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A REVIEW OF TYPE 3 COPPER ACTIVE SITES, THE PROTEINS THEY INHABIT, THEIR FUNCTIONS, AND THEIR BIOMIMETIC LIGANDS

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004780/00001

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Title: A REVIEW OF TYPE 3 COPPER ACTIVE SITES, THE PROTEINS THEY INHABIT, THEIR FUNCTIONS, AND THEIR BIOMIMETIC LIGANDS
Physical Description: Book
Language: English
Creator: Hayton, Janardana
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: Biomimetic
Class 3 Copper Proteins
Tyrosinase
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Class 3 copper proteins contain a dinuclear copper active site coordinated by three histidines apiece. These active sites bind dioxygen in a characteristic μ-η2:η2 configuration that results in a strongly antiferromagnetically coupled EPR silent dicopper-peroxo core. This active site functions as a respiratory oxygen carrier in arthropods and mollusks and it catalyzes the oxidation of monophenols and ortho-diphenols which results in a plethora of products that cause pigmentation, protect from pathogens, or act as pathogens through various biosynthetic pathways. These proteins are ubiquitous throughout life signifying their importance. This review aims to describe the function of class 3 protein active sites and surrounding residues of import, explain the biological function many of these proteins play, and illustrate ligands that have been synthesized in the context of the implications of their functionality. Finally, a novel ligand is proposed with reactions for its synthesis outlined and a proposed function described.
Statement of Responsibility: by Janardana Hayton
Thesis: Thesis (B.A.) -- New College of Florida, 2013
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Scudder, Paul

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Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2013 H4
System ID: NCFE004780:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004780/00001

Material Information

Title: A REVIEW OF TYPE 3 COPPER ACTIVE SITES, THE PROTEINS THEY INHABIT, THEIR FUNCTIONS, AND THEIR BIOMIMETIC LIGANDS
Physical Description: Book
Language: English
Creator: Hayton, Janardana
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2013
Publication Date: 2013

Subjects

Subjects / Keywords: Biomimetic
Class 3 Copper Proteins
Tyrosinase
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Class 3 copper proteins contain a dinuclear copper active site coordinated by three histidines apiece. These active sites bind dioxygen in a characteristic μ-η2:η2 configuration that results in a strongly antiferromagnetically coupled EPR silent dicopper-peroxo core. This active site functions as a respiratory oxygen carrier in arthropods and mollusks and it catalyzes the oxidation of monophenols and ortho-diphenols which results in a plethora of products that cause pigmentation, protect from pathogens, or act as pathogens through various biosynthetic pathways. These proteins are ubiquitous throughout life signifying their importance. This review aims to describe the function of class 3 protein active sites and surrounding residues of import, explain the biological function many of these proteins play, and illustrate ligands that have been synthesized in the context of the implications of their functionality. Finally, a novel ligand is proposed with reactions for its synthesis outlined and a proposed function described.
Statement of Responsibility: by Janardana Hayton
Thesis: Thesis (B.A.) -- New College of Florida, 2013
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Scudder, Paul

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2013 H4
System ID: NCFE004780:00001


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A REVIE W OF TYPE 3 COPPER ACTIVE SITES, THE PROTEINS THEY INHABIT, THEIR FUNCTIONS, AND THEIR BIOMIMETIC LIGANDS BY JANARDANA DAS HAY T ON A Thesis Submitted to the Division of Natural Sciences New College of Florida in partial fulfillment of the requirement for the degree Bachelor of Arts in Natural Science Under the sponsorship of Professor Paul H. Scudder Sarasota, Florida May, 2013

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ii ACKNOWLEDGMENTS Thanks committee for being on my committee! Thanks parents for raising me and stuff! Thanks Colin for giving me a job! Thanks Clare, Brie, Mia, Salome, Elena, Young Mike, Daphne, Laura, R osanna, and Amelia for being there for me through this process! Special thanks to Jehan, Ashley, and Helena who all saw me stress cry and put up with it! could have!

