Effects of Ionic Liquids on Metalloproteins

In the past decade, innovative protein therapies and bio-similar industries have grown rapidly. Additionally, ionic liquids (ILs) have been an area of great interest and rapid development in industrial processes over a similar timeline. Therefore, there is a pressing need to understand the structure and function of proteins in novel environments with ILs. Understanding the short-term and long-term stability of protein molecules in IL formulations will be key to using ILs for protein technologies. Similarly, ILs have been investigated as part of therapeutic delivery systems and implicated in numerous studies in which ILs impact the activity and/or stability of protein molecules. Notably, many of the proteins used in industrial applications are involved in redox chemistry, and thus often contain metal ions or metal-associated cofactors. In this review article, we focus on the current understanding of protein structure-function relationship in the presence of ILs, specifically focusing on the effect of ILs on metal containing proteins.


Introduction
Proteins are long chain polymers of amino acids connected by peptide bonds. These polypeptide chains are interlinked with hydrogen bonding, which leads to the formation of secondary structures in proteins and subsequent further organization of these secondary structure elements form tertiary structures [1]. Protein function is governed by the specific three-dimensional structure the protein adopts by arranging the appropriate functional groups in the proper orientation. Proteins are involved in multiple processes in the living cell and are located on the extracellular surface, intracellular region, and in the cell membrane [2]. Some examples of proteins that are commonly found in biological systems are hormones, antibodies, enzymes (biological catalysts), transporters, and receptors [3]. Because of these biological functions, proteins are also used as components of industrial processes and as therapeutic agents using specific formulations [4]. Industrial processes utilize a variety of proteins such as metalloproteases, laccases, cellulases, lipases, phosphatases, and amylases for numerous applications [5]. Therapeutically, proteins such as immunoglobulins, erythropoietin, interferons, insulin, and anti-clotting proteins are widely used in the clinic [5]. Depending on the structure of the protein, they are only stable in specific physiochemical environments, and therefore it is important to evaluate the effects of various physical and chemical conditions for developing a robust formulation [6].
In some cases, protein structures are associated with metal ions, including, but not limited to, Ca 2+ , Mg 2+ Cu 2+ , Fe 2+ , and Zn 2+ , and this class of proteins is referred to as metalloproteins [7]. Nearly 50% of the existing proteins in nature are metalloproteins [7]. Metal ions within metalloproteins play a key functional role in many biological redox processes and can provide structural stability to the protein [8]. Metal ions within these proteins

Ionic Liquids (ILs)
ILs are organic salts with melting points below 100 • C. In 1992 the first IL stable in air and ambient moisture was reported [93]. After that, ILs have been developed as an alternative to organic solvents and used in many more applications. ILs are useful industrial and laboratory solvents. The molecular composition of ILs is a combination of different cations and anions that leads to countless potential ionic liquid species. ILs have a wide range of physicochemical properties, including low vapor pressure, high thermal stability, high conductivity, non-flammability, and varying degrees of biocompatibility [94]. Therefore, they could be used as reaction media for synthesis and can be recycled multiple times, which underpins the "green" reputation of these solvents [95]. ILs have the ability to act as a host and can interact with both host and guest molecules via a combination of electrostatic, hydrogen bonding, πand van der Waals interactions [96]. The noncovalent interactions within ILs are easily broken and, therefore, are commonly used to dissolve recalcitrant materials [96]. ILs are currently being used in many different applications, including electrochemistry, energy, organic synthesis, and catalysis, as well as in biotechnology [97][98][99][100].

Ionic Liquid Interactions with Biomolecules
In nature, biomolecules are surrounded by charged species, including proteins, polysaccharides, nucleic acids, inorganic ions, and small organic molecules. Although proteins have evolved to function in these ion-rich environments, not all ionic species have identical effects on proteins. Specifically, there has been a significant amount of study regarding the ability of ionic species to stabilize or destabilize proteins in solution. This ranking of ions based on the effects on protein solubility, known as the Hofmeister series, is a core component of understanding protein behavior in complex ionic solutions [101][102][103]. Importantly, extensive study of the Hofmeister series has determined that the anionic component of the salt generally has a larger effect on protein solubility [101][102][103]. Mechanistically, ions in the Hofmeister series are thought to change the ordering and interactions of the bulk water around the protein rather than more direct protein interactions, which then impacts protein hydration and stability [102,[104][105][106]. Numerous ILs have been studied from the context of the Hofmeister series, especially since many commercially available ILs have simple anions or cations as part of the IL pair [107,108]. These studies include direct influences of ILs on biopolymers but also more fundamental studies of IL properties in solution, including physicochemical parameters such as ion hydration number, which appears to be an important factor in IL-biomolecule interactions [102,107,[109][110][111][112]. When considering the descriptions of IL-protein interactions below, the IL composition and ion placement in the Hofmeister series, when known, should be considered in the reader's interpretations.
The unique properties of IL have made them very useful as potential solvents for protein preservation, media for enzymatic reactions, as well as applications in the field of bioconversion and protein production/purification [42,113,114]. ILs are also found to enhance the solubility of certain proteins, mainly through the prevention of aggregation [115][116][117]. Furthermore, enhanced solubility of proteins in ILs can also help achieve highly supersaturated solutions, which were successfully used as an additive in media to promote protein crystallization. ILs were shown to influence the crystallization of multiple proteins as well as improving the size of the crystal formed (helping crystal growth), quality of crystals, and enhances the reproducibility of the crystallization process [118,119]. In addition, IL/aqueous bi-phasic systems were also used for the extraction of proteins from biological fluids [116,120]. These are a few representative instances where ILs can enhance protein stability and activity. However, not all ILs are compatible with proteins. Many ILs have been shown to destabilize protein structure and activity. The physicochemical properties of ILs such as polarity, alkyl chain length, hydrophobicity, and viscosity all have different effects on protein stability [42]. Therefore, a rational selection of IL for a specific protein under investigation is necessary before using it as a solvent for that application. Furthermore, there is only limited knowledge regarding the mechanism of protein stabi-lization or destabilization in the presence of ILs, and therefore, research is still needed to understand how they interact with proteins based on the chemistry of ILs [121].
There has been great interest in recent years to use ILs in various industries because of the beneficial properties and the desire to stabilize protein functionality over wider ranges of reaction conditions. Specifically, how these ILs interact with biomolecules and what cation-anion combinations may impact biomolecular functions are of great interest for industrial applications. Numerous groups have studied the interactions of proteins with a wide variety of ILs, resulting in some ILs enhancing protein activity and stabilizing protein structures, with others disrupting protein structures [5,122,123]. The disruptive ILs are effectively a destabilizing agent, acting as a denaturant. Exploiting the ability of some ILs to enhance protein denaturation can yield greater insights into these protein-IL interactions. In one study, ribonuclease A was used to understand the effect of ILs on protein stability and aggregation. Ribonuclease A, a small enzyme, was examined in the presence of ILs such as choline dihydrogen phosphate ( [Dhp] promoted the stability of the native state and increases the chances of refolding, which prevents protein aggregation [124]. In another study, human serum albumin (HSA), was studied in the presence of the ILs 1-butyl-3-methylimidazolium tetrafluoroborate ( 4 ] was shown to induce swelling of HSA loop 1, causing it to be 0.6 nm wider compared to what it is in water, although [Chol] [Dhp] was not able to impart a similar effect [125]. While this is one example, there are numerous reports in the literature comparing numerous proteins with an even greater number of ILs.

