements (i.e., switch‐1) bind both ATP and a divalent metal to coordinate ATP hydrolysis. ATP hydrolysis at the active site is linked via allosteric communication to the actin polymer binding site and lever arm movement, thus coupling the free energy of ATP hydrolysis to force generation. How active site motifs are functionally linked to actin binding and the power stroke is still poorly understood. We hypothesize that destabilizing switch‐1 movement at the active site will negatively affect the tight coupling of the ATPase catalytic cycle to force production. Using a metal‐switch system, we tested the effect of interfering with switch‐1 coordination of the divalent metal cofactor on force generation. We found that while ATPase activity increased, motility was inhibited. Our results demonstrate that a single atom change that affects the switch‐1 interaction with the divalent metal directly affects actin binding and productive force generation. Even slight modification of the switch‐1 divalent metal coordination can decouple ATP hydrolysis from motility. Switch‐1 movement is therefore critical for both structural communication with the actin binding site, as well as coupling the energy of ATP hydrolysis to force generation.

Intrinsic electronic conductivity of individual atomically resolved amyloid crystals reveals micrometer-long hole hopping via tyrosines

Proteins are commonly known to transfer electrons over distances limited to a few nanometers. However, many biological processes require electron transport over far longer distances. For example, soil and sediment bacteria transport electrons, over hundreds of micrometers to even centimeters, via putative filamentous proteins rich in aromatic residues. However, measurements of true protein conductivity have been hampered by artifacts due to large contact resistances between proteins and electrodes. Using individual amyloid protein crystals with atomic-resolution structures as a model system, we perform contact-free measurements of intrinsic electronic conductivity using a four-electrode approach. We find hole transport through micrometer-long stacked tyrosines at physiologically relevant potentials. Notably, the transport rate through tyrosines (105 s-1) is comparable to cytochromes. Our studies therefore show that amyloid proteins can efficiently transport charges, under ordinary thermal conditions, without any need for redox-active metal cofactors, large driving force, or photosensitizers to generate a high oxidation state for charge injection. By measuring conductivity as a function of molecular length, voltage, and temperature, while eliminating the dominant contribution of contact resistances, we show that a multistep hopping mechanism (composed of multiple tunneling steps), not single-step tunneling, explains the measured conductivity. Combined experimental and computational studies reveal that proton-coupled electron transfer confers conductivity; both the energetics of the proton acceptor, a neighboring glutamine, and its proximity to tyrosine influence the hole transport rate through a proton rocking mechanism. Surprisingly, conductivity increases 200-fold upon cooling due to higher availability of the proton acceptor by increased hydrogen bonding.

DpgC-Catalyzed Peroxidation of 3,5-Dihydroxyphenylacetyl-CoA (DPA-CoA): Insights into the Spin-Forbidden Transition and Charge Transfer Mechanisms*.

Despite being a very strong oxidizing agent, most organic molecules are not oxidized in the presence of O2 at room temperature because O2 is a diradical whereas most organic molecules are closed-shell. Oxidation then requires a change in the spin state of the system, which is forbidden according to non-relativistic quantum theory. To overcome this limitation, oxygenases usually rely on metal or redox cofactors to catalyze the incorporation of, at least, one oxygen atom into an organic substrate. However, some oxygenases do not require any cofactor, and the detailed mechanism followed by these enzymes remains elusive. To fill this gap, here the mechanism for the enzymatic cofactor-independent oxidation of 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) is studied by combining multireference calculations on a model system with QM/MM calculations. Our results reveal that intersystem crossing takes place without requiring the previous protonation of molecular oxygen. The characterization of the electronic states reveals that electron transfer is concomitant with the triplet-singlet transition. The enzyme plays a passive role in promoting the intersystem crossing, although spontaneous reorganization of the water wire connecting the active site with the bulk presets the substrate for subsequent chemical transformations. The results show that the stabilization of the singlet radical-pair between dioxygen and enolate is enough to promote spin-forbidden reaction without the need for neither metal cofactors nor basic residues in the active site.

Computational analysis of the metal selectivity of matrix metalloproteinase 8.

