Role of Phoh and Tyrosine in Selective Oxidation of Hydrocarbons

catalysts

Article Role of PhOH and Tyrosine in Selective Oxidation of Hydrocarbons

Ludmila Matienko * , Vladimir Binyukov † , Elena Mil and Alexander Goloshchapov

Emanuel Institute of Biochemical Physics, Russian Academy of Science, 4 Kosygin Street, 119334 Moscow, Russia; [email protected] (V.B.); [email protected] (E.M.); [email protected] (A.G.) * Correspondence: [email protected] or [email protected] † Deceased.

Abstract: Earlier, we established that nickel or iron heteroligand complexes, which include PhOH (nickel complexes) or tyrosine residue (nickel or iron complexes), are not only hydrocarbon oxidation catalysts (in the case of PhOH), but also simulate the active centers of enzymes (PhOH, tyrosine). The AFM method established the self-organization of nickel or iron heteroligand complexes, which included tyrosine residue or PhOH, into supramolecular structures on a modified silicon surface. Supramolecular structures were formed as a result of H-bonds and other non-covalent intermolecular interactions and, to a certain extent, reflected the structures involved in the mechanisms of reactions of homogeneous and enzymatic catalysis. Using the AFM method, we obtained evidence at the model level in favor of the involvement of the tyrosine fragment as one of the possible regulatory factors in the functioning of Ni(Fe)ARD dioxygenases or monooxygenases of the family of cytochrome P450. The principles of actions of these oxygenases were used to create highly efficient catalytic systems for the oxidation of hydrocarbons. Keywords: AFM; self-organization; PhOH; Tyr fragment; Ni(or Fe)-acac complexes; nanostructures; Citation: Matienko, L.; Binyukov, V.; Ni(Fe)ARD dioxygenases; cytochrome P450 monooxygenases; H-bonds; hemin; L-histidine; L-tyrosine Mil, E.; Goloshchapov, A. Role of PhOH and Tyrosine in Selective Oxidation of Hydrocarbons. Catalysts 2021, 11, 1032. https://doi.org/ 1. Introduction 10.3390/catal11091032 The development of industrial processes for the oxidation of hydrocarbons is determined by the ability of researchers to control these processes. An effective method of Academic Editor: Sónia Carabineiro controlling the rate and mechanism of hydrocarbon oxidation is the use of a catalyst. Currently, economically justified works in the field of homogeneous catalytic oxidation Received: 5 July 2021 of hydrocarbons in the world are carried out in two directions: free radical chain oxidation, Accepted: 22 August 2021 Published: 26 August 2021 catalyzed by complexes of transition metals, and catalysis by metal complexes that simulate the action of enzymes [1]. The latter direction has not yet experienced proper development

Publisher’s Note: MDPI stays neutral due to a low degree of conversion of hydrocarbons, which is associated with the rapid with regard to jurisdictional claims in deactivation of catalysts. published maps and institutional affil- The attention of researchers studying the mechanisms of action of axial modifying iations. ligands that control the activity of metal complex catalysts is, as a rule, focused on the steric and electron-donating properties of ligands. The influences in the outer coordination sphere, the role of hydrogen bonds, and other non-covalent interactions have been studied to a much lesser extent. The formation of supramolecular structures due to hydrogen bonds is characteristic of the catalysis mechanism. In addition, hydrogen bonds are known to Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. play an important role in biology. The facts of the formation of supramolecular structures This article is an open access article open up the possibility of assessing the existence of non-covalent interactions [1]. distributed under the terms and For the first time, we used atomic force microscopy (AFM) to investigate the possi- conditions of the Creative Commons bility of assessing H-bonds and the formation of supramolecular nanostructures based Attribution (CC BY) license (https:// on heteroligand complexes of nickel and iron, which are effective catalysts for the oxida- creativecommons.org/licenses/by/ tion of ethylbenzene to α-phenyl ethyl hydroperoxide and are simultaneously models of 4.0/). Ni(Fe)ARD dioxygenase [2,3].

Catalysts 2021, 11, 1032. https://doi.org/10.3390/catal11091032 https://www.mdpi.com/journal/catalysts Catalysts 2021, 11, 1032 2 of 12

In this self-review article, we discuss the participation of phenol and supramolecular structures in the mechanisms of selective catalysis of hydrocarbons, as well as, at the model level, the probable role of the Tyr fragment and supramolecular structures in the mechanisms of action of Ni(Fe)ARD dioxygenases and cytochrome P450 monooxygenases using the AFM method. We present for the ﬁrst time the AFM image of supramolecular structures based on {Hem+PhOH+His} complexes, as well as the design of the structures of {Hem+Tyr+His} complexes based on the obtained AFM data. The data collected can be considered a step towards understanding regulator activity of Tyr (and His) in the active sites of enzymes Ni(Fe)ARD dioxygenases or the family of cytochrome P450-dependent monooxygenases, the action principles of which were used to create highly efﬁcient catalytic systems.