Interaction of Ionic Liquids with Metalloproteins
Due to the sheer number of unique proteins found in nature, combined with the everincreasing number of ILs, it is unlikely there will be a set of hard and fast rules that define all IL-protein interactions. As a result, it is important to begin to focus on the interpretation and analysis by refining the types of molecules being investigated. This review focuses on understanding the impact of various ILs on metalloproteins such as laccase, myoglobin, alcohol dehydrogenase, and horseradish peroxidase (HRP).

Effect of ILs on Laccase
Laccase is a metal containing protein containing four copper ions in its active center [126,127]. Laccase was originally isolated from the Japanese lacquer tree Rhus vernicifera. After that, laccases were also found in multiple different plant sources like Rhus succedanea, Acer pseudoplatanus, Pinus taeda, Populus euramericana, Liriodendron tulipifera, and Nicotiana tabacum [128][129][130][131][132][133]. Laccases found from these sources are monomeric proteins that have molecular weights between 90-130 kDa [54]. Notably, they are also highly glycosylated, with carbohydrate content between 22-45% [134,135]. In addition to plant sources, fungi are a common source of laccase, and most fungi produce different laccase isoforms and isoenzymes. One of the most commonly studied forms of laccase is derived from the Trametes versicolor fungus [136][137][138][139]. The T. versicolor laccase contains two copper sites, a mono-copper and a tri-copper site ( Figure 1). The Cu 2+ at the mono-copper site is coordinated by two His and one Cys residue, while the Cu 2+ atoms at the tri-copper site involve coordination of at least 3 His residues and multiple carboxyl containing residues (Asp and Glu) [127,140,141]. Recent studies show laccase is also present in bacteria, although these proteins are less well studied [142][143][144].
Trametes versicolor fungus [136][137][138][139]. The T. versicolor laccase contains two copper sites, a mono-copper and a tri-copper site ( Figure 1). The Cu 2+ at the mono-copper site is coordinated by two His and one Cys residue, while the Cu 2+ atoms at the tri-copper site involve coordination of at least 3 His residues and multiple carboxyl containing residues (Asp and Glu) [127,140,141]. Recent studies show laccase is also present in bacteria, although these proteins are less well studied [142][143][144].  [126,145]). The structure was visualized using Visual Molecular Dynamics (VMD) software. (A) 3D structure of laccase. The N-and C-termini are shown as red and blue spheres, respectively, while the copper ions are shown in orange (partially occluded in the structure). (B) structural geometry of the mono-copper site with chelating residues highlighted.
In nature, laccases typically oxidize phenolic compounds and reduce molecular oxygen into water after several rounds of catalysis [146]. This is typically involved in the synthesis or degradation of naturally occurring plant lignins [147]. Laccase has found utility in bioremediation of waste products from numerous industries, remediation of excess pesticides and herbicides, as well as cleaning of wastewater streams [148]. Additionally, many synthetic organic compounds can be substrates for laccase. Organic substrates of laccase are categorized into three groups: ortho-, meta-, or para-substituted compounds (all with a lone pair of electrons). In most cases of laccase, ortho-substituted compounds work as the better substrate over para-or meta-substituted compounds [144,149]. One of the most useful synthetic laccase substrates is 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), which is a colorimetric substrate allowing spectroscopic monitoring of laccase activity. ABTS was used in monitoring the oxidation of non-phenolic lignin structures, which gave the impetus to find new laccase mediators [150,151]. A particularly interesting application of laccase is in the detoxification of chlorophenol-containing wastewater, which is achieved by laccase-mediated polymerization via radical coupling [152,153]. The industrial applications of laccase, coupled with the straightforward monitoring with ABTS, have made it a very attractive system to study with ILs. A brief summary of studies that have been published on laccase with ILs can be found in Table 1.  [126,145]). The structure was visualized using Visual Molecular Dynamics (VMD) software. (A) 3D structure of laccase. The N-and C-termini are shown as red and blue spheres, respectively, while the copper ions are shown in orange (partially occluded in the structure). (B) structural geometry of the mono-copper site with chelating residues highlighted.
In nature, laccases typically oxidize phenolic compounds and reduce molecular oxygen into water after several rounds of catalysis [146]. This is typically involved in the synthesis or degradation of naturally occurring plant lignins [147]. Laccase has found utility in bioremediation of waste products from numerous industries, remediation of excess pesticides and herbicides, as well as cleaning of wastewater streams [148]. Additionally, many synthetic organic compounds can be substrates for laccase. Organic substrates of laccase are categorized into three groups: ortho-, meta-, or para-substituted compounds (all with a lone pair of electrons). In most cases of laccase, ortho-substituted compounds work as the better substrate over para-or meta-substituted compounds [144,149]. One of the most useful synthetic laccase substrates is 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), which is a colorimetric substrate allowing spectroscopic monitoring of laccase activity. ABTS was used in monitoring the oxidation of non-phenolic lignin structures, which gave the impetus to find new laccase mediators [150,151]. A particularly interesting application of laccase is in the detoxification of chlorophenol-containing wastewater, which is achieved by laccase-mediated polymerization via radical coupling [152,153]. The industrial applications of laccase, coupled with the straightforward monitoring with ABTS, have made it a very attractive system to study with ILs. A brief summary of studies that have been published on laccase with ILs can be found in Table 1.          [157]. However, the authors did not specifically investigate the mechanism of IL inhibition of enzymatic activity [157].
Solution pH is another parameter that is important to understand the stability of laccase in ILs. The isoelectric point (pI) of laccase is 4.6 [160,161] and based on the nature of the IL it would affect its interaction with laccase. For example, the fluorescence intensity of laccase was found to decrease in presence of the IL [TMA][TfO] more at pH 3.6 than at pH 5 [155]. On the other hand, at pH 5.8, the fluorescence intensity of laccase was found to increase in the presence of [TMA][TfO]. At pH 3.6, there is greater contribution from CF 3 SO 3 − anion with respect to its interaction with the laccase interaction and as a chaotropic anion it has higher preference to bind with the protein-water interface and destabilize the enzyme (Hofmeister effect) [108,162]. However, at pH > pI (pH 5.8) the cation [TMA] + is more active in terms of ordering the water structure surrounding enzyme and makes laccase more compact, resulting in increased fluorescence intensity from the greater shielding of buried Trp residues by the bulk polar aqueous milieu [155]. In another study, the effect of laccase activity in the presence of three 1-ethyl-3-methyl imidazolium ILs (with anions [MDEGSO 4 ], [EtSO 4 ] and [MeSO 3 ]) was determined at pH 5, 7, and 9. The results show that at pH 7 and 9, the activity of laccase does not change with the addition of ILs. However, at pH 5 the laccase showed significantly reduced activity overall, but the IL-laccase samples showed a smaller loss of activity, that is, the laccase + IL mixtures performed better than laccase alone at pH 5 [163].
Above  6 ] was used as the IL, laccase activity was found to be higher for the water-in-IL microemulsion compared to pure IL or water-saturated IL [42].
In addition, ILs can impact the biocatalytic activity of the laccase. For instance, aqueous biphasic systems containing IL cholinium dihydrogen citrate ([Chol][DHCit]) have been shown to enhance the extraction efficiency of the enzyme and increase the biocatalytic activity by 50% [164].