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iii TABLE OF CONTENTS .1

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iv TABLE OF FIGURES Figure 1: General Reactions, (Ginsbach Figure 2: Hemocyanin Structures, (Decker, H. e Figure 3: Tyrosinase with Caddie Prot .9 Figure 3: Dicopper Adducts, (Karlin, K. D. et al., 2012 ) Figure 4: Catechol Oxidase Mechanism, (Eiken C. et al., 1999 ) Figure 5: Tyrosinase Mechanism, (Rolff, M., Schot Figure 6: Hydroxyanilinase Mechanism, (Ginsbach Figure 7: Hydroxyanilinase Enzyme Pathway, (N Figure 8: Aurone Biosynthesis Pathway, (Elumalai P. and Liu, H Figure 9: Thioether Bond, (Klabunde, T Figure 10: 3D Variance Map of Hemocyanin, (C Figure 11: Melanin Biosynthesis, (Falguer Figure 12: Bispidine Ligands, (Comba, P. et al., Figure 13: Bispidine Side Reaction, (Comba, Figure 14: Macrocyclic Ligands, (Banu, K. Figure 15: Trinuclear Ligand, (Mutti, F. G et Figure 16: Ligand with Dual Activity, (Rolff, M., Scho Figure 17: Michael Addition Product, (Casel Figure 18: 4 Pyridyl Ligand Synthesis, (Karlin, K. D. et al., 20 1 Figure 19: Side on Adduct Forming Ligand, (Karl Figure 20: 4 Pyridyl Ligands, (Karlin, K. Figure 21: Bidentate Alkylamine Ligands, (Manda Figure 22: Bidentate Alkylamine Ligand Reaction, ( Figure 23: Bidentate Alkylamine Mechanism, (Ma Figure 24: Theoretical Study Ligands, (Marti Figur e 25: Binding Energies of Dicopper Ligands, (Mar Figure 26: Binding Energy of Mononuclear Copper Ligand (Martinez, A. et al., 2012).51 Figure 27: N 4 Ligands, (Ramadan, A. E. Figure 28: Mononuclear Copper Mechanism, (Ramadan, A. Figure 29: Benzimidazolyl Ligand, (Baksh Figure 30: Benzimidazolyl Mechanism with Phosphate, Figure 31: Benzimidazolyl M echanism with Acetate, (Bakshi, R. et al., Figure 32: Tris Imidazole Ligand, (Kujime, M. Figure 33: Linked Imidazole, (McKie, Figure 34: Proposed L .58

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v A REVIEW OF TYPE 3 COPPER ACTIVE SITES, THE PROTEINS THEY INHABIT, THEIR FUNCTIONS, AND THEIR BIOMIMETIC LIGANDS Janardana Hayton New College of Florida, 2013 ABSTRACT Class 3 copper proteins contain a dinuclear copper active site coordinated by three histidines apiece. These active sites bind dioxygen in a characteristic 2 2 configuration that results in a strongly antiferromagnetically coupled EPR silent dicopper p eroxo core. This active site functions as a respiratory oxygen carrier in arthropods and mollusks and it catalyzes the oxidation of monophenols and ortho diphenols which results in a plethora of products that cause pigmentation, protect from pathogens, or act as pathogens through various biosynthetic pathways. These proteins are ubiquitous throughout life signifying their importance. This review aims to describe the function of class 3 protein active sites and surrounding residues of import, explain the bio logical function many of these proteins play, and illustrate ligands that have been synthesized in the context of the implications of their functionality. Finally, a novel ligand is proposed with reactions for its synthesis outlined and a proposed function described. Paul H. Scudder Natural Sciences

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1 Introduction Metalloproteins have a variety of functions that are integra l to many biological processes. Class 3 copper proteins belong to a category of metalloprotein that is characterized by the active site which binds dioxygen as peroxide to a dicopper core coordinated by six histidine amino acid residues (Rolff M., Schottenheim, J. et al., 2011 ). This c onserved active site allows for oxygen transport (hemocyanin) and enzymatic activity (c atechol oxidase, tyrosinase, hydroxyanilinase and others ) and is present ubiquitously. The structure of class 3 copper proteins has been elucidated in several species a nd the peroxo dicopper binding scheme is generally agreed upon but there are many specifics left to discover. Class 3 copper proteins differ in function and although they have similar active sites, there is much variation in the rest of the protein struct ure. Catechol oxidases and tyrosinases are both integral in the biosynthetic process of producing melanin. They have been found to reside freely dissolved in cytosol (bacteria), within chloroplasts (plants), within special cells that are released when need ed (arthropods), or within membrane s in melanocytes (mammals and vertebrates at large) (Rolff M., Schottenheim, J. et al., 2011 ). Enzymatic class 3 proteins create oxidized polyphenols which are potentially toxic alkylating agents ; therefore measures are taken to keep them out of the cytoplasm in higher organisms (Jacobson, E. S., 2000). Hemocyanins are oxygen carrying proteins in mollusks and arthropods that have a very similar oxygen binding pocket to other class 3 copper proteins but their major functio n is not as an enzyme in vivo Hemocyanins are found freely dissolved in the hemolymph in some arthropods and mollusks (Cuff M. E. et al., 1998 ). Hydroxyanilinase s are a newly coined group of class 3 copper protein s that