2. Results 2 2.1. Triple Systems Ni(acac)2+L +PhOH That Are the Catalysts of Reaction of Selective Ethylbenzene Oxidation in α-Phenyl Ethyl Hydroperoxide Some of the most efficient catalytic systems for the oxidation of ethylbenzene to α-phenyl ethyl hydroperoxide (PEH), developed by L.I. Matienko (and also by L.A. 2 Mosolova), are three-component Ni-containing catalytic systems {Ni(acac)2+L +PhOH} (L2 = N-methyl-2-pirrolidone (NMP), HMPA, MSt (M = Li, Na)) [1,4]. Studies observed the synergistic effect of an increase in the selectivity (S) and conversion (C) of ethylbenzene oxidation into PEH with additions of a third component, phenol PhOH, to the 2 reaction, catalyzed by the {Ni(acac)2+L } two-component system. This fact indicated the 2 formation of active triple complexes {Ni(acac)2+L +PhOH} [1,4]. The reaction rate during oxidation practically did not change until the conversion of ethylbenzene C = 20–30% (S = 87–90%) [1]. At that point, unlike the catalysis in the majority of research using 2 binary systems {Ni(acac)2+L }, in the course of the reaction of ethylbenzene oxidation, catalyzed with triple systems, the byproducts AP (acetophenone) and MPC (methylphenyl- carbinol) were formed parallel to PEH throughout the process [1,4]. Researching kinetic data indicated the important role of intra- and intermolecular H-bonds in these catalytic reactions [1,4]. 2 As follows from the kinetic data, the higher efficiency of the {Ni(acac)2·L ·PhOH} 2 triple system in comparison with catalysis by the {Ni(acac)2·L } binary system is appar- 2 ently due to the fact that the {Ni(acac)2·L ·PhOH} system is more stable and apparently does not undergo oxidative transformation during the oxidation of ethylbenzene. When 2 catalyzed by a two-component system {Ni(acac)2·L }, during the ethylbenzene oxidation a selective form of the catalyst was formed as a result of electrophilic inclusion of molecular oxygen to the γ-C atom of one of the acac- ligands [1]. In this case, an active selective cata- 2 lyst was an intermediate form of oxidation of the complex Ni(acac)2·L ,-Ni2(acac)(OAc)3 2- 2 L •2H2O (“A”) (L = NMP, MSt: M = Na, Li). (This is stage II of the oxidation process. 2 In stage I, there were the prime complex Ni(acac)2·L forms.) The decrease in the S of the oxidation process into PEH was associated with the formation of Ni(OAc)2, a product of 2 the complete oxidation of Ni(acac)2·L with molecular oxygen. Ni(OAc)2 was an effective catalyst for the heterolysis of PEH into phenol and acetaldehyde products (stage III of the 2 process) [1]. We established that the conversion of complex Ni(acac)2·L into active form “A” (stage II of the oxidation process) proceeded with a similar mechanism of action for NiARD dioxygenase (see below) [1]. It is known that heteroligand complexes are more active in reactions with electrophiles than homoligand complexes [1]. It is possible that the stability of heteroligand complexes Ni2(OAc)3(acac)NMP•2H2O (“A”) with respect to electrophilic inclusion of O2 can be explained by the formation of supramolecular structures due to intermolecular hydrogen bonds (H2O-MP, H2O-acetate (or acac-) group) [1]. We synthesized complex “A”, and its structure was established using various physicochemical methods: mass spectrometry, electronic and IR spectroscopy, and elemental analysis [1]. It was confirmed by kinetic methods that complexes “A” actually catalyzed the selective oxidation of ethylbenzene Catalysts 2021, 11, x FOR PEER REVIEW 3 of 12

its structure was established using various physicochemical methods: mass spectrometry, electronic and IR spectroscopy, and elemental analysis [1]. It was confirmed by kinetic methods that complexes “A” actually catalyzed the selective oxidation of ethylbenzene in Catalysts 2021, 11, 1032 PEH [1]. Figure 1a shows the likely structure of the supramolecular binuclear complex3 of 12 Ni2(OAc)3(acac)NMP•2H2O (“A”). Using atomic force microscopy (AFM), we showed for the first time the self-organi- inzation PEH of [1 supramolecular]. Figure1a shows complexes the likely Ni structure2(OAc)3(acac)NMP of the supramolecular•2H2O due to binuclear H-bonds complex and perhaps other non-covalent interactions (Figure 1b) [2,3]. Ni2(OAc)3(acac)NMP•2H2O (“A”).

CH3 H3C CH O 3 O H O O Ni O O H Ni O H C 3 O O H O O H

N CH3 CH3

(a) (b)