Effect of ILs on Myoglobin
Myoglobin ( Figure 2) is a water-soluble globular protein of 150 amino acids involved in transport and storage of oxygen found in mammalian muscle tissues [165,166]. Like laccase, myoglobin is a metalloprotein having an iron atom incorporated in the heme group which together are involved in reversibly binding oxygen [167]. The heme binding site of the protein contains two His residues, one (proximal) is attached directly to the heme iron and the other (distal) is on the opposite face of the heme but does not bind the iron, instead being available for binding to O 2 . The presence of this iron imparts a reddish-brown color to the protein and yields an intense absorption band at~409 nm [168]. The heme group is buried under a hydrophobic pocket of the myoglobin in its native folded state, however, upon unfolding, the heme group is exposed to the aqueous environment, resulting in decrease in the absorption at~409 nm [168]. Because of these easily interrogated absorbance properties, myoglobin has been widely used as a model protein to understand folding and unfolding kinetics as a function of the varieties of conditions involving not only thermal, pH, and mechanical stress, but also a wide range of denaturants such as detergents, organic solvents, and ILs [169][170][171][172]. A brief summary of studies that have been published on myoglobin with ILs can be found in Table 2.    ) were shown to destabilize myoglobin structure. One hypothesis is that ILs affect the stability of a protein by altering the hydration layer (i.e., layer of water molecules around the protein). Specifically, in this case, the authors postulated that phosphate-containing ILs significantly interact with the myoglobin polypeptide chain and hence are repelled from the protein.
In addition, because of these repulsions this IL also helps to provide better structure to the hydration layer, improving the stability of the protein [172]. As the acetate ions have greater affinity toward the polypeptide chain of myoglobin, they penetrate deep inside the protein structure and interact with amino acids of the polypeptide. Therefore, acetate ions present in ILs also disturb the native hydrogen bonding pattern as well as interactions of the protein with the hydration layer, resulting in protein destabilization. Further, results have indicated that anionic variation in the ILs has greater impact on stability of myoglobin compared to the cationic variations (summarized in Table 3) [172]. coworkers; downloaded from rcsb.org (1WLA) [145,173]. The structure was visualized using VMD. (A) 3D structure of myoglobin. The N-and C-termini are shown as red and blue spheres respectively while the heme is shown in orange (partially occluded in the structure). (B) Structural geometry of the heme with the iron shown in black and chelating residues highlighted. coworkers; downloaded from rcsb.org (1WLA) [145,173]. The structure was visualized using VMD. (A) 3D structure of myoglobin. The N-and C-termini are shown as red and blue spheres respectively while the heme is shown in orange (partially occluded in the structure). (B) Structural geometry of the heme with the iron shown in black and chelating residues highlighted. myoglobin. The N-and C-termini are shown as red and blue spheres respectively while the heme is shown in orange (partially occluded in the structure). (B) Structural geometry of the heme with the iron shown in black and chelating residues highlighted.  Table 3. Effect of various ILs on the melting temperature from fluorescence and differential scanning calorimetry (DSC) along with secondary structure composition of myoglobin determined from Far-UV CD spectra (adapted from reference [172]).

