The study of acidic/basic nature of metallothioneins and other metal-binding biomolecules in the soluble hepatic fraction of the northern pike (Esox lucius).

Since fish metalloproteins are still not thoroughly characterized, the aim of this study was to investigate the acidic/basic nature of biomolecules involved in the sequestration of twelve selected metals in the soluble hepatic fraction of an important aquatic bioindicator organism, namely the fish species northern pike (Esox lucius). For this purpose, the hyphenated system HPLC-ICP-MS was applied, with chromatographic separation based on anion/cation-exchange principle at physiological pH (7.4). The results indicated predominant acidic nature of metal-binding peptides/proteins in the studied hepatic fraction. More than 90 % of Ag, Cd, Co, Cu, Fe, Mo, and Pb were eluted with negatively charged biomolecules, and >70 % of Bi, Mn, and Zn. Thallium was revealed to bind equally to negatively and positively charged biomolecules, and Cs predominantly to positively charged ones. The majority of acidic (negatively charged) metalloproteins/peptides were coeluted within the elution time range of applied standard proteins, having pIs clustered around 4-6. Furthermore, binding of several metals (Ag, Cd, Cu, Zn) to two MT-isoforms was assumed, with Cd and Zn preferentially bound to MT1 and Ag to MT2, and Cu evenly distributed between the two. The results presented here are the first of their kind for the important bioindicator species, the northern pike, as well as one of the rare comprehensive studies on the acidic/basic nature of metal-binding biomolecules in fish, which can contribute significantly to a better understanding of the behaviour and fate of metals in the fish organism, specifically in liver as main metabolic and detoxification organ.

Acclimation to medium-level non-lethal iron limitation: Adjustment of electron flow around the PSII and metalloprotein expression in Trichodesmium erythraeum IMS101.

The aim of this study was to investigate how acclimation to medium-level, long-term, non-lethal iron limitation changes the electron flux around the Photosystem II of the oceanic diazotroph Trichodesmium erythraeum IMS101. Fe availability of about 5× and 100× lower than a replete level, i.e. conditions common in the natural environment of this cyanobacterium, were applied in chemostats. The response of the cells was studied not only in terms of growth, but also mechanistically, measuring the chlorophyll fluorescence of dark-adapted filaments via imaging fluorescence kinetic microscopy (FKM) with 0.3 ms time resolution. Combining these measurements with those of metal binding to proteins via online coupling of metal-free HPLC (size exclusion chromatography SEC) to sector-field ICP-MS allowed to track the fate of the photosystems, together with other metalloproteins. General increase of fluorescence has been observed, with the consequent decrease in the quantum yields φ of the PSII, while the efficiency ψ of the electron flux between PSII and the PSI remained surprisingly unchanged. This indicates the ability of Trichodesmium to cope with a situation that makes assembling the many iron clusters in Photosystem I a particular challenge, as shown by decreasing ratios of Fe to Mg in these proteins. The negative effect of Fe limitation on PSII may also be due to its fast turnover. A broader view was obtained from metalloproteomics via HPLC-ICP-MS, revealing a differential protein expression pattern under iron limitation with a drastic down-regulation especially of iron-containing proteins and some increase in low MW metal-binding complexes.

Be2+ Causes Hypersensitivity but Mg2+ and Ca2+ Do Not─Favorable Metal Coordination Is the Key for Differential Allosteric Modulation and Binding Affinities.

Although the ion selectivity of metalloproteins has been well established, selective metal antigen recognition by immunoproteins remains elusive. One such case is the recognition of the Be2+ ion against its heavier congeners, Mg2+ and Ca2+, by the human leukocyte antigen immunoprotein (HLA-DP2), leading to immunotoxicity. Integrating with our previous mechanistic study on Be2+ toxicity, herein, we have explored the basis of characteristic nontoxicity of Mg2+ and Ca2+ ions despite their in vivo abundance. The ion binding cleft of the HLA-DP2-peptide complex is composed of four acidic residues, p4D and p7E from the peptide and ß26E and ß69E from the protein. While the tetrahedral coordination site of the smaller Be2+ ion is located deep inside the cavity, hexa- to octa-coordination sites of Mg2+ and Ca2+ ions are located closer to the protein surface. The intrinsic high coordination number of Mg2+/Ca2+ ions induces allosteric modifications on the HLA-DP2_M2 surface, which are atypical for TCR recognition. Furthermore, the lower binding energy of larger Mg2+ and Ca2+ ions with the cavity residues can be correlated to the lower charge density and reduced covalent bonding nature as compared to those of the smaller Be2+ ion. In short, weak binding of Mg2+ and Ca2+ ions and the unfavorable allosteric surface modifications are probably the major determinants for the absence of Mg2+/Ca2+ ion-mediated hypersensitivity in humans.