Figure 1. (a) Structure of the complex Ni2(OAc)3(acac)NMP·2H2O (“A”); (b) the AFM 2D and 3D images of nanoparticles Figure 1. (a) Structure of the complex Ni2(OAc)3(acac)NMP∙2H2O (“A”); (b) the AFM 2D and 3D images of nanoparticles based on Ni (OAc) (acac)NMP·2H O (2 µm × 2 µm). based on Ni22(OAc)33(acac)NMP∙2H2O (2μm × 2μm). Using atomic force microscopy (AFM), we showed for the first time the self-organization The stability and efficiency of triple complexes {Ni(acac)2∙L2∙PhOH}, catalyzing the of supramolecular complexes Ni (OAc) (acac)NMP•2H O due to H-bonds and perhaps oxidation of ethylbenzene in PEH,2 could3 be associated with2 the formation of supramolec- other non-covalent interactions (Figure1b) [2,3]. ular structures due to intermolecular hydrogen bonds (phenol-carboxylate bonds), as well The stability and efficiency of triple complexes {Ni(acac) ·L2·PhOH}, catalyzing the as other non-covalent interactions (Matienko LI, Mosolova LA:2 [1,4]). This assumption, oxidation of ethylbenzene in PEH, could be associated with the formation of supramolecu- based on kinetic data, is confirmed by UV spectroscopy data, which indicate the intra- lar structures due to intermolecular hydrogen bonds (phenol-carboxylate bonds), as well 2 3 3 asand other outer-sphere non-covalent coordination interactions of the (Matienko activating LI, ligands Mosolova L = NMP LA: [ 1and,4]). L This(in the assumption, case of L based= L-tyrosine) on kinetic with data, Ni(acac) is confirmed2 [4]. by UV spectroscopy data, which indicate the intra- and outer-sphereThe possibility coordination of the formation of the activatingof intermolecular ligands Lbonds2 = NMP (H-bonds and L 3and(in theother case non- of covalent3 interactions) and supramolecular structures based on triple complexes L = L-tyrosine) with Ni(acac)2 [4]. 2 2 {Ni(acac)The2 possibility∙L ∙PhOH} of(L the = N-methyl-2-pyrrolidone formation of intermolecular (NMP), bonds HMPA, (H-bonds MSt (M and = otherLi, Na)), non- is covalentevidenced interactions) by the data we and obtained supramolecular using the AFM structures method (Figure based 2). on The triple supramolecular complexes Catalysts 2021, 11, x FOR PEER REVIEWstructures · were2· formed2 because of fixation of the {Ni(acac)2∙L2∙PhOH} systems on a4 spe-of 12 {Ni(acac)2 L PhOH} (L = N-methyl-2-pyrrolidone (NMP), HMPA, MSt (M = Li, Na)), iscially evidenced prepared by thesilicon data surface we obtained due to usingH-bonds the AFMand subsequent method (Figure spontaneous2). The supramolec- self-organization in nanostructures due to intermolecular H-bonds and, possibly,2 other non-covalent ular structures were formed because of fixation of the {Ni(acac)2·L ·PhOH} systems on ainteractions specially prepared [5]. silicon surface due to H-bonds and subsequent spontaneous self- organization in nanostructures due to intermolecular H-bonds and, possibly, other non- covalent interactions [5].

(a) (b)

FigureFigure 2. 2.( a(a)) AFM AFM 3D 3D image image of of structures structures with (5.0 h~80–100 × 5.0 (µm)) nm, with formed h∼80–100 on a modiﬁed nm, formed silicon on surface, a modified based silicon on triple surface, complexes based μ μ ∼ Ni(acac)on triple2· NMPcomplexes·PhOH Ni(acac) (3.5 µm2∙NMP× 3.5∙µPhOHm); (b (3.5) AFMm 3D× 3.5 imagem); of(b) structures AFM 3D image (30 µm of× structures30 µm) with (30 h~300× 30 (µm)) nm, basedwith h on300 triple nm, complexesbased on triple Ni(acac) complexes2·NaSt·PhOH. Ni(acac)2∙NaSt∙PhOH.

The formation of supramolecular structures due to H-bonds was also observed in the case of binary complexes {Ni(acac)2∙L2}. However, these nanoparticles differed in shape from structures based on three-component systems {Ni(acac)2∙L2 L3} and had a lower height of h~8 nm (Figures 2 and 3) [5].

2.2. Possible Effect of Tyr Fragment Hydrogen bonds have importance in catalysis and biology. The variation of H-bond cleavage energies is from~15–40 kcal/mol (the strongest interactions) to ~4 kcal/mol (the weakest ones). Strong hydrogen bonds are covalent in nature, and a larger electrostatic contribution is attributed to weak hydrogen bonds [6,7]. Due to the relatively large phenol-amphipathic side chain, tyrosine residues located in different regions of the protein are involved in the formation of various hydrogen bonds [8] and in conformation and molecular recognition [9]. The role of tyrosine in enzymatic reactions is no less important. Involvement of tyrosine residues in the mechanism of action of heme oxygenase (HO) was studied. It is known that the hemeless HO enzyme catalyzes the decomposition of protoporphyrin IX (Por)Fe(III) to biliverdin, CO, and Fe(II). In [10], in the mechanism of action of isoform-1 of human hemoxygenase (hHO-1), the role of tyrosyl radicals, which are formed after the reaction of conversion of Fe(III)(Por) to Fe(IV) = O(Por (+)), is discussed. The Tyr fragment can take part in O2 activation with an iron-containing catalyst. It is assumed that binding through the Tyr fragment can lead to a decrease in the rate of О2 oxidation of the substrate in catalysis by homoprotocatechuate 2.3-dioxygenase [11]. The roles of Tyr and His fragments in the transfer of the methyl group from S-adenosylmethionine (AdoMet) in dopamine are discussed in [12,13]. The QM calculations suggest that carbonyl group of His-293 engaged in substantially stronger CH•••O hydrogen bonding to AdoMet than the tyrosine side chain. It was assumed, based on a model of methyltransferase and a structural study, that methyl CH•••O H-bonding represents a function of AdoMet-dependent methyltransferases as the universal mechanism for methyl transfer [13]. Tyr149 was found in astacin endopeptidases and serralisines. Tyr149, donating a proton, bonded with zinc as the fifth ligand. This switch played an important role, taking part in the stabilization of the transition state when the substrate bound to the enzyme [14].

