Redox-reversible siderophore-based catalyst anchoring within cross-linked artificial metalloenzyme aggregates enables enantioselectivity switching.

The immobilisation of artificial metalloenzymes (ArMs) holds promise for the implementation of new biocatalytic reactions. We present the synthesis of cross-linked artificial metalloenzyme aggregates (CLArMAs) with excellent recyclability, as an alternative to carrier-based immobilisation strategies. Furthermore, iron-siderophore supramolecular anchoring facilitates redox-triggered cofactor release, enabling CLArMAs to be recharged with alternative cofactors for diverse selectivity.

Lanmodulin's EF 2-3 Domain: Insights from Infrared Spectroscopy and Simulations.

Lanmodulins are small, ∼110-residue proteins with four EF-hand motifs that demonstrate a picomolar affinity for lanthanide ions, making them efficient in the recovery and separation of these technologically important metals. In this study, we examine the thermodynamic and structural complexities of lanthanide ion binding to a 41-residue domain, EF 2-3, that constitutes the two highest-affinity metal-binding sites in the lanmodulin protein from Methylorubrum extorquens. Using a combination of circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we characterize the metal binding capabilities of EF 2-3. ITC demonstrates that binding occurs between peptide and lanthanides with conditional dissociation constants (Kd) in the range 20-30 µM, with no significant differences in the Kd values for La3+, Eu3+, and Tb3+ at pH 7.4. In addition, CD spectroscopy suggests that only one binding site of EF 2-3 undergoes a significant conformational change in the presence of lanthanides. 2D IR spectroscopy demonstrates the presence of both mono- and bidentate binding configurations in EF 2-3 with all three lanthanides. MD simulations, supported by Eu3+ luminescence measurements, explore these results, suggesting a competition between water-lanthanide and carboxylate-lanthanide interactions in the EF 2-3 domain. These results underscore the role of the core helical bundle of the protein architecture in influencing binding affinities and communication between the metal-binding sites in the full-length protein.

A database overview of metal-coordination distances in metalloproteins.

Metalloproteins are ubiquitous in all living organisms and take part in a very wide range of biological processes. For this reason, their experimental characterization is crucial to obtain improved knowledge of their structure and biological functions. The three-dimensional structure represents highly relevant information since it provides insight into the interaction between the metal ion(s) and the protein fold. Such interactions determine the chemical reactivity of the bound metal. The available PDB structures can contain errors due to experimental factors such as poor resolution and radiation damage. A lack of use of distance restraints during the refinement and validation process also impacts the structure quality. Here, the aim was to obtain a thorough overview of the distribution of the distances between metal ions and their donor atoms through the statistical analysis of a data set based on more than 115 000 metal-binding sites in proteins. This analysis not only produced reference data that can be used by experimentalists to support the structure-determination process, for example as refinement restraints, but also resulted in an improved insight into how protein coordination occurs for different metals and the nature of their binding interactions. In particular, the features of carboxylate coordination were inspected, which is the only type of interaction that is commonly present for nearly all metals.

Integration of metalloproteome and immunoproteome reveals a tight link of iron-related proteins with COVID-19 pathogenesis and immunity.

Increasing clinical data show that the imbalance of host metallome is closely associated with different kinds of disease, however, the intrinsic mechanisms of action of metals in immunity and pathogenesis of disease remain largely undefined. There is lack of multiplexed profiling system to integrate the metalloproteome-immunoproteome information at systemic level for exploring the roles of metals in immunity and disease pathogenesis. In this study, we build up a metal-coding assisted multiplexed proteome assay platform for serum metalloproteomic and immunoproteomic profiling. By taking COVID-19 as a showcase, we unbiasedly uncovered the most evident modulation of iron-related proteins, i.e., Ft and Tf, in serum of severe COVID-19 patients, and the value of Ft/Tf could work as a robust biomarker for COVID-19 severity stratification, which overtakes the well-established clinical risk factors (cytokines). We further uncovered a tight association of transferrin with inflammation mediator IL-10 in COVID-19 patients, which was proved to be mainly governed by the monocyte/macrophage of liver, shedding light on new pathophysiological and immune regulatory mechanisms of COVID-19 disease. We finally validated the beneficial effects of iron chelators as anti-viral agents in SARS-CoV-2-infected K18-hACE2 mice through modulation of iron dyshomeostasis and alleviating inflammation response. Our findings highlight the critical role of liver-mediated iron dysregulation in COVID-19 disease severity, providing solid evidence on the involvement of iron-related proteins in COVID-19 pathophysiology and immunity.