Coordination chemistry of mitochondrial copper metalloenzymes: exploring implications for copper dyshomeostasis in cell death.

Mitochondria, fundamental cellular organelles that govern energy metabolism, hold a pivotal role in cellular vitality. While consuming dioxygen to produce adenosine triphosphate (ATP), the electron transfer process within mitochondria can engender the formation of reactive oxygen species that exert dual roles in endothelial homeostatic signaling and oxidative stress. In the context of the intricate electron transfer process, several metal ions that include copper, iron, zinc, and manganese serve as crucial cofactors in mitochondrial metalloenzymes to mediate the synthesis of ATP and antioxidant defense. In this mini review, we provide a comprehensive understanding of the coordination chemistry of mitochondrial cuproenzymes. In detail, cytochrome c oxidase (CcO) reduces dioxygen to water coupled with proton pumping to generate an electrochemical gradient, while superoxide dismutase 1 (SOD1) functions in detoxifying superoxide into hydrogen peroxide. With an emphasis on the catalytic reactions of the copper metalloenzymes and insights into their ligand environment, we also outline the metalation process of these enzymes throughout the copper trafficking system. The impairment of copper homeostasis can trigger mitochondrial dysfunction, and potentially lead to the development of copper-related disorders. We describe the current knowledge regarding copper-mediated toxicity mechanisms, thereby shedding light on prospective therapeutic strategies for pathologies intertwined with copper dyshomeostasis. [BMB Reports 2023; 56(11): 575-583].

Role of the SLC22A17/lipocalin-2 receptor in renal endocytosis of proteins/metalloproteins: a focus on iron- and cadmium-binding proteins.

The transmembrane protein SLC22A17 [or the neutrophil gelatinase-associated lipocalin/lipocalin-2 (LCN2)/24p3 receptor] is an atypical member of the SLC22 family of organic anion and cation transporters: it does not carry typical substrates of SLC22 transporters but mediates receptor-mediated endocytosis (RME) of LCN2. One important task of the kidney is the prevention of urinary loss of proteins filtered by the glomerulus by bulk reabsorption of multiple ligands via megalin:cubilin:amnionless-mediated endocytosis in the proximal tubule (PT). Accordingly, overflow, glomerular, or PT damage, as in Fanconi syndrome, results in proteinuria. Strikingly, up to 20% of filtered proteins escape the PT under physiological conditions and are reabsorbed by the distal nephron. The renal distal tubule and collecting duct express SLC22A17, which mediates RME of filtered proteins that evade the PT but with limited capacity to prevent proteinuria under pathological conditions. The kidney also prevents excretion of filtered essential and nonessential transition metals, such as iron or cadmium, respectively, that are largely bound to proteins with high affinity, e.g., LCN2, transferrin, or metallothionein, or low affinity, e.g., microglobulins or albumin. Hence, increased uptake of transition metals may cause nephrotoxicity. Here, we assess the literature on SLC22A17 structure, topology, tissue distribution, regulation, and assumed functions, emphasizing renal SLC22A17, which has relevance for physiology, pathology, and nephrotoxicity due to the accumulation of proteins complexed with transition metals, e.g., cadmium or iron. Other putative renal functions of SLC22A17, such as its contribution to osmotic stress adaptation, protection against urinary tract infection, or renal carcinogenesis, are discussed.

Chemoproteomics Reveals Disruption of Metal Homeostasis and Metalloproteins by the Antibiotic Holomycin.

The natural product holomycin contains a unique cyclic ene-disulfide and exhibits broad-spectrum antimicrobial activities. Reduced holomycin chelates metal ions with a high affinity and disrupts metal homeostasis in the cell. To identify cellular metalloproteins inhibited by holomycin, reactive-cysteine profiling was performed using isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). This chemoproteomic analysis demonstrated that holomycin treatment increases the reactivity of metal-coordinating cysteine residues in several zinc-dependent and iron-sulfur cluster-dependent enzymes, including carbonic anhydrase II and fumarase A. We validated that holomycin inhibits fumarase A activity in bacterial cells and diminishes the presence of iron-sulfur clusters in fumarase A. Whole-proteome abundance analysis revealed that holomycin treatment induces zinc and iron starvation and cellular stress. This study suggests that holomycin inhibits bacterial growth by impairing the functions of multiple metalloenzymes and sets the stage for investigating the impact of metal-binding molecules on metalloproteomes by using chemoproteomics.