Sample
Fluorescence In work from Zhang et al. it was demonstrated that variation in the cations can also influence the stability of myoglobin [178]. They demonstrated that GuHCl-induced denaturation midpoints of myoglobin was not altered when interacted with phosphate buffer having 150 mM of various ILs differing only in their anions such as BF 4 − , NO 3 − , Cl − , and Br − , while keeping the same cation 1-butyl-3-methylimidazolium (BMIM + ) [178]. Furthermore, they have shown that increasing length of alkyl chain of imidazolium cation in the ILs affects denaturation of the myoglobin and the denaturation midpoint were found to be [ [123]. The results from this study indicated that these four ILs accelerate myoglobin unfolding kinetics not only due to changes in the aqueous solution ionic strength, but also due to IL-specific interactions [123]. While, in another study, [EMIM][Ac] did not impact myoglobin stability, but the IL [BMIM][BF 4 ] drastically reduced the free energy required for myoglobin unfolding and hence significantly destabilized the myoglobin structure [62].
In addition, impact of ILs on the detergent-mediated denaturation of myoglobin was also evaluated. According to one study, inclusion of a series of ILs such as 1-butyl-3methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF 4 ) in aqueous solution had negligible impact on the detergent N,N-dimethyl-N-dodecylglycine betaine induced denaturation and heme-loss from myoglobin [177]. In another study, the effect of alkylated imidazolium chlorides based ILs such as [ was tested on unfolding of myoglobin in the presence of different detergents such as N,N-dimethyl-N-dodecylglycine betaine (zwitterionic; Empigen BB ® , and EBB), tetradecyltrimethylammonium bromide (cationic; TTAB), and sodium dodecyl sulfate (anionic; SDS) [78]. It was observed that, presence of ILs does not affect the EBB-and TTAB-induced dissociation of heme; however, SDS-induced dissociation is affected by presence of ILs. Furthermore, it was found that, heme dissociation follow a cooperative process at low IL concentration, while at high IL concentration the heme dissociation occur via more complex pattern, which could be due to micellization of the ILs or their direct interactions with the myoglobin [78].

Effects of IL on Azurin
The blue copper protein, azurin, is part of the azurin-nitrate reductase redox protein complex. This protein is involved in denitrification metabolism in bacteria [87,179]. The presence of copper is necessary for protein stability. It is a small protein that can be produced from two bacterial strains-Pseudomonas aeruginosa and Alcaligenes denitrificans [180]. Azurin's structure from P. aeruginosa consists of a hydrophobic alpha helix, six short beta sheets and a random-coil that allows for copper-binding [87,181,182] (Figure 3). Notably, azurin exhibits the most blue-shifted Trp emission spectrum from naturally derived proteins, arising from the single Trp residue at position 48 [183]. This is attributed to the very hydrophobic interior of the protein, which also includes the copper binding site. The Cu 2+ is coordinated by Gly45, His46, Asn47, Cys112, Phe114, His117, and Met121 [183]. A brief summary of studies that have been published on azurin with ILs can be found in Table 4. beta sheets and a random-coil that allows for copper-binding [87,181,182] (Figure 3). Notably, azurin exhibits the most blue-shifted Trp emission spectrum from naturally derived proteins, arising from the single Trp residue at position 48 [183]. This is attributed to the very hydrophobic interior of the protein, which also includes the copper binding site. The Cu 2+ is coordinated by Gly45, His46, Asn47, Cys112, Phe114, His117, and Met121 [183].
A brief summary of studies that have been published on azurin with ILs can be found in Table 4. ILs can affect the protein structure and its stability based on several characteristics. As a protein with a mixed structure, azurin's stability is affected differently in the presence of ILs.  [Cl] is stronger than the other ILs presented in this study, which were consisting of smaller alkyl chains and a decreased level of hydrophobicity [87]. It is important to note that at the concentrations tested, the [OMIM] [Cl] has been shown to form micelles, These micelle structures likely impact the interactions with the protein, and can potentially form mixed structures with the protein. [87].
In a study by Fujita   ILs can affect the protein structure and its stability based on several characteristics. As a protein with a mixed structure, azurin's stability is affected differently in the presence of ILs.  [Cl] is stronger than the other ILs presented in this study, which were consisting of smaller alkyl chains and a decreased level of hydrophobicity [87]. It is important to note that at the concentrations tested, the [OMIM] [Cl] has been shown to form micelles, These micelle structures likely impact the interactions with the protein, and can potentially form mixed structures with the protein. [87].
In a study by Fujita et al.,  , it was found that these proteins, when dissolved, do not have any disturbance to the active sites found in the proteins. Notably, not all proteins tested were soluble under these conditions. Among those that were soluble, the retention of structural elements was supported by spectral signatures in Raman and CD spectra. Notably, resonance raman spectra showed the peaks near 260 cm −1 and 400 cm −1 for Cu-N and Cu-S, respectively, which was consistent with the spectra for azurin in its native conformation. This indicated that the protein retained its structure and function when dissolved with the IL [184]. structural elements was supported by spectral signatures in Raman and CD spectra. Notably, resonance raman spectra showed the peaks near 260 cm −1 and 400 cm −1 for Cu-N and Cu-S, respectively, which was consistent with the spectra for azurin in its native conformation. This indicated that the protein retained its structure and function when dissolved with the IL [184].
In another study the same IL, hydrated [Chol] [Dhp], was studied to understand the interaction between the IL and azurin, specifically focusing on the redox reaction rate for azurin (dissolved in the IL) and the SAM-AuNP electrode. In the presence of this IL, it was found that the proteins were able to maintain their structure, showing long term and thermal stability. Similar to the previous study explained above, it was found that the active site of the protein was maintained in the presence of the IL using Raman spectroscopy. It was also found that electron transfer rate constant (ks) between azurin and the electrode in the IL (202 s −1 ) was found to be larger than that of the ammonium acetate buffer solution (44 s −1 ) and the reason for this difference could possibly be due to protein shrinkage. Both the buffer and the IL showed that electron transfer reactions were possible at a fast rate; this would mean that this fast rate would be much more stable over a broad range of temperature values and a longer time period for the IL [185]. Temperature-dependent fluorescence and IR Spectroscopy, IR and VCD Spectroscopy temperature jump kinetics ILs affected the protein structure by destabilizing it; however, the degree to which the protein unfolded is dependent on the ionic liquid in terms of hydrophobicity and alkyl chain length. [174] Azurin II was purified and expressed from Alcaligenes Xylosoxidans (Az). Pseudoazurin was isolated from Achromobacter cyclastes IAM 1013 (Paz). CD, Raman spectroscopy, enzyme activity The protein and IL did not have an interaction that caused a disturbance in the structure or function of the protein. [123] Azurin from P. aeruginosa Raman spectroscopy, direct electrochemistry of azurin performed on self-assembled monolayer (SAM)-AuNP Electrode The protein maintained its structure and its active site in the presence of the IL. Fast and stable electron transfer reactions could occur over a range of temperature values at longer periods of time. [175,185]