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2 have been isolated from two diffe rent open reading frame s in Streptomyces murayamaensis There have been two enzymes found to be hydroxyanilinases, GriF and NspF (Noguchi, A. et al., 2010). They react preferably with ortho aminophenols but GriF has slight diphenolase activity while NspF h as a little monophenolase activity (Suzuki, H. et al., 2006; Ginsbach, J. W. et al., 2012). Unlike the other class 3 copper proteins, hydroxyanilinases have yet to have their structure elucidated and have only been found in Streptomyces murayamaensis Aureusidin synthase is another newly characterized enzyme that is thought to be a class 3 copper protein homologue that catalyzes the synthesis of aurones, a yellow pigment found in some plants (Elumalai, P. and Liu, H., 2011). The relative newness of th e discovery of hydroxyanilinases and aureusidin synthase along with their lack of 3 D structure make a comparison of their homology to other proteins in this group theoretical and possibly premature. The structural differences that cause different activity in the other proteins however, have been postulated for some time. Tyrosinases catalyze the oxidation of monophenols and ortho diphenols whereas catechol oxidase can only catalyze the oxidation of ortho diphenols (Mandal S. et al., 2012 ). This differenc e in selectivity is thought to be due in part to an amino acid difference near the active site of the two enzymes. Catec h ol oxidase has a phenylalanine which blocks one of the two coppers in the active site while tyrosinase has an isoleucine or a valine wh ich is less bulky and therefore allows access (Decker, H. et al., 2006; Sendovski, M. et al., 2011) The copper that is blocked off may be the reason catechol oxidase cannot catalyze oxidation of monophenols while tyrosinases can (Decker H. et al., 2006 ). This is exemplified by the binding coordination that shows monophenolase

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3 activity to require both copper atoms binding the substrate while diphenolase activity only requires one copper atom to bind the substrate ( Eiken C. et al., 1999 ; Rolff M., Schotte nheim, J. et al., 2011). The difference in binding specificity is not universally agreed upon in regards to how substrates interact with the active site The overall rate by which catechol oxidase catalyzes the production of ortho quinones is known to be m uch quicker than that of tyrosinase (Ramadan, A. E. M. et al., 2012) This suggests that catechol oxidases have traded in the ability to catalyze reactions with a wider variety of substrates to have a better reaction turnover rate. As a respiratory protei n, hemocyanin is constantly binding oxygen for storage and transport. The structural difference that impairs hemocyanin from executing catacolase, tyrosinase, or hydroxyanilinase activity is a C terminal domain that sterically hinders the binding of substr ates (Decker H. et al., 2006 ; Gerdemann C. et al., 2002 ). As hemocyanins regularly occur as oligomers, modifications that cause structural changes (Terwilliger, N. B., 2007). D espite the newness of hydroxyanilinases, they have been compared most closely in sequence and activity to tyrosinases. Some tyrosinases have a copper chelating caddy protein which delivers the copper atoms to the active site. This has been found to be simi lar to GriF which has a caddy located on the open reading frame GriE (Suzuki, H. et al., 2006). Once more is known more is known about hydroxyanilinases the divergence of activity can be better mapped. Class 3 copper proteins are often categorized in an ascending order of activity: hemocyanin can only bind dioxygen, catechol oxidase binds dioxygen and has