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Scheme 1. The structure of NiARD with Tyr residue in the outer coordination sphere of a nickel ion [18].

Using the AFM method, we obtained convincing evidence on the model level in favor Catalysts 2021, 11, 1032 of the involvement of a tyrosine fragment in the stabilization of primary NiARD com- 4 of 12 plexes as one of the possible regulatory factors in the Ni(Fe)ARD action. For the first time, we observed self-organization of model triple complexes including Tyr and His fragments {Ni(acac)2•NMP•Tyr}, {Ni(acac)2•His•Tyr} (His = L-histidine, Tyr = L-tyrosine) into very stable supramolecular structures due to H-bonds and possibly other non-covalent inter- Theactions formation (Figure 3 of) [5]. supramolecular structures due to H-bonds was also observed in the 2 case of binary complexes {Ni(acac)2·L }. However, these nanoparticles differed in shape 2 3 from structures based on three-component systems {Ni(acac)2·L L } and had a lower height of h~8 nm (Figures2 and3)[5].

(a) (b)

Figure 3. (a) 3D AFM image of nanostructures based on systems {Ni(acac)2 His Tyr} (h = 45 nm) (4.5 µm × 4.5 µm); Figure 3. (a) 3D AFM image of nanostructures based on systems {Ni(acac)2 His Tyr} (h = 45 nm) (4.5m × 4.5m); (b) 3D (b) 3D AFM imageAFM of image nanostructures of nanostructures based based on on systems systems {Ni(acac) {Ni(acac)2NMPTyr}2·NMP (h = 25· nm)Tyr} (2 (hm =× 2 25m) nm).μm (2 µm × 2 µm). 2.2. Possible Effect of Tyr Fragment Hydrogen bonds have importance in catalysis and biology. The variation of H-bond cleavage energies is from~15–40 kcal/mol (the strongest interactions) to ~4 kcal/mol (the weakest ones). Strong hydrogen bonds are covalent in nature, and a larger electrostatic contribution is attributed to weak hydrogen bonds [6,7]. Due to the relatively large phenol-amphipathic side chain, tyrosine residues located in different regions of the protein are involved in the formation of various hydrogen bonds [8] and in conformation and molecular recognition [9]. The role of tyrosine in enzymatic reactions is no less important. Involvement of tyrosine residues in the mechanism of action of heme oxygenase (HO) was studied. It is known that the hemeless HO enzyme catalyzes the decomposition of protoporphyrin IX (Por)Fe(III) to biliverdin, CO, and Fe(II). In [10], in the mechanism of action of isoform-1 of human hemoxygenase (hHO-1), the role of tyrosyl radicals, which are formed after the reaction of conversion of Fe(III)(Por) to Fe(IV) = O(Por (+)), is discussed. The Tyr fragment can take part in O2 activation with an iron-containing catalyst. It is assumed that binding through the Tyr fragment can lead to a decrease in the rate of O2 oxidation of the substrate in catalysis by homoprotocatechuate 2.3-dioxygenase [11]. The roles of Tyr and His fragments in the transfer of the methyl group from S-adenosylmethionine (AdoMet) in dopamine are discussed in [12,13]. The QM calculations suggest that carbonyl group of His-293 engaged in substantially stronger CH•••O hydrogen bonding to AdoMet than the tyrosine side chain. It was assumed, based on a model of methyltransferase and a structural study, that methyl CH•••O H-bonding represents a function of AdoMet-dependent methyltransferases as the universal mechanism for methyl transfer [13]. Tyr149 was found in astacin endopeptidases and serralisines. Tyr149, donating a proton, bonded with zinc as the ﬁfth ligand. This switch played an important role, taking part in the stabilization of the transition state when the substrate bound to the enzyme [14]. Mononuclear monooxygenase non-heme iron enzyme, that is tyrosine hydroxylase (an enzyme in the central nervous system), catalyzed the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine. This reaction limited the rate of biosynthesis of cate- cholamine neurotransmitters. Catalysis was conducted with the participation of tyrosine, tetrahydrobiopterin, and O2. The active center of the enzyme included the ferrous ion, coordinated by the facial side chains of 2 histidines and glutamate [15]. Additional protection of enzymes during oxidative transformations can be provided by the presence of chains of nearby tyrosine (Tyr) and tryptophan (Trp) residues. Redox Catalysts 2021, 11, 1032 5 of 12

residues Tyr and Trp can carry out the transfer of oxidizing equivalents of cpd I on the surface of proteins. This protective mechanism may be important for the catalysis of P450 [16,17].