Electron-Transferring Metalloenzymes and their Potential Biotechnological Applications.

Modern societies rely heavily on centralized industrial processes to generate a multitude of products ranging from electrical energy to synthetic chemical building blocks to construction materials. To date, these processes have relied extensively on energy produced from fossil fuels, which has led to dramatically increased quantities of greenhouse gases (including carbon dioxide) being released into the atmosphere; the effects of the ensuing change to our climate are easily observed in day-to-day life. Some of the reactions catalyzed by these industrial processes can be catalyzed in nature by metal-containing enzymes (metalloenzymes) that have evolved over the course of up to 3.8 billion years to do so under mild physiological conditions using Earth-abundant metals. While such metalloenzymes could in principle facilitate the implementation of carbon-neutral processes around the globe, either in "bio-inspired" catalyst design or even by direct exploitation, many remaining questions surrounding their mechanisms often preclude both options. Here, our recent efforts in understanding and applying metalloenzymes that catalyze reactions such as dinitrogen reduction to ammonia or proton reduction to molecular hydrogen are discussed. In closing, an opinion on the question: "Can these types of enzymes really be used in new biotechnologies?" is offered.

Flexibility of Binding Site is Essential to the Ca2+ Selectivity in EF-Hand Calcium-Binding Proteins.

High binding affinity and selectivity of metal ions are essential to the function of metalloproteins. Thus, understanding the factors that determine these binding characteristics is of major interest for both fundamental mechanistic investigations and guiding of the design of novel metalloproteins. In this work, we perform QM cluster model calculations and quantum mechanics/molecular mechanics (QM/MM) free energy simulations to understand the binding selectivity of Ca2+ and Mg2+ in the wild-type carp parvalbumin and its mutant. While a nonpolarizable MM model (CHARMM36) does not lead to the correct experimental trend, treatment of the metal binding site with the DFTB3 model in a QM/MM framework leads to relative binding free energies (ΔΔGbind) comparable with experimental data. For the wild-type (WT) protein, the calculated ΔΔGbind is ∼6.6 kcal/mol in comparison with the experimental value of 5.6 kcal/mol. The good agreement highlights the value of a QM description of the metal binding site and supports the role of electronic polarization and charge transfer to metal binding selectivity. For the D51A/E101D/F102W mutant, different binding site models lead to considerable variations in computed binding affinities. With a coordination number of seven for Ca2+, which is shown by QM/MM metadynamics simulations to be the dominant coordination number for the mutant, the calculated relative binding affinity is ∼4.8 kcal/mol, in fair agreement with the experimental value of 1.6 kcal/mol. The WT protein is observed to feature a flexible binding site that accommodates a range of coordination numbers for Ca2+, which is essential to the high binding selectivity for Ca2+ over Mg2+. In the mutant, the E101D mutation reduces the flexibility of the binding site and limits the dominant coordination number of Ca2+ to be seven, thereby leading to reduced binding selectivity against Mg2+. Our results highlight that the binding selectivity of metal ions depends on both the structural and dynamical properties of the protein binding site.

Discovery of Novel Metalloenzyme Inhibitors Based on Property Characterization: Strategy and Application for HDAC1 Inhibitors.

Metalloenzymes are ubiquitously present in the human body and are relevant to a variety of diseases. However, the development of metalloenzyme inhibitors is limited by low specificity and poor drug-likeness associated with metal-binding fragments (MBFs). A generalized drug discovery strategy was established, which is characterized by the property characterization of zinc-dependent metalloenzyme inhibitors (ZnMIs). Fifteen potential Zn2+-binding fragments (ZnBFs) were identified, and a customized pharmacophore feature was defined based on these ZnBFs. The customized feature was set as a required feature and applied to a search for novel inhibitors for histone deacetylase 1 (HDAC1). Ten potential HDAC1 inhibitors were recognized, and one of them (compound 9) was a known potent HDAC1 inhibitor. The results demonstrated the effectiveness of our strategy to identify novel inhibitors for zinc-dependent metalloenzymes.

Mononuclear binding and catalytic activity of europium(III) and gadolinium(III) at the active site of the model metalloenzyme phosphotriesterase.

Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61 Šresolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.

Multiscale modeling of unfolding and bond dissociation of rubredoxin metalloprotein.