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4 diphenolase activity, tyrosinase binds dioxygen and has both mono and diphenolase activity, and hydr oxyanilinase binds dioxygen and can catalyze both tyrosinase reactions while having a unique reaction with ortho aminophenols. If aureusidin synthase turns out to be a class 3 copper protein, its existence marks the first divergence of this copper functionality. The general reactions of the three most studied class 3 copper proteins are outlined in Figure 1. Figure 1: Overview of the functions of established class 3 copper proteins (Ginsbach J. W. et al., 2012 ). The oxygen carrying function of class 3 copper proteins is thought to have been adapted from proteins that acted as mono and diphenol oxidases (Burmester, T., 2001). This

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5 Protein Structure and Differences Using spectroscopy, the structures of various catechol oxidases, tyrosinases, and hemocyanins have been determined. A structure of hydroxyanilinases has yet to be obtained however it has been characterized genetically and bioche mically to be very similar to tyrosinase with subtle amino acid differences near the active site (Ginsbach J. W. et al., 2012 ). Similarly, aureusidin synthase has only been comparatively characterized and not yet modeled, and unlike hydroxyanilinase, it i s more divergent, and thus result s in its unique enzymatic ability (Elumalai, P. and Liu, H., 2011). Overall sequence differences are vast across the class of protein. An example is the sequence difference between catechol oxidase from Ipomoea batatas and a functional unit from Octopus dofleini hemocyanin in which comparative modeling shows a 27% sequence similarity ( Gerdemann C. et al., 2002 ). Ipomoea batatas catechol oxidase and the bacterial tyrosinase from Streptomyces castaneoglobisporus have 17 21% sequence identity with the N terminal residues that make up the functional tyrosinase unit from Agaricus bisporus (Ismaya, W. T. et al., 2013). Despite the small amounts of sequence homology, the tertiary structure of the active protein subunit, on ce hindering N or C terminal domains are removed, are remarkably similar. Most of the proteins have been found to occur in vivo as assemblies with vast differences in quaternary structures. The number of subunits that make up the quaternary structure of he mocyanin varies from species to species and there are major tertiary differences between mollusk and arthropod organization (Cuff, M. E. et al., 1998). There is very little similarity in the

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6 prim ary structure of each type of hemocyanin except for a 42 ami no acid region that coordinates CuB. Arthropod hemocyanins are ~75 kDa folded polypeptide subunits organized into hexamers or groups of hexamers (1x6mer, 2x6mer, 4x6mer, 6x6mer, and 8x6mer) Arthropod hemocyanins that have had their crystal structure solve d include 48 meric Limulus polyphemus the 36 meric Scutigera coleoptrata and the 24 meric Pandinus imperato r proteins (Jaenicke, E. et al., 2012). Molluscan hemocyanins have larger ~400 kDa subunits arranged as decamers, di decamers, or even larger assemblies that form a hollow cylindrical figuration The quaternary structures of different hemocyanins have a single isoform when the protein is a sing le decamer or one, two, and three isoforms in different didecamers (Velkova, L. et al., 2010). A generalized structure scheme for hemocyanin is shown in Figure 2.

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7 Figure 2: Overview of hemocyanin structure in both arthropods and mollusks. The above view of the molluscan hemocyanin show s it to have a hollow center (Decker, H. et al., 2007). Each subunit contains 7 or 8 oxygen binding sites and, when disassociated from the quaternary structure, appears as beads on a string structures (Cuff, M. E. et al., 1998). Subunit interactions in hemocyanins cause conformational changes that give them the highest oxygen binding cooperativity found in nature (Jaenicke, E. et al., 2012). Tyrosinase has been found in most cases to exist as a monomer, but in some cases they are dimers or hexamers (Decker, H. et al., 2006). There is also an association with a caddy protein that impacts activity by a mechanism not yet fully understood (Rolff, M.,

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8 Schottenheim, J. et al., 2011). Tyrosinase crystal structure was difficult to acquire due to hydrophobicity and its transmembrane location H owever, utilizing the caddie protein, a crystal structure was first obtained (Matoba, Y. et al., 2006). Tyrosi nases have been found to be ~65 kDa or ~43 kDa in size in button mushrooms with some of the ~43 kDa proteins being associated with a ~14 kDa caddy protein or a subunit with unknown function to form tetramers (Ismaya, W. T. et al., 2013). The subunit with u nknown function contains a lectin like fold Since the subunit has no carbohydrate binding domains it does not assis t the protein in associating with membrane bound glycoproteins (Ismaya, W. T. et al., 2013). The presence of calcium ions has been correlate d to the assembly of the quaternary tyrosinase tetramer in Agaricus bisporus (Ismaya, W. T. et al., 2013). Catechol oxidase from the sweet potato is ~39 kDa and no associated caddy protein has been found (Eiken, C. et al., 1999). Figure 3 shows the structu re of a bacterial tyrosinase.