An Antibody Engineered Metalloenzyme for Mediating Cell-Cell Communication and Activation of Immuno- and Chemotherapy.

Artificial metalloenzymes (ArMs) are gaining much attention in life sciences. However, the function of the present ArMs for disease treatment is still in its infancy, which may impede the possible therapeutic potential. Herein, we construct an antibody engineered ArM by using the Fc region of IgG and bioorthogonal chemistry, which endows the ArM with the capability of manipulating cell-cell communication and bioorthogonal catalysis for tumor immuno- and chemotherapy. Specially, Fc-Pd ArM is modified on the cancer cell surface by metabolic glycoengineering to catalyze the bioorthogonal activation of prodrug for tumor chemotherapy. More importantly, the antibody-based ArM can mediate cell-cell communication between cancer cells and NK cells, activating the ADCC effect for immunotherapy. In vivo antitumor applications suggest that the ArM can not only eliminate primary tumor but also inhibit tumor lung metastasis. Our work provides a new attempt to develop artificial metalloenzymes with cell-cell communication the ability for bioorthogonal catalysis and combination therapy.

Cellular production of a de novo membrane cytochrome.

Heme-containing integral membrane proteins are at the heart of many bioenergetic complexes and electron transport chains. The importance of these electron relay hubs across biology has inspired the design of de novo proteins that recreate their core features within robust, versatile, and tractable protein folds. To this end, we report here the computational design and in-cell production of a minimal diheme membrane cytochrome which successfully integrates into the cellular membrane of live bacteria. This synthetic construct emulates a four-helix bundle found in modern respiratory complexes but has no sequence homology to any polypeptide sequence found in nature. The two b-type hemes, which appear to be recruited from the endogenous heme pool, have distinct split redox potentials with values close to those of natural membrane-spanning cytochromes. The purified protein can engage in rapid biomimetic electron transport with small molecules, with other redox proteins, and with biologically relevant diffusive electron carriers. We thus report an artificial membrane metalloprotein with the potential to serve as a functional electron transfer module in both synthetic protocells and living systems.

E74-like factor 1 contributes to nerve trauma-induced nociceptive hypersensitivity through transcriptionally activating matrix metalloprotein-9 in dorsal root ganglion neurons.

ABSTRACT: Nerve trauma-induced alternations of gene expression in the neurons of dorsal root ganglion (DRG) participate in nerve trauma-caused nociceptive hypersensitivity. Transcription factors regulate gene expression. Whether the transcription factor E74-like factor 1 (ELF1) in the DRG contributes to neuropathic pain is unknown. We report here that peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve or unilateral fourth lumbar spinal nerve ligation led to the time-dependent increases in the levels of Elf1 mRNA and ELF1 protein in injured DRG, but not in the spinal cord. Preventing this increase through DRG microinjection of adeno-associated virus 5 expressing Elf1 shRNA attenuated the CCI-induced upregulation of matrix metallopeptidase 9 (MMP9) in injured DRG and induction and maintenance of nociceptive hypersensitivities, without changing locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking this increase through DRG microinjection of AAV5 expressing full-length Elf1 upregulated DRG MMP9 and produced enhanced responses to mechanical, heat, and cold stimuli in naive mice. Mechanistically, more ELF1 directly bond to and activated Mmp9 promoter in injured DRG neurons after CCI. Our data indicate that ELF1 participates in nerve trauma-caused nociceptive hypersensitivity likely through upregulating MMP9 in injured DRG. E74-like factor 1 may be a new target for management of neuropathic pain.

Studying Peptide-Metal Ion Complex Structures by Solution-State NMR.

Metal chelation can provide structural stability and form reactive centers in metalloproteins. Approximately one third of known protein structures are metalloproteins, and metal binding, or the lack thereof, is often implicated in disease, making it necessary to be able to study these systems in detail. Peptide-metal complexes are both present in nature and can provide a means to focus on the binding region of a protein and control experimental variables to a high degree. Structural studies of peptide complexes with metal ions by nuclear magnetic resonance (NMR) were surveyed for all the essential metal complexes and many non-essential metal complexes. The various methods used to study each metal ion are presented together with examples of recent research. Many of these metal systems have been individually reviewed and this current overview of NMR studies of metallopeptide complexes aims to provide a basis for inspiration from structural studies and methodology applied in the field.