Effect of ILs on Other Metal Containing Proteins
Impact of ILs has also been evaluated on other metal containing proteins such as horseradish peroxidase, alcohol dehydrogenase etc. As above, the primary purpose for tably, resonance raman spectra showed the peaks near 260 cm −1 and 400 cm −1 for Cu-N and Cu-S, respectively, which was consistent with the spectra for azurin in its native conformation. This indicated that the protein retained its structure and function when dissolved with the IL [184]. In another study the same IL, hydrated [Chol] [Dhp], was studied to understand the interaction between the IL and azurin, specifically focusing on the redox reaction rate for azurin (dissolved in the IL) and the SAM-AuNP electrode. In the presence of this IL, it was found that the proteins were able to maintain their structure, showing long term and thermal stability. Similar to the previous study explained above, it was found that the active site of the protein was maintained in the presence of the IL using Raman spectroscopy. It was also found that electron transfer rate constant (ks) between azurin and the electrode in the IL (202 s −1 ) was found to be larger than that of the ammonium acetate buffer solution (44 s −1 ) and the reason for this difference could possibly be due to protein shrinkage. Both the buffer and the IL showed that electron transfer reactions were possible at a fast rate; this would mean that this fast rate would be much more stable over a broad range of temperature values and a longer time period for the IL [185]. Temperature-dependent fluorescence and IR Spectroscopy, IR and VCD Spectroscopy temperature jump kinetics ILs affected the protein structure by destabilizing it; however, the degree to which the protein unfolded is dependent on the ionic liquid in terms of hydrophobicity and alkyl chain length. [174] Azurin II was purified and expressed from Alcaligenes Xylosoxidans (Az). Pseudoazurin was isolated from Achromobacter cyclastes IAM 1013 (Paz). CD, Raman spectroscopy, enzyme activity The protein and IL did not have an interaction that caused a disturbance in the structure or function of the protein. [123] Azurin from P. aeruginosa Raman spectroscopy, direct electrochemistry of azurin performed on self-assembled monolayer (SAM)-AuNP Electrode The protein maintained its structure and its active site in the presence of the IL. Fast and stable electron transfer reactions could occur over a range of temperature values at longer periods of time. [175,185]

Effect of ILs on Other Metal Containing Proteins
Impact of ILs has also been evaluated on other metal containing proteins such as horseradish peroxidase, alcohol dehydrogenase etc. As above, the primary purpose for CD, Raman spectroscopy, enzyme activity The protein and IL did not have an interaction that caused a disturbance in the structure or function of the protein. [123] Azurin from P. aeruginosa In another study the same IL, hydrated [Chol] [Dhp], was studied to understand the interaction between the IL and azurin, specifically focusing on the redox reaction rate for azurin (dissolved in the IL) and the SAM-AuNP electrode. In the presence of this IL, it was found that the proteins were able to maintain their structure, showing long term and thermal stability. Similar to the previous study explained above, it was found that the active site of the protein was maintained in the presence of the IL using Raman spectroscopy. It was also found that electron transfer rate constant (ks) between azurin and the electrode in the IL (202 s −1 ) was found to be larger than that of the ammonium acetate buffer solution (44 s −1 ) and the reason for this difference could possibly be due to protein shrinkage. Both the buffer and the IL showed that electron transfer reactions were possible at a fast rate; this would mean that this fast rate would be much more stable over a broad range of temperature values and a longer time period for the IL [185]. Temperature-dependent fluorescence and IR Spectroscopy, IR and VCD Spectroscopy temperature jump kinetics ILs affected the protein structure by destabilizing it; however, the degree to which the protein unfolded is dependent on the ionic liquid in terms of hydrophobicity and alkyl chain length. [174] Azurin II was purified and expressed from Alcaligenes Xylosoxidans (Az). Pseudoazurin was isolated from Achromobacter cyclastes IAM 1013 (Paz). Raman spectroscopy, direct electrochemistry of azurin performed on self-assembled monolayer (SAM)-AuNP Electrode The protein maintained its structure and its active site in the presence of the IL. Fast and stable electron transfer reactions could occur over a range of temperature values at longer periods of time. [175,185]

Effect of ILs on Other Metal Containing Proteins
Impact of ILs has also been evaluated on other metal containing proteins such as horseradish peroxidase, alcohol dehydrogenase etc. As above, the primary purpose for Raman spectroscopy, direct electrochemistry of azurin performed on self-assembled monolayer (SAM)-AuNP Electrode The protein maintained its structure and its active site in the presence of the IL. Fast and stable electron transfer reactions could occur over a range of temperature values at longer periods of time. [175,185] In another study the same IL, hydrated [Chol] [Dhp], was studied to understand the interaction between the IL and azurin, specifically focusing on the redox reaction rate for azurin (dissolved in the IL) and the SAM-AuNP electrode. In the presence of this IL, it was found that the proteins were able to maintain their structure, showing long term and thermal stability. Similar to the previous study explained above, it was found that the active site of the protein was maintained in the presence of the IL using Raman spectroscopy. It was also found that electron transfer rate constant (k s ) between azurin and the electrode in the IL (202 s −1 ) was found to be larger than that of the ammonium acetate buffer solution (44 s −1 ) and the reason for this difference could possibly be due to protein shrinkage. Both the buffer and the IL showed that electron transfer reactions were possible at a fast rate; this would mean that this fast rate would be much more stable over a broad range of temperature values and a longer time period for the IL [185].