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9 Figure 3: The structure of tyrosinase from Strptomyces castaneoglobisporus illustrating the dissociation of the caddie protein. The indicated Y98 is a tyrosine on the caddie protein that is thought to occupy the enzyme active site (Rolff, M., Schottenheim, J. et al., 2011). Not all reported tyrosinases have an associated caddie protein. It is possible that depending on the species and function, the tyrosinase may need a caddie to control whether or not it is activated while other enzymes use other mechanisms to activate it as needed. Hemocyanin in arthropods evolved separately f rom molluscan hemocyanin, and due to its conserved nature; it is used to trace phylogeny. Most arthropod taxa use

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10 hemocyanin as an oxygen carrier, and how many subunits the protein has can be related to evolutionary divergence (Rehm, P. et al, 2012). As pr eviously mentioned, some arthropods have an activated hemocyanin like protein that essentially functions as a tyrosinase. The fact that enzymatic activity is inducible in hemocyanin to the point where some hemocyanins actually are classified as tyrosinases make the system in place for differentiating these similar class 3 copper proteins convoluted. A different classification should be used to separate the arthropod activated hemocyanin, since it is still the same protein despite acquiring enzymatic capabil ities, and tyrosinases which do also exist in arthropods and carry out the same function as the activated hemocyanin. Protein Structure Determination Methods Since many class 3 copper proteins receive post translational modifications or oligomerize, it i s difficult to accurately discern their crystal structures. Two popular processes that have been used to find the protein crystal structures are cryo electron microscopy and the hanging drop vapor diffusion method. Hanging drop vapor diffusion has been use d to create crystals of hemocyanin, tyrosinases, and catechol oxidase. X ray crystallography or 3 D modeling is then done taking into account the amino acid sequence, spectrographic data, and electron density maps. Active Site Structure and Reaction Mechanisms All class 3 copper proteins have the same dicopper peroxo coordination chemistry in common. The two copper atoms in the core are strongly antiferromagnetically coupled and are electron paramagnetic resonance ( EPR ) spectroscopy silent (Mandal, S. et al.,

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11 2012; Martinez, A. et al., 2012). Along with this it is generally agreed upon that they 2 2 configuration These two parameters are indicative of class 3 copper proteins. 2 2 ( side on), bis 1,2 peroxo (end on) shown in Figure 4 Figure 4 : Three k nown [Cu 2 O 2 ] 2+ adducts (Karlin, K. D. et al., 2012 ). While catechol oxidases and hemocyanins use the 2 2 peroxo dicopper binding configuration solely, it is debated that tyrosinases use the equilibrium bis oxo configuration to catalyze the monophenol oxidation unique to tyrosinases (Mandal, S. et al., 2012). In the small amount of available work pertaining to hydroxyanilinase, it is thought to bind dioxygen as 2 2 (Ginsbach, J. W. et al., 2012). Hydroxyanilinase and the monophenol oxidase step done by tyrosinase both donate an oxygen atom from the peroxo core to create the product. It would not be prude nt to suppose that the dicopper peroxo core adduct would be the same in these two cases, however, what is happening specifically has not yet been agreed upon. The first f orm is the native met form in which the copper atoms are oxidized (Cu II ) and the metal center is bridged by a hydroxyl group or water (Eiken, C. et al., 1999). The

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12 coordination environment for each copp er atom is roughly trigonal pla n a r. The second form is an intermediate oxygenated oxy [Cu II O 2 Cu II ] form that is bridged by a water molecule which makes the coordination sphere four coordinate trigonal pyramidal for each cupric metal ion. The third form is the reduced deoxy ( Cu I ) form where the copper atoms are farther apart and coordination sites are distorted trigonal pyramidal (CuA) and square planar with an unoccupied site (CuB) (Eiken C. et al., 1999 ; Banu K. S. et al., 2008 ). Having two metal ions in close proximity increases t he electron accepting capacity of the enzyme or biomimetic ligand thus prompting the initia l electron donor step (Martinez A. et al., 2012 ). The reaction mechanism of catechol oxida se is shown in Figure 5