Stealthy microbes: How Neisseria gonorrhoeae hijacks bulwarked iron during infection.

Transition metals are essential for metalloprotein function among all domains of life. Humans utilize nutritional immunity to limit bacterial infections, employing metalloproteins such as hemoglobin, transferrin, and lactoferrin across a variety of physiological niches to sequester iron from invading bacteria. Consequently, some bacteria have evolved mechanisms to pirate the sequestered metals and thrive in these metal-restricted environments. Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, causes devastating disease worldwide and is an example of a bacterium capable of circumventing human nutritional immunity. Via production of specific outer-membrane metallotransporters, N. gonorrhoeae is capable of extracting iron directly from human innate immunity metalloproteins. This review focuses on the function and expression of each metalloprotein at gonococcal infection sites, as well as what is known about how the gonococcus accesses bound iron.

Molybdenum Cofactor Deficiency in Humans.

Molybdenum cofactor (Moco) deficiency (MoCD) is characterized by neonatal-onset myoclonic epileptic encephalopathy and dystonia with cerebral MRI changes similar to hypoxic-ischemic lesions. The molecular cause of the disease is the loss of sulfite oxidase (SOX) activity, one of four Moco-dependent enzymes in men. Accumulating toxic sulfite causes a secondary increase of metabolites such as S-sulfocysteine and thiosulfate as well as a decrease in cysteine and its oxidized form, cystine. Moco is synthesized by a three-step biosynthetic pathway that involves the gene products of MOCS1, MOCS2, MOCS3, and GPHN. Depending on which synthetic step is impaired, MoCD is classified as type A, B, or C. This distinction is relevant for patient management because the metabolic block in MoCD type A can be circumvented by administering cyclic pyranopterin monophosphate (cPMP). Substitution therapy with cPMP is highly effective in reducing sulfite toxicity and restoring biochemical homeostasis, while the clinical outcome critically depends on the degree of brain injury prior to the start of treatment. In the absence of a specific treatment for MoCD type B/C and SOX deficiency, we summarize recent progress in our understanding of the underlying metabolic changes in cysteine homeostasis and propose novel therapeutic interventions to circumvent those pathological changes.

Evaluation of acute toxicity of cancer-targeting albumin-based artificial metalloenzymes.

Recently, the development of abiotic metal-mediated drug delivery has been significant growth in the fields of anticancer approach and biomedical application. However, the intrinsic toxicity of abiotic metal catalysts makes in vivo use difficult. Our group developed a system of cancer-targeting albumin-based artificial metalloenyzmes (ArMs) capable of performing localized drug synthesis and selective tagging therapy in vivo for cancer therapy. The toxicity of the system at higher concentrations was investigated in vitro and in vivo in the study to demonstrate its safety for potential application in clinical trials. In cell-based experiments, the study revealed that the cytotoxicity of metal catalysts anchored within the binding cavity of the cancer-targeting ArMs could be significantly reduced compared to free-in-solution metal catalysts. Moreover, the in vivo data demonstrated that the cancer-targeting ArMs did not cause considerable damage in organs or change in the hematological parameters in a single-dose (160 mg/Kg) toxicity study in rats. Therefore, the system is safe, highlighting that it could be used in clinical trials for cancer treatment.

MTM1 displays a new function in the regulation of nickel resistance in Saccharomyces cerevisiae.

Nickel (Ni) is an essential yet toxic trace element. Although a cofactor for many metalloenzymes, nickel function and metabolism is not fully explored in eukaryotes. Molecular biology and metallomic methods were utilized to explore the new physiological functions of nickel in Saccharomyces cerevisiae. Here we showed that MTM1 knockout cells displayed much stronger nickel tolerance than wild-type cells and mitochondrial accumulations of Ni and Fe of mtm1Δ cells dramatically decreased compared to wild-type cells when exposed to excess nickel. Superoxide dismutase 2 (Sod2p) activity in mtm1Δ cells was severely attenuated and restored through Ni supplementation in media or total protein. SOD2 mRNA level of mtm1Δ cells was significantly higher than that in the wild-type strain but was decreased by Ni supplementation. MTM1 knockout afforded resistance to excess nickel mediated through reactive oxygen species levels. Meanwhile, additional Ni showed no significant effect on the localization of Mtm1p. Our study reveals the MTM1 gene plays an important role in nickel homeostasis and identifies a novel function of nickel in promoting Sod2p activity in yeast cells.