2.2.1. Catalysis with Acireductone Dioxygenases Ni(Fe)ARD 2 Earlier, we established that the conversion of complex Ni(acac)2·L into active form “A” during ethylbenzene oxidation with O2 proceeded with a mechanism similar of action of NiARD dioxygenase mechanism [1]. This, as well as the established phenomenal effect of phenol on the oxidation of ethylbenzene with O2, catalyzed by nickel systems 2 {Ni(acac)2+L }[1], suggested the involvement of the tyrosine fragment in the action of acireductone dioxygenase NiARD. Acireductone dioxygenases Ni(Fe)ARD are involved in the methionine salvage pathway (MSP), which is the universal recycling pathway for the conversion of sulfur-containing metabolites to methionine. Ni(Fe)ARD represent an unusual case of catalysis. Depending on the nature of the metal ion in the active site, nickel–iron enzymes differ in their mechanism of action. They catalyze the conversion of the same substrates (1,2-dihydroxy-3-oxo-5 (methylthio), pent-1-ene (β-diketone acireductone), and molecular oxygen) into various products [18]. FeARD catalyzes the penultimate step in the metabolic pathway of acireductone oxidation in formate and 2-keto-4- (thiomethyl) butyrate, which is a precursor of methionine. The NiARD-catalyzed reaction pathway does not produce methionine, but this reaction produces CO, which is a neurotransmitter. CO has been identiﬁed as an anti-apoptotic molecule in mammals. Recently, it was found that a human enzyme regulated the activity of matrix metalloproteinase I (MMP-I), which is involved in tumor metastasis, by binding to the cytoplasmic transmembrane tail peptide MMP-I [19]. We assumed that the different activities of NiARD and FeARD with respect to the same substrates (acireductone and O2) might be connected to the formation of different macrostructures due to intermolecular H-bonds. We also assumed the role of the Tyr fragment in the second coordination sphere (see Scheme1[ 18]). In the case of catalysis by Catalysts 2021, 11, x FOR PEER REVIEWNi-dioxygenase, NiARD, the Tyr fragment, participating in the mechanism,6 couldof 12 reduce

the NiARD activity.

SchemeScheme 1. 1. TheThe structure structure of of NiARD NiARD with with Tyr Tyr residue residue in th ine the outer outer coordination coordination sphere sphere of a ofnickel a nickel ion ion [18]. [18]. Using the AFM method, we obtained convincing evidence on the model level in favorUsing of the the involvement AFM method, ofwe a obtained tyrosine convincing fragment evidence in the stabilization on the model level of primary in favor NiARD complexesof the involvement as one ofof thea tyrosine possible fragment regulatory in the factors stabilization in the Ni(Fe)ARDof primary NiARD action. com- For the ﬁrst time,plexes weas one observed of the possible self-organization regulatory fact ofors model in the tripleNi(Fe)ARD complexes action. For including the first time, Tyr and His we observed self-organization of model triple complexes including Tyr and His fragments fragments {Ni(acac)2•NMP•Tyr}, {Ni(acac)2•His•Tyr} (His = L-histidine, Tyr = L-tyrosine) {Ni(acac)2•NMP•Tyr}, {Ni(acac)2•His•Tyr} (His = L-histidine, Tyr = L-tyrosine) into very stable supramolecular structures due to H-bonds and possibly other non-covalent interactions (Figure 3) [5].

(a) (b)

Figure 3. (a) 3D AFM image of nanostructures based on systems {Ni(acac)2 His Tyr} (h = 45 nm) (4.5μm × 4.5μm); (b) 3D AFM image of nanostructures based on systems {Ni(acac)2⋅NMP⋅Tyr} (h = 25 nm) (2μm × 2μm).µm

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Catalysts 2021, 11, x FOR PEER REVIEW 7 of 12 into very stable supramolecular structures due to H-bonds and possibly other non-covalent interactions (Figure3)[5]. Self-assembly of complexes of iron into supramolecular structures can favor the Self-assembly of complexes of iron into supramolecular structures can favor the for- formationmation of methionine.of methionine. UsingUsing the AFMthe AFM method, method, wewe established the theself-organization self-organization of complexes of complexes III III IIIIII Fe x(acac)Fe xy(acac)18C6y18C6m(Hm2(HO)2O)n,n and, andFe Fe xx(acac)(acac)y(His)y(His)m(Tyr)m(Tyr)n(H2O)np(H, which2O)p ,simulated which simulated the active the active center ofcenter FeARD, of FeARD, into supramolecularinto supramolecular hollow hollow ring-shaped ring-shaped cellular cellular structures structures (Figure (Figure 4a,b), 4a,b), resembling the tubular shape of the tubulin protein. These structures had an average resemblingdiameter the of tubular 20–30 nm, shape which is of approximately the tubulin the protein. same diameter These as that structures of the tube had of the an average diametertubulin of 20–30 protein. nm, In whichthe case is of approximately the two-component the systems same {Fe(acac) diameter3+His}, asthat we observed of the tube of the tubulinthe protein. formation In of the less case distinct of the structures two-component that were less systemsstable. UV {Fe(acac)spectroscopy3+His}, data have we observed the formationrevealed ofthe less outer-sphere distinct coordination structures of that L-histidine were less(H-bond) stable. with UV Fe(acac) spectroscopy3 [20]. data have revealed the outer-sphere coordination of L-histidine (H-bond) with Fe(acac)3 [20].