Effect of ILs on Other Metal Containing Proteins
Impact of ILs has also been evaluated on other metal containing proteins such as horseradish peroxidase, alcohol dehydrogenase etc. As above, the primary purpose for these studies was to understand the how the ILs will influence folding and/or unfolding behavior of these proteins.
Horseradish peroxidase (HRP) is an enzyme having two different metal ions namely, a ferrous ion incorporated in a heme group and a calcium ion (Figure 4). Notably, the hemeiron is directly involved in the catalytic reaction center, while the calcium is structural [186]. The effect of various ILs on activity of the HRP was evaluated using chromogenic substrates. In one study, the effect of various ILs as well as hemin and calcium cofactors were evaluated for effects on the refolding properties of HRP. This study used ILs with varying anions such as EMIM with Ac − , BF 4 − , Cl − , ES − , and TfO − , as well as with different alkyl chain lengths such as EMIM + , BMIM + , HMIM + , and OMIM + [187]. Among various tested anions, Cl − based ILs showed highest enzyme activity, while, among various ILs having different alkyl chain lengths, EMIM showed highest enzyme activity [187]. Notably, in the presence of IL [BMIM] [PF 6 ], the activity of HRP was also shown to be enhanced [188]. Moreover, HRP immobilized on a sol-gel matrix prepared from [BMIM] [BF 4 ] and silica was shown to have 30-fold higher activity compared to that of the enzyme immobilized on only silica gel [189]. 15 of 27 these studies was to understand the how the ILs will influence folding and/or unfolding behavior of these proteins.
Horseradish peroxidase (HRP) is an enzyme having two different metal ions namely, a ferrous ion incorporated in a heme group and a calcium ion (Figure 4). Notably, the heme-iron is directly involved in the catalytic reaction center, while the calcium is structural [186]. The effect of various ILs on activity of the HRP was evaluated using chromogenic substrates. In one study, the effect of various ILs as well as hemin and calcium cofactors were evaluated for effects on the refolding properties of HRP. This study used ILs with varying anions such as EMIM with Ac − , BF4 − , Cl − , ES − , and TfO − , as well as with different alkyl chain lengths such as EMIM + , BMIM + , HMIM + , and OMIM + [187]. Among various tested anions, Cl − based ILs showed highest enzyme activity, while, among various ILs having different alkyl chain lengths, EMIM showed highest enzyme activity [187]. Notably, in the presence of IL [BMIM][PF6], the activity of HRP was also shown to be enhanced [188]. Moreover, HRP immobilized on a sol-gel matrix prepared from [BMIM][BF4] and silica was shown to have 30-fold higher activity compared to that of the enzyme immobilized on only silica gel [189].  [145,186]. The structure was visualized using VMD. The N-and C-termini are shown as red and blue spheres respectively while the calcium ions are shown in green, the heme in orange and the heme-iron in black.
A tailor-made IL specifically designed to work with HRP was also developed, which has the cation tetrakis(2-hydroxyethyl)ammonium 2 possible anions:Cl − or [CF3SO3] [190]. This tailor-made IL has a structure similar to TRIS (buffer), possessing four hydroxyethyl moieties. Improvement in the enzyme activity was observed with the tailor-made ILs compared to that of the common, commercially available ILs such as [ [191]. Furthermore, [BMIM][BF4] is also capable of enhancing the reaction yield and purity for the reactions converting water insoluble phenolic compounds to a novel compound 4-phenylphenol ortho dimer [2,2′bi-(4-phenylphenol)] [192]. However, the enzymatic catalysis was sensitive to solution pH with the best catalytic activity observed with [BMIM][BF4] (90% v/v IL in water) at pH > 9. The enzyme activity was found to decrease as the pH was shifted toward neutral and as pH decreases further, the [BMIM][BF4] exerts inhibitory action on the HRP attributed to  [145,186]. The structure was visualized using VMD. The N-and C-termini are shown as red and blue spheres respectively while the calcium ions are shown in green, the heme in orange and the heme-iron in black.
A tailor-made IL specifically designed to work with HRP was also developed, which has the cation tetrakis(2-hydroxyethyl)ammonium 2 possible anions:Cl − or [CF 3 SO 3 ] [190]. This tailor-made IL has a structure similar to TRIS (buffer), possessing four hydroxyethyl moieties. Improvement in the enzyme activity was observed with the tailor-made ILs compared to that of the common, commercially available ILs such as [ 4 ] is capable of improving the thermal stability of the horseradish peroxidase when used at a concentration of 5-10% (v/v) [191]. Furthermore, [BMIM] [BF 4 ] is also capable of enhancing the reaction yield and purity for the reactions converting water insoluble phenolic compounds to a novel compound 4-phenylphenol ortho dimer [2,2 -bi-(4-phenylphenol)] [192]. However, the enzymatic catalysis was sensitive to solution pH with the best catalytic activity observed with [BMIM] [BF 4 ] (90% v/v IL in water) at pH > 9. The enzyme activity was found to decrease as the pH was shifted toward neutral and as pH decreases further, the [BMIM] [BF 4 ] exerts inhibitory action on the HRP attributed to the tetrafluoroborate anion releasing fluoride ions which bind with the heme iron group [192].
Alcohol dehydrogenase is another commonly studied metalloenzyme which has zinc ions in the active structure. The Saccharomyces cerevisiae alcohol dehydrogenase has a homotetrameric structure with each subunit having a zinc ion in the catalytic center ( Figure 5) [193]. The major function of this enzyme is to carry out oxidation of alcohols using the co-substrate β-nicotinamide adenine dinucleotide (NAD + ). This is a thoroughly studied model system that, in yeast, converts acealdehyde into ethanol along with formation of NADH and H + . The active site contains the Zn 2+ atoms coordinated by Cys, His, and Glu residues [194]. In one study, the activity and stability of the yeast alcohol dehydrogenase was evaluated in solutions containing various ILs including 1methylimidazolium chloride ( [Cl] with the adenine moiety of NAD + was proposed to allow interaction with the active site and hence stabilize the enzyme at higher temperature [195]. In another study, the effect of [BMIM][PF 6 ] on the yeast alcohol dehydrogenase was investigated and the data indicated a rapid decrease in the activity of the enzyme as a function of [BMIM][PF 6 ] concentration [196]. the tetrafluoroborate anion releasing fluoride ions which bind with the heme iron group [192]. Alcohol dehydrogenase is another commonly studied metalloenzyme which has zinc ions in the active structure. The Saccharomyces cerevisiae alcohol dehydrogenase has a homotetrameric structure with each subunit having a zinc ion in the catalytic center ( Figure  5) [193]. The major function of this enzyme is to carry out oxidation of alcohols using the co-substrate β-nicotinamide adenine dinucleotide (NAD + ). This is a thoroughly studied model system that, in yeast, converts acealdehyde into ethanol along with formation of NADH and H + . The active site contains the Zn 2+ atoms coordinated by Cys, His, and Glu residues [194]. In one study, the activity and stability of the yeast alcohol dehydrogenase was evaluated in solutions containing various ILs including 1-methylimidazolium chloride ( [Cl] with the adenine moiety of NAD + was proposed to allow interaction with the active site and hence stabilize the enzyme at higher temperature [195]. In another study, the effect of [BMIM][PF6] on the yeast alcohol dehydrogenase was investigated and the data indicated a rapid decrease in the activity of the enzyme as a function of [BMIM][PF6] concentration [196]. The crystal structure was solved by Ramaswamy and coworkers; downloaded from rcsb.org (5ENV) ( [145,193]. The structure was visualized using VMD. The N-and C-termini are shown as red and blue spheres respectively while the zinc ions are shown in black. The structure represents one monomer of a homotetramer.