Bicyclostreptins are radical SAM enzyme-modified peptides with unique cyclization motifs.

Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins-peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic ß-ether and a heterocyclic sp3-sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon. Both reactions are installed, in that order, by two radical S-adenosylmethionine (RaS) metalloenzymes. Bicyclostreptins are produced at nM concentrations and are potent growth regulation agents in Streptococcus thermophilus. Our results add a distinct and unusual chemotype to the growing family of ribosomal peptide natural products, expand the already impressive catalytic scope of RaS enzymes, and provide avenues for further biological studies in human-associated streptococci.

Differences in the Second Coordination Sphere Tailor the Substrate Specificity and Reactivity of Thiol Dioxygenases.

In recent years, considerable progress has been made toward elucidating the geometric and electronic structures of thiol dioxygenases (TDOs). TDOs catalyze the conversion of substrates with a sulfhydryl group to their sulfinic acid derivatives via the addition of both oxygen atoms from molecular oxygen. All TDOs discovered to date belong to the family of cupin-type mononuclear nonheme Fe(II)-dependent metalloenzymes. While most members of this enzyme family bind the Fe cofactor by two histidines and one carboxylate side chain (2-His-1-carboxylate) to provide a monoanionic binding motif, TDOs feature a neutral three histidine (3-His) facial triad. In this Account, we present a bioinformatics analysis and multiple sequence alignment that highlight the significance of the secondary coordination sphere in tailoring the substrate specificity and reactivity among the different TDOs. These insights provide the framework within which important structural and functional features of the distinct TDOs are discussed.The best studied TDO is cysteine dioxygenase (CDO), which catalyzes the conversion of cysteine to cysteine sulfinic acid in both eukaryotes and prokaryotes. Crystal structures of resting and substrate-bound mammalian CDOs revealed two surprising structural motifs in the first- and second coordination spheres of the Fe center. The first is the presence of the abovementioned neutral 3-His facial triad that coordinates the Fe ion. The second is the existence of a covalent cross-link between the sulfur of Cys93 and an ortho carbon of Tyr157 (mouse CDO numbering scheme). While the exact role of this cross-link remains incompletely understood, various studies established that it is needed for proper substrate Cys positioning and gating solvent access to the active site. Intriguingly, bacterial CDOs lack the Cys-Tyr cross-link; yet, they are as active as cross-linked eukaryotic CDOs.The other known mammalian TDO is cysteamine dioxygenase (ADO). Initially, it was believed that ADO solely catalyzes the oxidation of cysteamine to hypotaurine. However, it has recently been shown that ADO additionally oxidizes N-terminal cysteine (Nt-Cys) peptides, which indicates that ADO may play a much more significant role in mammalian physiology than was originally anticipated. Though predicted on the basis of sequence alignment, site-directed mutagenesis, and spectroscopic studies, it was not until last year that two crystal structures, one of wild-type mouse ADO (solved by us) and the other of a variant of nickel-substituted human ADO, finally provided direct evidence that this enzyme also features a 3-His facial triad. These structures additionally revealed several features that are unique to ADO, including a putative cosubstrate O2 access tunnel that is lined by two Cys residues. Disulfide formation under conditions of high O2 levels may serve as a gating mechanism to prevent ADO from depleting organisms of Nt-Cys-containing molecules.The combination of kinetic and spectroscopic studies in conjunction with structural characterizations of TDOs has furthered our understanding of enzymatic sulfhydryl substrate regulation. In this article, we take advantage of the fact that the ADO X-ray crystal structures provided the final piece needed to compare and contrast key features of TDOs, an essential family of metalloenzymes found across all kingdoms of life.

Thermodynamics of iron, tetrahydrobiopterin, and phenylalanine binding to phenylalanine hydroxylase from Chromobacterium violaceum.

Phenylalanine hydroxylase (PheH) is a pterin-dependent, mononuclear nonheme iron(II) oxygenase that uses the oxidative power of O2 to hydroxylate phenylalanine to form tyrosine. PheH is a member of a superfamily of O2-activating enzymes that utilizes a common metal binding motif: the 2-His-1-carboxylate facial triad. Like most members of this superfamily, binding of substrates to PheH results in a reorganization of its active site to allow O2 activation. Exploring the energetics of each step before O2 activation can provide mechanistic insight into the initial steps that support the highly specific O2 activation pathway carried out by this metalloenzyme. Here the thermal stability of PheH and its substrate complexes were investigated under an anaerobic environment by using differential scanning calorimetry. In context with known binding constants for PheH, a thermodynamic cycle associated with iron(II), tetrahydrobiopterin (BH4), and phenylalanine binding to the active site was generated, showing a distinctive cooperativity between the binding of BH4 and Phe. The addition of phenylalanine and BH4 to PheH·Fe increased the stability of this enzyme (ΔTm of 8.5 (±0.7) °C with an associated δΔH of 43.0 (±2.9) kcal/mol). The thermodynamic data presented here gives insight into the complicated interactions between metal center, cofactor, and substrate, and how this interplay sets the stage for highly specific, oxidative C-H activation in this enzyme.