(a) (b)

III Figure 4. (a) The AFM 3D image of nanoparticles based on Fe x(acac)y18C6m(H2O)n;(b) the AFM 3D image of nanoparticles Figure 4. (a) TheIII AFM 3D image of nanoparticles based on FeIIIx(acac)y18C6m(H2O)n; (b) the AFM 3D image of nanoparticles based on triple system Fe x(acac)yIII(His)m(Tyr)n(H2O)p. based on triple system Fe x(acac)y(His)m(Tyr)n(H2O)p.

The mechanismThe mechanism of action of action of of FeARD, FeARD, presumably, presumably, included included the stage the of stage O2-activation of O2-activation II II → IIIIII − − II II of Fe of+ Fe O2+O→2 FeFe –O−2 .O This2 .oxygen This activation oxygen mechanism activation is proposed mechanism for Fe -acetylacetone is proposed for Fe - acetylacetonedioxygenase dioxygenase (Dke1) [21]. (Dke1) The oxygen [21]. activati The oxygenon, and activation,the subsequent and reactions the subsequent (the regi- reactions (the regioselectiveoselective inclusion inclusion of activated of activated O2 to the O acireductone,to the acireductone, and the reactions and the leading reactions to the leading to formation of methionine) can be facilitated2 by the formation of structures similar to tubu- the formationlin tubules. of methionine) In fact, structures can similar be facilitated to these were by theformed formation as a result of of structures self-organization similar to tubulin tubules.of complexes In fact, structuresFeIIIx(acac)y18C6 similarm(H2O) ton [1,5], these and were FeIII formedx(acac)y(His) asm a(Tyr) resultn(H2 ofO)p self-organization (Figure 4) of III III complexeson the Fe surfacex(acac) duey to18C6 H-bonds.m(H2 O)n [1,5], and Fe x(acac)y(His)m(Tyr)n(H2O)p (Figure4) on the surface due to H-bonds. 2.2.2. Catalysis with the Family of Cytochrome P450-Dependent Monooxygenases 2.2.2. CatalysisThe original with the molecular Family structure of Cytochrome of myoglobin P450-Dependent (Mb) was determined Monooxygenases in 1958. Since then, scientists have used advances in structural biology, biophysics, computational Thechemistry, original and molecular spectroscopy structure to obtain ofnecess myoglobinary information (Mb) about was the determined effect of various in 1958. Since then, scientistsstructural have factors used on the advances reactivity inof metal structural cofactors. biology, These studies biophysics, have shown computational that the chemistry, andcoordination spectroscopy spheres to of obtain the active necessary centers of information proteins play about a decisive the role effect in determining of various structural the properties of metal cofactors [22]. factors on theThe reactivity family of cytochrome of metal cofactors.P450-depende Thesent monooxygenases studies have (cytochrome shown that P450) the are coordination sphereshemoproteins of the active with centers significantly of proteins different play functions. a decisive Proteins role cont inaining determining heme are wide- the properties of metalspread cofactors and found [22]. in almost all organisms, from bacteria to humans. These proteins are Theinvolved family in many of cytochrome metabolic reactions. P450-dependent They have different monooxygenases mechanisms of action (cytochrome and par- P450) are hemoproteinsticipate in the withoxidation signiﬁcantly of numerous differentcompounds functions.[23]. Proteins containing heme are widespread and found in almost all organisms, from bacteria to humans. These proteins are involved in many metabolic reactions. They have different mechanisms of action and participate in the oxidation of numerous compounds [23]. In nature, various types of binding of amino acids to metalloporphyrins are observed. It is known, for example, that Tyr (tyrosine) forms hydrogen bonds with His (histidine) [1,24,25]. For the ﬁrst time, we used the AFM method to study the formation of supramolecular structures based on metal complexes of porphyrin with amino acids, tyrosine, and histi- Catalysts 2021, 11, x FOR PEER REVIEW 8 of 12