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13 Figure 5 : Catechol oxidase enzymatic pathway with PTU as an i nhibitor also shown The binding of peroxide to regenerate the active site allows for the reaction of ortho catechol to ortho quinone to occur twice (Eiken C. et al., 1999 ). The mechanism for catechol oxidase illustrates the produc t being made twice for each cycle from met to deoxy to oxy forms of the enzyme. The oxy form is preferred for enzyme activity to occur but stoichiometric amount of product s form immediately after catechol is added in the absence of dioxygen (Eiken C. et al., 1999 ). The deoxy form has no activity for two main reasons: the intermetallic distance is increased and the reduction of the copper atoms lowers their oxidative capabilities. The binding of the inhibitor phenylthiourea (PTU) displaces the hydroxyl, w ater, or dioxygen in the active site and pushes the copper atoms ~1 further away from each other (Klabunde, T. et al., 1998).

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14 The reaction mechanism for tyrosinase is shown in Figure 6 Figure 6 : Proposed mechanism of tyrosinases depicting both mono and diphenolase activity The reaction begins with the binding of peroxide to the deoxy (here called desoxy ) form of the active site (Rolff, M., Schottenheim, J. et al., 2011). The mechanism of tyrosinase shows the met and oxy form making quinone products, while the oxy form is the only one that mediates the production of the catechol intermediate. The mechanism in Figure 6 shows the catechol to bind both coppers however this is not agreed upon. The monophenolase activity has b een found to be slower than the diphenolase activity which is supported by this mechanism, and there is a different kinetic rate constant when there are intermediates present along with the initial substrate tyrosine (Falguera, V. et al., 2010). The oxy fo rm is required for monophenolase

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15 activity since the product requires one of the oxygen atoms in the dicopper peroxide core to go forward (Matoba, Y. et al., 2006). The substrates bind to only one of the copper atoms at a time in catechol oxidase and the m onophenol oxidation of tyrosinase while the diphenol oxidation done by tyrosinase utilizes both copper atoms (Eiken C. et al., 1999 ; Rolff M., Schottenheim, J. et al., 2011 ) The active site has a flexible arginine residue located near the entrance of th e active site that is thought to play a role in substrate binding (Sendovski, M. et al., 2011). The mechanism behind hydroxyanilinase shown in Figure 7 resembles the diphenol oxidation carried out by tyrosinase or catechol oxidase. Figure 7 : The propos ed mechanism of hydroxyanilinase with axial histidines removed for clarity (Ginsbach, J. W. et al., 2012). The activity of hydroxyanilinase with the o nitrosophenol in the resting form yields an ortho quinone imine through a two electron process (Ginsbach J. W. et al., 2012). The L

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16 in Figure 7 refers to either water or hydroxide occupying the copper core that is homologous to the unoccupied met form in tyrosinase or catechol oxidase. This conversion into ortho quinone imine is carried out by GriF or NspF. The second reaction shown is mediated by NspF and results in a C nitroso product which proceeds through an electrophilic mechanism (Suzuki, H. et al., 2006; Ginsbach, J. W. et al., 2012). The two hydroxyanilinase enzymes discovered so far play roles in b iosyntheses in Streptomyces murayamaensis GriF is responsible for the formation of a phenoxazinone chromophore in the grixazone biosynthetic pathway (Suzuki, H. et al., 2006). Grixazone is a mix of grixazones A and B where A is a novel compound and B has been patented as a parasiticide (Suzuki, H. et al., 2006). Together grixazone is a yellow pigment. NspF is implemented in the final step of 4 hydroxy 3 nitrosobenzamide biosynthesis, the biosynthetic pathway shown in Figure 8 Figure 8 : The enzymatic pathway utilizing hydroxyanilinase (Noguchi, A. et al., 2010).