Mechanical properties of proteins that have a crucial effect on their operation. This study used a molecular dynamics simulation package to investigate rubredoxin unfolding on the atomic scale. Different simulation techniques were applied, and due to the dissociation of covalent/hydrogen bonds, this protein demonstrates several intermediate states in force-extension behavior. A conceptual model based on the cohesive finite element method was developed to consider the intermediate damages that occur during unfolding. This model is based on force-displacement curves derived from molecular dynamics results. The proposed conceptual model is designed to accurately identify bond rupture points and determine the associated forces. This is achieved by conducting a thorough comparison between molecular dynamics and cohesive finite element results. The utilization of a viscoelastic cohesive zone model allows for the consideration of loading rate effects. This rate-dependent model can be further developed and integrated into the multiscale modeling of large assemblies of metalloproteins, providing a comprehensive understanding of mechanical behavior while maintaining a reduced computational cost.

Design of a gold nanoparticles site in an engineered lipase: an artificial metalloenzyme with enantioselective reductase-like activity.

The conjugation of gold complexes with proteins has proved to be interesting and effective in obtaining artificial metalloenzymes as catalysts with improved properties such as higher stability, activity and selectivity. However, the design and precise regulation of their structure as protein nanostructured forms level remains a challenge. Here, we have designed and constructed a gold nanoparticles-enzyme bioconjugate, by tailoring the in situ formation of gold nanoparticles (AuNPs) at two specific sites on the structure of an alkalophilic lipase from Geobacillus thermocatenulatus (GTL). For this purpose, two genetically modified variants of GTL were created by inserting a unique cysteine residue into the catalytic active site by replacing the active serine (GTL-114) and into the lid site (GTL-193). The enzyme, after a first protein-gold coordination, induced the in situ formation of AuNPs, generating a homogeneous artificial enzyme. The size and morphology of the nanoparticles in the AuNPs-enzyme conjugate have been controlled by specific pH conditions in synthesis and the specific protein region where they are formed. Reductase activity of all of them was confirmed in the hydrogenation of nitroarenes in aqueous media. The protein area seemed to be key for the AuNPs, with the best TOF values obtained for the bioconjugates with AuNPs in the lid site. Finally, the protein environment and the asymmetric properties of the AuNPs were tested in the reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. A high reductive conversion and an enantiomeric excess of up to 39% towards (R)-1-phenylethanol was found using Au-Mt@GTL-114 pH 10 as a catalyst. Moderate enantioselectivity towards the opposite isomer was also observed using the Au-Mt@GTL-193 pH 10 conjugate.

H2 production under stress: [FeFe]­hydrogenases reveal strong stability in high pressure environments.

Hydrogenases are a diverse group of metalloenzymes that catalyze the conversion of H2 into protons and electrons and the reverse reaction. A subgroup is formed by the [FeFe]­hydrogenases, which are the most efficient enzymes of microbes for catalytic H2 conversion. We have determined the stability and activity of two [FeFe]­hydrogenases under high temperature and pressure conditions employing FTIR spectroscopy and the high-pressure stopped-flow methodology in combination with fast UV/Vis detection. Our data show high temperature stability and an increase in activity up to the unfolding temperatures of the enzymes. Remarkably, both enzymes reveal a very high pressure stability of their structure, even up to pressures of several kbars. Their high pressure-stability enables high enzymatic activity up to 2 kbar, which largely exceeds the pressure limit encountered by organisms in the deep sea and sub-seafloor on Earth.

Unlocking Precision Docking for Metalloproteins.

Metalloproteins play a fundamental role in molecular biology, contributing to various biological processes. However, the discovery of high-affinity ligands targeting metalloproteins has been delayed due, in part, to a lack of suitable tools and data. Molecular docking, a widely used technique for virtual screening of small-molecule ligand interactions with proteins, often faces challenges when applied to metalloproteins due to the particular nature of the ligand metal bond. To address these limitations associated with docking metalloproteins, we introduce a knowledge-driven docking approach known as "metalloprotein bias docking" (MBD), which extends the AutoDock Bias technique. We assembled a comprehensive data set of metalloprotein-ligand complexes from 15 different metalloprotein families, encompassing Ca, Co, Fe, Mg, Mn, and Zn metal ions. Subsequently, we conducted a performance analysis of our MBD method and compared it to the conventional docking (CD) program AutoDock4, applied to various metalloprotein targets within our data set. Our results demonstrate that MBD outperforms CD, significantly enhancing accuracy, selectivity, and precision in ligand pose prediction. Additionally, we observed a positive correlation between our predicted ligand free energies and the corresponding experimental values. These findings underscore the potential of MBD as a valuable tool for the effective exploration of metalloprotein-ligand interactions.

Studying the role of Bombyx mori molybdenum cofactor sulfurase in Bombyx mori nucleopolyhedrovirus infection.