Metalloenzymes involved in carotenoid biosynthesis in plants.

Carotenoids are a family of pigment compounds, a subset of which are precursors for vitamin A biosynthesis. These pigments are derived from isopentenyl pyrophosphate (IPP), with geranylgeranyl diphosphate being the first metabolite unique to carotenoid biosynthesis in plants, algae, fungi, some bacteria, and arthropods. This chapter highlights the metal-dependent enzymes involved in synthesizing carotenoids in plants and the current state of knowledge of their cofactors and mechanisms. Emphasis is given to spectroscopic methods used to characterize metal centers. The recently discovered heme-dependent isomerase Z-ISO is presented as a case study in how to interrogate a metalloenzyme. Use of UV-vis, electron paramagnetic resonance, and magnetic circular dichroism spectroscopies of a metal center at various oxidation states and with external small molecule probes (CN-, CO, and NO) can provide information about the nature of the metal center, the identity of its ligands, and its mechanism of action. Z-ISO is a histidine/cysteine ligated heme-dependent enzyme that is only active in the ferrous state and possesses redox-linked ligand switching. The choice and design of experiments are discussed as well as the conclusions that can be drawn.

Design, synthesis, and in silico studies of tetrahydropyrimidine analogs as urease enzyme inhibitors.

The urease enzyme, a metalloenzyme having Ni2+ ions, is recognized in some bacteria, fungi, and plants. Particularly, it is vital to the progress of infections induced by pathogenic microbes, such as Proteus mirabilis and Helicobacter pylori. Herein, we reported the synthesis of a series of tetrahydropyrimidine derivatives and evaluated their antiurease activity. Finally, quantitative and qualitative analyses of the derivatives were performed via in silico studies. Urease inhibitory activity was determined as the reaction of H. pylori urease with different concentrations of compounds, and thiourea was used as a standard compound. Docking and dynamics methodologies were applied to study the interactions of the best compounds with the amino acids in the active site. All compounds showed good to excellent antiurease activity. The potent compounds were not cytotoxic against the HUVEC normal cell line. Based on the docking study, compound 4e with the highest urease inhibitory activity (IC50 = 6.81 ± 1.42 µM) showed chelates with both Ni2+ ions of the urease active site. Further, compound 4f displayed a very good inhibitory activity (IC50 = 8.45 ± 1.64 µM) in comparison to thiourea (IC50 = 22.03 ± 1.24 µM). The molecular docking and dynamics simulation results were correlated with the in vitro assay results. Moreover, the derivatives 4a-n followed Lipinski's rule-of-five and had drug-likeness properties.

Long-Range Charge Delocalization Mediates the Ultrafast Ligand-to-Metal Charge Transfer Dynamics at the Cu2+-Active Site in Azurin.

The blue color in metalloprotein azurin has traditionally been attributed to the intense cysteine-to-Cu2+ ligand-to-metal charge transfer transition centered at 628 nm. Although resonance Raman measurements of the Cu2+ active site have implied that the LMCT transition electronically couples to the protein scaffold well beyond its primary metal-ligand coordination shell, the structural extent of this electronic coupling and visualization of the protein-mediated charge transfer dynamics have remained elusive. Here, using femtosecond broadband transient absorption and impulsive Raman spectroscopy, we provide direct evidence for a rapid relaxation between two distinct charge transfer states, having different spatial delocalization, within ∼300 fs followed by recombination of charges in subpicosecond time scales. We invoke the formation of a protein-centered radical cation, possibly Trp48 or a Phe residue, within 100 fs substantiating the long-range electronic coupling for the first time beyond the traditional copper active site. The Raman spectra of the excited CT state show the presence of protein-centric vibrations along with the vibrational modes assigned to the copper active site. Our results demonstrate a large delocalization length scale of the initially populated CT state, thereby highlighting the possibility of exploiting azurin photochemistry for energy conversion techniques.