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17 This is important since it is the first enzyme found to catalyze C nitrosation in natural compounds (Noguchi, A. et al., 2010). C nitroso aromatic compounds that have been synthesized chemically have been found to be HIV 1 inhibitors (Noguchi, A. et al., 2 010). This act of C nitrosation is the first to be seen in a naturally occurring enzymatic pathway which opens up the possibilities of using enzymes in drug production. Aureusidin synthase is a suspected homolog to class 3 copper proteins. Interestingly it does not catalyze the monophenolase and diphenolase reactions that catechol oxidase and tyrosinase execute (Elumalai, P. and Liu, H., 2011). Furthermore this enzyme binds PTU which is something that all active type 3 copper cores have in common. While a crystal structure has not been found, comparative modeling shows aureusidin synthase to have many indicative copper 3 protein traits, such as the six histidines that complex with the two copper atoms and the thioether bridge. Aureusidin synthase is a glyc oprotein with a vastly different N terminal domain than other class 3 copper proteins that catalyzes the synthesis, as illustrated in Figure 9 of aurones (a yellow pigment found in decorative flowers and other plants) from chalcones. Figure 9 : Aurone b iosynthesis pathway. THC refers to 2 ,4 ,6 ,4 tetrahydroxychalcone (Elumalai, P. and Liu, H., 2011). The amino acids around the active site of aureusidin synthase prevent monophenolase and diphenolase reactions with typical substrates. Future work and a be tter understanding

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18 of the enzyme structure may explain why this is the case. T he active site flexibility seen in the other enzymes remains (Elumalai, P. and Liu, H., 2011). This flavonoid pigment biosynthetic pathway shows that some plants have adapted th e conserved enzyme motif to suit their needs, in this case create a pigment that makes the plant more attractive to pollinators. Additionally tyrosinases have been found to play a role in the biosynthesis of another pigment group, betalains. Tyrosinase in this pathway creates a DOPA intermediate that instead of undergoing the diphenolase reaction, is acted upon by another enzyme to create this red and yellow pigment that gives beetroots their distinctive color (Gandia Herrero, F. and Garcia Carmona, F., 20 12). This alternate role in pigment biosynthesis shows that there are various side functions that this class of proteins may be facilitating that are not yet known. Hemocyanins are found in many arthropods and mollusks as non heme oxygen carrying proteins. The etymology of the word hemocyanin is from the Greek for blue blood. Since it is a non heme protein, the hemo root is slightly misleading (Cuff, M. E. et al., 1998). Unlike other known oxygen carriers, the metal center contains copper instead of iron ( C uff M. E. et al., 1998). The mechanism behind peroxide binding has largely been speculated in mollusk and arthropod hemocyanin. When peroxide binds, the protein goes from being colorless to blue. When looking at hemocyanin found in Scyllarides latus oxyg en binding affinity has been found to increase with temperature and the effect of the temperature increase as well as affinit y itself increased due to urate in vivo (Sanna, M. T. et al., 2004). The concentration of calcium in vitro causes a positive binding affinity at low concentrations and a negative binding affinity at high concentrations. Physiological calcium levels in vivo saturate the binding sites yet has a positive effect on

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19 oxygen binding suggesting multiple calcium bindin g sites with selective activity (Sanna, M. T. et al., 2004). In Rapana thomasiana an arthropod, there is an extra path by which the peroxide travels to the active site which is through a tunnel on the outside of a functional unit that closes upon active site binding. This has been illustrated by conformational changes found that show a ~thirteen amino acid helix move to disallow anything else from reaching the active site from that end (Perbandt M. et al., 2003 ). No such tunnel exists in mollusks. As pre viously mentioned, subunit interactions in hemocyanins cause conformational changes that make oxygen binding cooperative (Jaenicke, E. et al., 2012). This conformation change in arthropod hemocyanin may be connected with the reason oligomers have less poly phenolase activity since cooperative conformational changes are less pronounced in monomeric units. Comparing oxygen binding at different temperatures in different hemocyanins has shown that each hemocyanin has evolved to function for the environment the species inhabits. For example, the stone crab Paralithodes camtschatica (2x6mer) and tarantula Eurypelma californicum respectively (Decker H. et al., 2007 ). In their natural environments they release oxygen adaptation to live at colder temperatures. An uncommon feature in many class 3 copper proteins is a thioether bond between one of the histidines that holds CuA in place and a nearby cysteine residue