The effect of variation of the anionic and cationic moieties in the ILs has also been investigated on the stability of the yeast alcohol dehydrogenases [197]. Regarding [197]. On the other hand, for variation in the cation, the enzyme deactivating order was found to be [Chol] + > [EMIM] + > [Et4N] + > Figure 5. 3D Structure of alcohol dehydrogenase from Saccharomyces cerevisiae. The crystal structure was solved by Ramaswamy and coworkers; downloaded from rcsb.org (5ENV) ( [145,193]. The structure was visualized using VMD. The N-and C-termini are shown as red and blue spheres respectively while the zinc ions are shown in black. The structure represents one monomer of a homotetramer. The effect of variation of the anionic and cationic moieties in the ILs has also been investigated on the stability of the yeast alcohol dehydrogenases [197]. Regarding [197]. In addition, the effect of ILs on a bacterial alcohol dehydrogenase obtained from Thermoanaerobacter brockii (TBADH) was also investigated. Specifically, the impact of ILs such as [ 4 ]. This study also showed that in ILs with similar anions, the activity depends on the alkyl chain length of imidazolium as well as structural similarity of cations to that of the substrate; because of this structure similarity these ILs to that of the enzyme subtract they act as an enzyme inhibitor [198]. As a result of the structural similarity of MIM ILs to that of substrate (NADP+), it was proposed that reduction in activity caused by this IL and the related [BMIM] were due to direct substrate competition rather than kosmotropic interactions with bulk water [198].
Glucose isomerase is a homotetrameric metalloenzyme with four catalytic centers and promiscuous functionality ( Figure 6) [199]. The enzyme catalyzes reversible isomerizations of D-glucose to D-fructose as well as D-xylose to D-xylulose. Each of the catalytic centers has two subunits that form a pocket-like shape and have two divalent metal ion binding sites. Glucose isomerase is usually associated with metal ions like Mg 2+ , Co 2+ , or Mn 2+ , or a combination of these [200]. The active site contains the metal ions and several critical carboxyl containing residues (Asp and Glu) as well as a His residue involved in proton transfer. Glucose isomerase is a very important industrial enzyme for petroleum and food applications as it is used for production of ethanol for fuel as well as high fructose corn syrup [200]. One study compared effects of various ILs on the activity of glucose isomerase toward converting glucose to fructose [201] . This study also showed that in ILs with similar anions, the activity depends on the alkyl chain length of imidazolium as well as structural similarity of cations to that of the substrate; because of this structure similarity these ILs to that of the enzyme subtract they act as an enzyme inhibitor [198]. As a result of the structural similarity of MIM ILs to that of substrate (NADP+), it was proposed that reduction in activity caused by this IL and the related [BMIM] were due to direct substrate competition rather than kosmotropic interactions with bulk water [198]. Glucose isomerase is a homotetrameric metalloenzyme with four catalytic centers and promiscuous functionality ( Figure 6) [199]. The enzyme catalyzes reversible isomerizations of D-glucose to D-fructose as well as D-xylose to D-xylulose. Each of the catalytic centers has two subunits that form a pocket-like shape and have two divalent metal ion binding sites. Glucose isomerase is usually associated with metal ions like Mg 2+ , Co 2+ , or Mn 2+ , or a combination of these [200]. The active site contains the metal ions and several critical carboxyl containing residues (Asp and Glu) as well as a His residue involved in proton transfer. Glucose isomerase is a very important industrial enzyme for petroleum and food applications as it is used for production of ethanol for fuel as well as high fructose corn syrup [200]. One study compared effects of various ILs on the activity of glucose isomerase toward converting glucose to fructose [201]. showed the highest fructose production (of about 52%) in comparison to other ILs, when the final water content was kept at 21% w/w. In addition, [DBEA][Oc] was the only IL which was also able to produce mannose at 2% w/w, while all other ILs showed intermediate fructose production. These results indicate that the presence of ILs can significantly affect enzyme activity/stability and it is important to screen multiple ILs to find the one which provides optimum results [201].  [145,199] The structure was visualized using VMD. The N-and C-termini are shown as red and blue spheres respectively while the manganase ions are shown in tan and the magnesium ions shown in cyan. The structure represents one monomer of a homodimer.
ILs have also shown to impact the crystallization and X-ray diffraction resolution for glucose isomerase [202]. For instance, in a study by Judge et  . Among all ILs the triisobutyl (methyl) phosphonium p-toluenesulfonate was shown to produce bigger crystals with a change in the morphology of glucose isomerase crystals compared to control samples without ILs [202]. However, proper optimization of the IL concentration during the crystallization is necessary because in some cases higher amounts of IL might negatively impact the crystal. For example, when crystallization of glucose isomerase was carried out with [BMIM][Cl] at 0 M, 0.2 M, and 0.4 M, plate-like crystals of glucose isomerase were obtained only with 0.2 M IL, while the samples with no IL gave salt precipitates and samples with 0.4 M IL did not yield any crystals or precipitates [203]. Furthermore, a synergistic effect was observed when ILs were combined with other techniques that also promote enzyme activity. For instance, the activity of immobilized glucose isomerase and reaction yield for glucose conversion to fructose was found to be highest when [EMIM] [Cl] was used in combination of ultrasound irradiation, compared to use of only the IL or ultrasound irradiation individually [204].