Molybdenum cofactor sulfurase (MoCoS) is a key gene involved in the uric acid metabolic pathway that activates xanthine dehydrogenase to synthesise uric acid. Uric acid is harmful to mammals but plays crucial roles in insects, one of which is the immune responses. However, the function of Bombyx mori MoCoS in response to BmNPV remains unclear. In this study, BmMoCoS was found to be relatively highly expressed in embryonic development, gonads and the Malpighian tubules. In addition, the expression levels of BmMoCoS were significantly upregulated in three silkworm strains with different levels of resistance after virus infection, suggesting a close link between them. Furthermore, RNAi and overexpression studies showed that BmMoCoS was involved in resistance to BmNPV infection, and its antivirus effects were found to be related to the regulation of uric acid metabolism, which was uncovered by inosine- and febuxostat-coupled RNAi and overexpression. Finally, the BmMoCoS-mediated uric acid pathway was preliminarily confirmed to be a potential target to protect silkworms from BmNPV infection. Overall, this study provides new evidence for elucidating the molecular mechanism of silkworms in response to BmNPV infection and new strategies for the prevention of viral infections in sericulture.

Metal-binding amino acid ligands commonly found in metalloproteins differentially fractionate copper isotopes.

Copper (Cu) is a cofactor in numerous key proteins and, thus, an essential element for life. In biological systems, Cu isotope abundances shift with metabolic and homeostatic state. However, the mechanisms underpinning these isotopic shifts remain poorly understood, hampering use of Cu isotopes as biomarkers. Computational predictions suggest that isotope fractionation occurs when proteins bind Cu, with the magnitude of this effect dependent on the identity and arrangement of the coordinating amino acids. This study sought to constrain equilibrium isotope fractionation values for Cu bound by common amino acids at protein metal-binding sites. Free and bound metal ions were separated via Donnan dialysis using a cation-permeable membrane. Isotope ratios of pre- and post-dialysis solutions were measured by MC-ICP-MS following purification. Sulfur ligands (cysteine) preferentially bound the light isotope (63Cu) relative to water (Δ65Cucomplex-free = - 0.48 ± 0.18‰) while oxygen ligands favored the heavy isotope (65Cu; + 0.26 ± 0.04‰ for glutamate and + 0.16 ± 0.10‰ for aspartate). Binding by nitrogen ligands (histidine) imparted no isotope effect (- 0.01 ± 0.04‰). This experimental work unequivocally demonstrates that amino acids differentially fractionate Cu isotopes and supports the hypothesis that metalloprotein biosynthesis affects the distribution of transition metal isotopes in biological systems.

Overlooked Hydrogen Bond in a Blue Copper Protein Uncovered by Neutron and Sub-Ångström Resolution X-ray Crystallography.

Metalloproteins play fundamental roles in organisms and are utilized as starting points for the directed evolution of artificial enzymes. Knowing the strategies of metalloproteins, by which they exquisitely tune their activities, will not only lead to an understanding of biochemical phenomena but also contribute to various applications. The blue copper protein (BCP) has been a renowned model system to understand the biology, chemistry, and physics of metalloproteins. Pseudoazurin (Paz), a blue copper protein, mediates electron transfer in the bacterial anaerobic respiratory chain. Its redox potential is finely tuned by hydrogen (H) bond networks; however, difficulty in visualizing H atom positions in the protein hinders the detailed understanding of the protein's structure-function relationship. We here used neutron and sub-ångström resolution X-ray crystallography to directly observe H atoms in Paz. The 0.86-Å-resolution X-ray structure shows that the peptide bond between Pro80 and the His81 Cu ligand deviates from the ideal planar structure. The 1.9-Å-resolution neutron structure confirms a long-overlooked H bond formed by the amide of His81 and the S atom of another Cu ligand Cys78. Quantum mechanics/molecular mechanics calculations show that this H bond increases the redox potential of the Cu site and explains the experimental results well. Our study demonstrates the potential of neutron and sub-ångström resolution X-ray crystallography to understand the chemistry of metalloproteins at atomic and quantum levels.

Significance of the Disulfide Bridge in the Structure and Stability of Metalloprotein Azurin.

Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a ß-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the ß-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual ß-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.

On the hunt for metalloenzyme inhibitors: Investigating the presence of metal-coordinating compounds in screening libraries and chemical spaces.