In nature, various types of binding of amino acids to metalloporphyrins are observed. It is known, for example, that Tyr (tyrosine) forms hydrogen bonds with His (histidine) Catalysts 2021, 11, 1032 7 of 12 [1,24,25]. For the first time, we used the AFM method to study the formation of supramolecular structures based on metal complexes of porphyrin with amino acids, tyrosine, and histidine,dine, which which are are part part of the of active the sites active of en siteszymes, of enzymes,in particular, in cytochrome particular, P450-depend- cytochrome P450-dependentent monooxygenases. monooxygenases. The fact of the The formation fact of the of formation such structures of such can structures be used can to beanalyze used tonon-covalent analyze non-covalent intermolecular intermolecular interactions interactions [26] in the [ 26mechanisms] in the mechanisms of enzymatic of enzymatic catalysis catalysis[27,28]. [27,28]. BasedBased onon publishedpublished datadata onon thethe regulatoryregulatory rolerole ofof HisHis andand TyrTyr fragments,fragments, asas wellwell asas porphyrinsporphyrins inin thethe functioningfunctioning ofof enzymesenzymes (heme(heme proteins)proteins) of thethe P450P450 familyfamily [[28],28], self-self- organizationorganization ofof supramolecularsupramolecular structuresstructures basedbased onon thethe modelsmodels ofof thethe P450P450 familyfamily cancan bebe expectedexpected [[29].29]. TheThe bindingbinding ofof porphyrinsporphyrins throughthrough H-bondsH-bonds isis aa commoncommon intermolecularintermolecular interactioninteraction inin nature.nature. The formation of aa dimerdimer basedbased onon aa carboxyliccarboxylic acidacid groupgroup isis anan exampleexample ofof thethe simplest artiﬁciallyartificially self-assemblingself-assembling supramolecular porphyrinporphyrin system [[24].24]. FigureFigure5 5a,ba,b show show that that the the AFM AFM images images of of stable stable nanostructures nanostructures of of model model systems systems {Hem+Tyr+His}{Hem+Tyr+His} had had the the form form of of triangular triangular prisms. prisms. These These structures, structures, apparently, apparently, were wereformed formed due to due the to coordination the coordination of intermolecular of intermolecular interactions interactions of the of thecomponents components of the of the{Hem•Tyr•His} {Hem•Tyr•His} complex complex during during spontaneous spontaneous self-organization self-organization with with the the participation participation of ofmainly mainly hydrogen hydrogen bonds bonds and and other other interm intermolecularolecular non-covalent non-covalent interactions. interactions.

(a) (b)

Figure 5. AFM two- (a) and three-dimensional (b) image of stable nanostructures based on model {Hem•Tyr•His} complexes. Figure 5. AFM two- (a) and three-dimensional (b) image of stable nanostructures based on model {Hem•Tyr•His} complexes. In the case of the {Hem+PhOH+His} complexes, i.e., substituting PhOH for Tyr, we also observed the formation of supramolecular structures. However, in this case, the structures based on {Hem+PhOH+His} differed in shape from nanostructures based on complexes {Hem•Tyr•His} (compare Figures5 and6).

Catalysts 2021, 11, x FOR PEER REVIEW 9 of 13

In the case of the {Hem+PhOH+His} complexes, i.e., substituting PhOH for Tyr, we also observed the formation of supramolecular structures. However, in this case, the struc- Catalysts 2021, 11, 1032 8 of 12 tures based on {Hem+PhOH+His} differed in shape from nanostructures based on complexes {Hem•Tyr•His} (compare Figures 5 and 6).

(a)

(b)

Figure 6. (a) AFM two-dimensional image of supramolecular structures based on {Hem+PhOH+His} complexes and a two-dimensional image proﬁle of these structures; (b) AFM three-dimensional image of supramolecular structures, based on {Hem+PhOH+His} complexes.

One of the possible schemes for the formation of triangular prisms due to H-bonds NH•••O or N•••HO [25,27] is shown in Figure7a. The combination of individual triangles into structures (such as Sierpinski triangular motifs [30], Figure7b) due to H-bonds and π–π- intermolecular interplanar interactions can lead to the formation of triangular prisms. Catalysts 2021, 11, x FOR PEER REVIEW 9 of 12

In the case of the {Hem+PhOH+His} complexes, i.e., substituting PhOH for Tyr, we also observed the formation of supramolecular structures. However, in this case, the structures based on {Hem+PhOH+His} differed in shape from nanostructures based on complexes {Hem•Tyr•His} (compare Figures 5 and 6).

Figure 6. (a) AFM two-dimensional image of supramolecular structures based on {Hem+PhOH+His} complexes and a two- dimensional image profile of these structures; (b) AFM three-dimensional image of supramolecular structures, based on {Hem+PhOH+His} complexes.

One of the possible schemes for the formation of triangular prisms due to H-bonds NH•••O or N•••HO [25,27] is shown in Figure 7a. The combination of individual trian- Catalysts 2021, 11, 1032 gles into structures (such as Sierpinski triangular motifs [30], Figure 7b) due to 9H-bonds of 12 and π–π- intermolecular interplanar interactions can lead to the formation of triangular prisms.

(a) (b)

Figure 7. The possibleFigure triangular7. The possible structure triangular of {Hem structure•Tyr•His} of {Hem•Tyr•His} complex that is complex formed that due tois formed H-bonds due NH to••• H-bondsO or N NH•••O•••HO or (a). The combinationN•••HO of individual (a). The combination triangles intoof individual structures triangles such asinto Sierpinski structures triangular such as Sierpinski motifs (triangularb). motifs (b).