Conclusions/Perspective
Depending on the physicochemical properties of ILs such as polarity, alkyl chain length in cation, anions in IL, hydrophobicity, and viscosity, ILs can have differential effects on protein stability. Some ILs have been shown to improve the stability of proteins, some are inert, and others disruptive to protein structure and function. Because of these unique properties, ILs have applications in multiple fields such as chemistry/synthesis, biotech, pharmaceutical, and the electronics industries. Specifically, ILs that have been shown to stabilize proteins can potentially be beneficial in developing formulations of protein therapeutics or in industrial processes using biocatalysts.
As the protein stabilization or destabilization is very specific to the chemistry of ILs, a rational selection of IL for protein under investigation is necessary before using it as a solvent for improving protein stability or activity. There is only limited knowledge regarding the mechanism of protein stabilization or destabilization in the presence of ILs and therefore research is still needed to understand fundamental chemistry of ILs and how they interact with proteins. This is a crucial step before ILs can be effectively incorporated into protein production, purification, or biocatalytic processes. These experiments, in total, should aim to develop a predictive model for IL-biomolecule systems which varies both the cation and anion of the IL based on the properties and functional environment of the protein. This is a critical but challenging process because of the variability in IL compositions, ongoing development of new ILs, and the variability and complexity between different proteins.
One approach which has been recently described is instead of single entities, mixtures of different ILs have also been used for obtaining better protein stability [205]. In addition to experimental approaches for evaluating the effect on ILs on the protein stability, various in silico analyses have also been performed. For instance, a study using molecular dynamics simulation analysis indicated that in the presence of ILs the bovine serum albumin does not destabilize the structure it adopts, which was also confirmed by experimental analysis [206]. These molecular dynamics simulations will undoubtedly help to narrow the field of potential IL candidates for specific protein and biomolecular applications.
Importantly, in the study of metalloproteins with ILs, there are still numerous questions regarding mechanism of IL-protein interactions. Most importantly, the majority of studies focus on the protein structure for obvious reasons. However, it leaves any direct interactions between ILs and the metal ions ambiguous. While in most cases it is clear from spectroscopic measurements that the metal ions are no longer properly coordinated in the protein structure, which was the initial driving force? Do IL interactions directly with the metal cause a destabilization in the protein or does destabilization of the protein cause a loss or displacement of the metal ion from the native binding site? While the latter is intuitive, there is only preliminary direct evidence. Additional studies that directly interrogate the metal sites such as vibrational methods and magnetic circular dichroism will help shed light on this question.
Another important aspect that must be considered when discussing IL-biomolecule applications is toxicity. The ability of a specific IL to stabilize a protein structure does not inherently mean it will be stabilizing to all proteins and may cause cytotoxic effects through other mechanisms. Similarly, there is no guarantee that because an IL is well tolerated by one organism that it will be equally biocompatible with all organisms. As such, the study of IL toxicity is an ongoing and rich area of research with numerous groups focused on this problem. Many studies have shown that some ILs can exhibit environmental toxicity or organismal cytotoxicity [207][208][209]. Alternatively, there are numerous examples in the literature of ILs that exhibit low levels of cytotoxicity, encouraging the investigation of these formulations for biological and pharmaceutical applications [207,208,[210][211][212][213]. Our own work has shown that the cytotoxicity of ILs with imidazolium-based cations is dependent on alkyl chain length but can be used synergistically with traditional antimicrobials well below the cytotoxicity window against human cells [214,215]. These findings parallel that of many other groups which have shown a link between lipophilicity and cytotoxicity for ILs [216,217]. However, in light of the vast number of IL species combined with the breadth of biological species, it is necessary to expand the throughput of screening IL toxicity. Many groups have employed computational QSAR approaches to build predictive models of IL toxicity to cells [216,[218][219][220]. These studies can potentially yield a great deal of insight for experimentalists in the design of IL formulations for specific applications.
Finally, the significance and importance of metalloproteins will continue to grow. Numerous industrial processes rely on metalloproteins for catalysis. These include enzymes such as metalloproteases, laccases, cellulases, lipases, phosphatases, and amylases [221,222]. Further, some of the metalloproteins are involved in the progression of the cancer and other diseases [223]. Once suitable ILs are identified and their effects on a given protein have been thoroughly evaluated, they can be successfully be used in combination with those targets to enhance or reduce activity. Because of having these beneficial properties, ILs have potential to serve as an ideal vehicle for protein therapeutics, a combinatorial therapeutic component, and an activity-enhancing additive in industrial processes in the near future.
Author Contributions: Writing-original draft preparation, A.Y.P. and K.S.J.; writing-review and editing, G.A.C., C.W., and T.D.V.; visualization, N.P. and C.W.; funding acquisition, T.D.V., C.W., and G.A.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Science Foundation, grant number DMR 1904797.

Conflicts of Interest:
The authors declare no conflict of interest.