Metalloenzymes play vital roles in various biological processes, requiring the search for inhibitors to develop treatment options for diverse diseases. While compound library screening is a conventional approach, the exploration of virtual chemical spaces housing trillions of compounds has emerged as an alternative strategy. In this study, we investigated the suitability of selected screening libraries and chemical spaces for discovering inhibitors of metalloenzymes featuring common ions (Mg2+, Mn2+, and Zn2+). First, metal-coordinating groups from ligands interacting with ions in the Protein Data Bank were extracted. Subsequently, the prevalence of these groups in two focused screening libraries (Life Chemicals' chelator library, comprising 6,428 compounds, and Otava's chelator fragment library, with 1,784 fragments) as well as two chemical spaces (GalaXi and REAL space, containing billions of virtual products) was investigated. In total, 1,223 metal-coordinating groups were identified, with about a quarter of these groups found within the examined libraries and spaces. Our results indicate that these can serve as valuable starting points for drug discovery targeting metalloenzymes. In addition, this study suggests ways to improve libraries and spaces for better success in finding potential inhibitors for metalloenzymes.

Expanding the catalytic landscape of metalloenzymes with lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) have an essential role in global carbon cycle, industrial biomass processing and microbial pathogenicity by catalysing the oxidative cleavage of recalcitrant polysaccharides. Despite initially being considered monooxygenases, experimental and theoretical studies show that LPMOs are essentially peroxygenases, using a single copper ion and H2O2 for C-H bond oxygenation. Here, we examine LPMO catalysis, emphasizing key studies that have shaped our comprehension of their function, and address side and competing reactions that have partially obscured our understanding. Then, we compare this novel copper-peroxygenase reaction with reactions catalysed by haem iron enzymes, highlighting the different chemistries at play. We conclude by addressing some open questions surrounding LPMO catalysis, including the importance of peroxygenase and monooxygenase reactions in biological contexts, how LPMOs modulate copper site reactivity and potential protective mechanisms against oxidative damage.

Structural, Biochemical, and Computational Characterization of Sulfamides as Bimetallic Peptidase Inhibitors.

The sulfonamide function is used extensively as a general building block in various inhibitory scaffolds and, more specifically, as a zinc-binding group (ZBG) of metalloenzyme inhibitors. Here, we provide biochemical, structural, and computational characterization of a metallopeptidase in complex with inhibitors, where the mono- and bisubstituted sulfamide functions are designed to directly engage zinc ions of a bimetallic enzyme site. Structural data showed that while monosubstituted sulfamides coordinate active-site zinc ions via the free negatively charged amino group in a canonical manner, their bisubstituted counterparts adopt an atypical binding pattern divergent from expected positioning of corresponding tetrahedral reaction intermediates. Accompanying quantum mechanics calculations revealed that electroneutrality of the sulfamide function is a major factor contributing to the markedly lower potency of bisubstituted compounds by considerably lowering their interaction energy with the enzyme. Overall, while bisubstituted uncharged sulfamide functions can bolster favorable pharmacological properties of a given inhibitor, their use as ZBGs in metalloenzyme inhibitors might be less advantageous due to their suboptimal metal-ligand properties.

Development of a Fluctuating Charge Model for Zinc-Containing Metalloproteins.

Metalloproteins widely exist in biology and play important roles in various processes. To accurately simulate metalloprotein systems, modeling polarization and charge transfer effects is vital. The fluctuating charge (FQ) model can efficiently generate atomic charges and simulate the charge transfer effect; it has been developed for a wide range of applications, but few models have been specifically tailored for metalloproteins. In this study, we present a fluctuating charge model specifically for zinc-containing metalloproteins based on the extended charge equilibration (EQeq) scheme. Our model was parametrized to reproduce CM5 charges instead of RESP/CHELPG charges because the former is less dependent on the conformation or basis set, does not suffer from unphysical charges for buried atoms, and is still being able to well reproduce the molecular dipoles. During our study, we found that adding the Pauling-bond-order-like term (referred to as the "+C term" in a previous study) between the zinc ion and ligating atoms significantly improves the model's performance. Although our model was trained for four-coordinated zinc sites only, our results indicated it can well describe the atomic charges in diverse zinc sites. Morever, our model was used to generate partial charges for the metal sites in three different zinc-containing metalloproteins (with four-, five-, and six-coordinated metal sites, respectively). These charges exhibited performance comparable to that of the RESP charges in molecular dynamics (MD) simulations. Additional tests indicated our model could also well reproduce the CM5 charges when geometric changes were involved. Those results indicate that our model can efficiently calculate the atomic charges for metal sites and well simulate the charge transfer effect, which marks an important step toward developing versatile polarizable force fields for metalloproteins.