Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys → T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.

Metalloproteins play a crucial role in regulating different aspects of the immune system in humans. They have various functions in immunity, including recognizing and presenting antigens, aiding in the movement and effectiveness of immune cells, and facilitating interactions between the host and pathogens. Understanding how these proteins work can help us develop new methods to control the immune response in different diseases. Metalloproteins contain metal ions in their structure, which allows them to perform these diverse functions. They encompass a wide range of enzymes, signaling molecules, and structural proteins that utilize metal ions as cofactors for their activities. Examples of metalloproteins include superoxide dismutase, catalase, and metalloproteases, which regulate oxidative stress, inflammation, and tissue remodelling processes associated with immune activation. By studying their functions and the effects of their dysfunction, researchers can develop strategies to improve immune function and combat various diseases. This review explores the diverse functions of metalloproteins in immune processes, highlighting their significance in both health and disease.

Several species during evolution suffered random mutations in response to various environmental factors, which resulted in the formation of venom in phylogenetically distant species. The composition of the venom of most species is poorly known. Snake venom is well characterized while most species have poorly known composition. In contrast, snake venoms are well characterized which proteins and peptides are the main active and most abundant constituents. 42 protein families have been identified, including metalloproteins known as metalloproteinases. These macromolecules are enzymes with zinc in their active site derived from the disintegrin A and metalloproteinase (ADAM) cellular family and are categorized into three classes (PI, PII and PIII) according to their domain organization. The snake venom metalloproteinases (SVMP) are cytotoxic, neurotoxic, myotoxic and/or hematotoxic with a crucial role in the defense and restraint of prey. In this scenario envenoming represents a danger to human health and has been considered a neglected disease worldwide, particularly in tropical and subtropical countries. Nevertheless, recently advances in \"omics\" technologies have demonstrated interesting biological activities of SVMPs such as antimicrobial, anticancer, against cardiovascular diseases and nervous system disorders. Metalloproteins have the therapeutic potential to be converted into drugs as other components of the venom have undergone this process (e.g., captopril, tirefiban and eptifibatide). So, this chapter is focused on the metalloproteins found in the secretions of venomous species, highlight some aspects such as structure, biological activity, pharmacological therapeutic potential and on.

The gut microbial metalloenzymes play an important role in maintaining the balance between gut microbial ecosystem, human physiologically processes and immune system. The metals coordinated into active site contribute in various detoxification and defense strategies to avoid unfavourable environment and ensure bacterial survival in human gut. Metallo-β-lactamase is a potent degrader of antibiotics present in periplasmic space of both commensals and pathogenic bacteria. The resistance to anti-microbial agents developed in this enzyme is one of the global threats for human health. The organophosphorus eliminator, organophosphorus hydrolases have evolved over a course of time to hydrolyze toxic organophosphorus compounds and decrease its effect on human health. Further, the redox stress responders namely superoxide dismutase and catalase are key metalloenzymes in reducing both endogenous and exogenous oxidative stress. They hold a great importance for pathogens as they contribute in pathogenesis in human gut along with reduction of oxidative stress. The in-silico study on these enzymes reveals the importance of point mutation for the evolution of these enzymes in order to enhance their enzyme activity and stability. Various mutation studies were conducted to investigate the catalytic activity of these enzymes. By using the \"directed evolution\" method, the enzymes involved in detoxification and defense system can be engineered to produce new variants with enhance catalytic features, which may be used to predict the severity due to multi-drug resistance and degradation pattern of organophosphorus compounds in human gut.

We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotype in tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactone scaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids and patient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.

TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue.

Enzymes are nature\'s ultimate machinery to catalyze complex reactions. Though enzymes are evolved to catalyze specific reactions, they also show significant promiscuity in reactions and substrate selection. Metalloenzymes contain a metal ion or metal cofactor in their active site, which is crucial in their catalytic activity. Depending on the metal and its coordination environment, the metal ion or cofactor may function as a Lewis acid or base and a redox center and thus can catalyze a plethora of natural reactions. In fact, the versatility in the oxidation state of the metal ions provides metalloenzymes with a high level of catalytic adaptability and promiscuity. In this chapter, we discuss different aspects of promiscuity in metalloenzymes by using several recent experimental and theoretical works as case studies. We start our discussion by introducing the concept of promiscuity and then we delve into the mechanistic insight into promiscuity at the molecular level.

Metalloproteins represents more than one third of human proteome, with huge variation in physiological functions and pathological implications, depending on the metal/metals involved and tissue context. Their functions range from catalysis, bioenergetics, redox, to DNA repair, cell proliferation, signaling, transport of vital elements, and immunity. The human metalloproteomic studies revealed that many families of metalloproteins along with individual metalloproteins are dysregulated under several clinical conditions. Also, several sorts of interaction between redox- active or redox- inert metalloproteins are observed in health and disease. Metalloproteins profiling shows distinct alterations in neurodegenerative diseases, cancer, inflammation, infection, diabetes mellitus, among other diseases. This makes metalloproteins -either individually or as families- a promising target for several therapeutic approaches. Inhibitors and activators of metalloenzymes, metal chelators, along with artificial metalloproteins could be versatile in diagnosis and treatment of several diseases, in addition to other biomedical and industrial applications.

Transition metal ions play crucial roles in the structure and function of numerous proteins, contributing to essential biological processes such as catalysis, electron transfer, and oxygen binding. However, accurately modeling the electronic structure and properties of metalloproteins poses significant challenges due to the complex nature of their electronic configurations and strong correlation effects. Multiconfigurational quantum chemistry methods are, in principle, the most appropriate tools for addressing these challenges, offering the capability to capture the inherent multi-reference character and strong electron correlation present in bio-inorganic systems. Yet their computational cost has long hindered wider adoption, making methods such as density functional theory (DFT) the method of choice. However, advancements over the past decade have substantially alleviated this limitation, rendering multiconfigurational quantum chemistry methods more accessible and applicable to a wider range of bio-inorganic systems. In this perspective, we discuss some of these developments and how they have already been used to answer some of the most important questions in bio-inorganic chemistry. We also comment on ongoing developments in the field and how the future of the field may evolve.

CH functionalization, a promising frontier in modern organic chemistry, facilitates the direct conversion of inert CH bonds into many valuable functional groups. Despite its merits, traditional homogeneous catalysis, often faces challenges in efficiency, selectivity, and sustainability towards this transformation. In this context, artificial metalloenzymes (ArMs), resulting from the incorporation of a catalytically-competent metal cofactor within an evolvable protein scaffold, bridges the gap between the efficiency of enzymatic transformations and the versatility of transition metal catalysis. Accordingly, ArMs have emerged as attractive tools for various challenging catalytic transformations. Additionally, the coming of age of directed evolution has unlocked unprecedented avenues for optimizing enzymatic catalysis. Taking advantage of their genetically-encoded protein scaffold, ArMs have been evolved to catalyze various CH functionalization reactions. This review delves into the recent developments of ArM-catalyzed CH functionalization reactions, highlighting the benefits of engineering the second coordination sphere around a metal cofactor within a host protein.