3. Discussion3. Discussion We used the AFM method to study the role of self-organized supramolecular nanostructures and phenol in mechanisms of catalysis of selective hydrocarbon oxidation and also the possible role of supramolecular nanostructures and tyrosine in mechanisms of enzymatic catalysis, in actions of dioxygenases Ni(Fe)ARD, and of enzymes of the P450 cytochrome family. The fact of the formation of self-organized supramolecular structures can be used to analyze non-covalent molecular interactions [27]. Using the AFM method, we obtained convincing evidence on the model level in favor of the involvement of a tyrosine fragment in the stabilization of primary NiARD complexes as one of the possible regulatory factors in the functioning of Ni(Fe)ARD. Self-assembly of iron complexes into supramolecular structures, such as tubulin tubules, can facilitate reactions leading to the formation of methionine (FeARD catalysis). We used the AFM method to study the formation of supramolecular structures, based on porphyrin metal complexes with amino acids, tyrosine, and histidine, which are part of the active centers of enzymes, in particular, the family of cytochrome P450-dependent monooxygenases. The nanostructures, based on the model {Hem•Tyr•His} complex in the form of triangular prisms, apparently, were formed through spontaneous self-organization due to the coordination of intermolecular interactions of the components of the complex {Hem•Tyr•His} with the participation of mainly hydrogen bonds and other intermolecular non-covalent interactions. We did not observe such structures in the case of complexes {Hem•PhOH•His}, i.e., when replac- ing Tyr with PhOH. A possible scheme for the formation of nanostructures based on the {Hem•Tyr•His} complex is proposed. The data obtained can be considered a step towards understanding the regulatory activity of Tyr (and His) with respect to the active centers of the enzymes Ni(Fe)ARD dioxygenases or the family of cytochrome P450-dependent monooxygenases, the principles of which were used to create highly efﬁcient catalytic systems.

4. Materials and Methods For the AFM study, we used the scanning probe microscope SOLVER P47 SMENA (Adm. Distr. Zelenograd of Moscow, 124490, Russia) at a frequency of 150 kHz using an NSG30 cantilever with a radius of curvature of 10 nm. We also used an NSG30_SS cantilever (NanosensorsTM Advanced TecTM AFM probes, CH-2000 Neuchatel, Switzerland) with a radius of curvature of 2 nm, a resonance frequency of 300 kHz, and a force con- stant of 22–100 N/m in tapping mode was used for AFM research of supramolecular structures. Sampling was carried out using a spin coating process, from a CHCl3 solu- 2 tion (Ni(acac)2·L ·PhOH complexes), an aqueous solution ({Fe(acac)3+His+Tyr} mixture), or a water-alcohol solution of the mixture {Hem+Tyr+His} (Hem = hemin, Tyr = L-tyrosine, Catalysts 2021, 11, 1032 10 of 12

His = L-histidine, molar ratio of 1:1:1). The measurement was conducted on air-dried samples. In the course of scanning the studied samples, it was found that the structures were ﬁxed on the surface rather strongly, perhaps due to the H-bonds. In fact, we registered the self-assembly of the three components of the system into nanostructures with the participation, we believe, of hydrogen bonds and perhaps other non-covalent interactions. The spontaneous process of self-organization of the nanostructures, based on researched systems on a specially prepared modiﬁed silicon surface, was due to the balance between molecular–surface and intermolecular interactions.

5. Conclusions Using the AFM method, we registered the self-assembly of three components of 2 the system ({Ni(acac)2+L +PhOH(Tyr)}, {Fe(acac)3+His+Tyr}, and {Hem+Tyr+His}) into nanostructures with the participation, we believe, of hydrogen bonds, as well as, possibly, other non-covalent interactions. The images that we see in the examples of complexes, which are models of active sites of enzymes, tell us that such an assembly was taking place. Apparently, this type of interaction in the active center of the enzyme cannot be ignored either. With the AFM method, we obtained convincing evidence at the model level in favor of the involvement of tyrosine (and histidine) fragments as the possible regulatory factors in the functioning of di- or monooxygenases, namely, Ni(Fe)ARD dioxygenases or enzymes in the family of the cytochrome P450-dependent monooxygenases. The principles of actions of these oxygenases were used to create highly efﬁcient catalytic systems for the oxidation of hydrocarbons.

Author Contributions: Data curation, L.M., A.G.; Methodology, V.B., E.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by Russian Academy of Sciences and Presidium of the Russian Academy of Sciences RAS 14P, State Register Number: AAAA-A17-117121920169-0. Data Availability Statement: All data generated or analyzed during this study are included in this published article. Acknowledgments: To A.V. Lobanov for the reagents provided and participation in the discussions. Conflicts of Interest: The authors declare no conflict of interest, financial or otherwise.

Abbreviations

AFM method Atomic Force Microscopy method (acac)− Acetylacetonate ion ARD Acireductone ligand 18C6 18-crown-6 CO Carbon monoxide Dke1 Fe-acetylacetone dioxygenase HMPA Hexamethylphosphorotriamide Hacac Acetylacetone Hem Hemin His L-histidine L2 Electron-donating mono-, or multidentate ligand MSt Stearates of Na, Li NMP N-methyl-2-pirrolidone Ni(Fe)ARD Ni(Fe) acireductone dioxygenases (OAc)− Acetate ion PhOH Phenol Tyr L-tyrosine UV Ultraviolet-spectroscopic data Catalysts 2021, 11, 1032 11 of 12

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