Matrix metalloproteinase (MMP) is a class of metalloenzyme that cleaves peptide bonds in extracellular matrices. Their functions are important in both health and disease of animals. Here using quantum mechanics simulations of the MMP8 protein, the coordination chemistry of different metal cofactors is examined. Structural comparisons reveal that Jhan-Teller effects induced by Cu(II) coordination distorts the wild-type MMP8 active site corresponding to a significant reduction in activity observed in previous experiments. In addition, further analysis suggests that a histidine to glutamine mutation at residue number 197 can potentially allow the MMP8 protein to utilize Cu(II) in reactions. Simulations also demonstrates the requirement of a conformational change in the ligand before enzymatic cleavage. The insights provided here will assist future protein engineering efforts utilizing the MMP8 protein.

Structural and biochemical characteristics of two Staphylococcus epidermidis RNase J paralogs RNase J1 and RNase J2

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

Directed Evolution of a Cp*RhIII -Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification.

Directed evolution of Cp*RhIII‐linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high‐throughput screening (HTS) platform that involves an affinity purification step employing a starch‐agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP‐tagged biohybrid catalysts in a 96‐well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII‐linked NB(T98H/L100K/K127E) variant with a 4.9‐fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.

Ruthenacyclic carbamoyl mimics of the [Fe]-hydrogenase active site: Derivatisation at the 4-position of the pyridinyl ring

The ruthenacyclic carbamoyl complexes [RuBr(2-NHC(O)C5H3N-4-R)(CO)2(MeCN)] in which the 4-position of the pyridinyl ring is derivatized, have been synthesized. These are ruthenium-based structural mimics of the natural [Fe]-hydrogenase metal cofactor. The derivative with R = H reacts with phosphine to form a disubstituted product [RuBr(2-NHC(O)C5H4N)(CO)(PPh2SPh)2].

Selective Metal Ion Utilization Contributes to the Transformation of the Activity of Yeast Polymerase η from DNA Polymerization toward RNA Polymerization.

Polymerase eta (Polη) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Polη in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Polη proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here, we show that manganese triggers drastic changes in the activity of Polη. Kinetics experiments indicate that manganese increases the efficiency of ribonucleoside incorporation into RNA by ~400–2000-fold opposite undamaged DNA, and ~3000 and ~6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination is almost lost during DNA synthesis by Polη with manganese. Moreover, Polη shows strong preference for manganese during RNA synthesis even at a 25-fold excess magnesium concentration. Based on this, we suggest that a new regulatory mechanism, selective metal cofactor utilization, modulates the specificity of Polη helping it to perform distinct activities needed for its separate functions during replication and transcription.

Insights into the Superoxide Dismutase Gene Family and Its Roles in Dendrobium catenatum under Abiotic Stresses.

Dendrobium catenatum is a member of epiphytic orchids with extensive range of pharmacological properties and ornamental values. Superoxide dismutase (SOD), a key member of antioxidant system, plays a vital role in protecting plants against oxidative damage caused by various biotic and abiotic stresses. So far, little is known about the SOD gene family in D. catenatum. In this study, eight SOD genes, including four Cu/ZnSODs, three FeSODs and one MnSOD, were identified in D. catenatum genome. Phylogenetic analyses of SOD proteins in D. catenatum and several other species revealed that these SOD proteins can be assigned to three subfamilies based on their metal co-factors. Moreover, the similarities in conserved motifs and gene structures in the same subfamily corroborated their classification and inferred evolutionary relationships. There were many hormone and stress response elements in DcaSODs, of which light responsiveness elements was the largest group. All DcaSODs displayed tissue-specific expression patterns and exhibited abundant expression levels in flower and leaf. According to public RNA-seq data and qRT-PCR analysis showed that the almost DcaSODs, except for DcaFSD2, were highly expressed under cold and drought treatments. Under heat, light, and salt stresses, DcaCSD1, DcaCSD2, DcaCSD3 were always significantly up-regulated, which may play a vital role in coping with various stresses. The expression levels of DcaFSD1 and DcaFSD2 were promoted by high light, suggesting their important roles in light response. These findings provided valuable information for further research on DcaSODs in D. catenatum.