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20 shown in Figure 10 This bond has been found in the first catechol oxidase from which a crystal structure was documented from Ipomoea batatas (Eiken C. et al., 1999; Decker H. et al., 2006 ). The thioether bond also occurs in a more recent categorized catechol oxidase from Vitis vinifera (Virador, V. M. et al., 2010). Mollusk hemocyanin has the same unusual thioether bond betwee n a histidine in the CuA cluster whereas it is absent in arthropod hemocyanins (Cuff, M. E. et al., 1998; Decker, H. et al., 2006). Tyrosinases often have the thioether bond but not in the case of humans and some fungal enzymes. The bond itself does not pl ay a direct role in substrate binding or the catalytic mechanism; however, it is thought to play a supporting role in the enzymatic reaction (Eiken, C. et al., 1999). Figure 10 : Electron density map of the oxidized catechol oxidase active site showing t he trigonal planar coordination sphere and the thioether that is seen in many type 3 copper proteins. The imaged was computed using SETOR from by Klabunde. The thioether bond occurs near CuA which is the copper least implemented during diphenolase activit y. It may play a role in altering the electronegativity of CuA

PAGE 26

21 which in the case of tyrosinases, may lead to CuA binding the substrate in the monophenolase reaction despite it having the room to bind either one of the coppers. Some tyrosinases do not have the thioether bond. These tyrosinases have more flexibility due to the histidine not being restrained by the thioether bond (Matoba, Y. et al., 2006). This active site flexibility surrounding CuA has been correlated with a higher monophenolase to diphenola se activity ratio. This supports the hypothesis that CuA is implicated in monophenolase substrate binding (Sendovski, M. et al., 2011). Steric hindrance in catechol oxidase is thought to be the reason the substrate only binds to CuB so a difference in elec tronegativity would not play a role in selectivity. The presence of the thioether bond in hemocyanins and catechol oxidases, which both do not require copper binding specificity, paired with the fact that it is conserved throughout class 3 copper proteins, means it has a purpose beyond aiding in activity. It may play a role in protein folding or possibly just occur coincidently. Mutants with the cysteine removed would not be enough to tell what the thioether linkage is used for in regards to activation (Vir ador, V. M. et al., 2010). In type 1 copper active sites, a cysteine residue along with histidines chelate the copper atom (Bertrand, T. et al., 2002). The proximity of a cysteine to the active site in type 3 copper proteins suggests a common ancestry of c opper protein active sites. Due to the active site similarities of class 3 copper proteins, being an inhibitor for one often denotes being an inhibitor for the others. Inhibitors are often used to characterize active sites. As shown with the proposed cate chol oxidase reaction mechanism, PTU is an inhibitor that binds directly to the copper core ( Eiken C. et al., 1999 ). Other known inhibitors that have been characterized include kojic acid, tropolone,

PAGE 27

22 and 4 hexylresorcinol. These however do not bind with t he copper center but instead are competitive inhibitors (Wright, J. et al., 2012). Furthermore, tropolone has been found to only associate with tyrosinase in its oxy form in Agaricus bisporus tyrosinase while kojic acid has been found to associate with the oxy form of tyrosinase from Bacillus megaterium (Ismaya, W. T. et al., 2013). Enzymatic Activity Variations Catechols are important intermediates in many industrial products. Work has been done to alter amino acid residues around the active site to create a tyrosinase that has a greater monophenolase activity in comparison to its diphenolase activity so catechol could be salvaged before reacting (Goldfeder M. et al., 2013 ). Creating enzyme mutants has been success ful at increasing catechol producing c apabilities. For example, by replacing a valine near the active site with phenylalanine or glycine, the tyrosinase of Bacillus megaterium has been seen to increase the ratio of mono/diphenolase catalytic rate by 9 and 4.4 fold respectively (Goldfeder, M. e t al., 2013). Ionic liquids have been found to alter certain tyrosinase selectivity. Water miscible ionic liquids have been found to increase the monophenolase/diphenolase ratio of activity by several folds. SDS has been found to generally increase the ac tivity of tyrosinases by modulating the protein structure without destroying it (Goldfeder, M., Egozy, M., et al., 2013). The orientation of substrate binding is important in tyrosinases since they catalyze two similar but unique reactions. The fact that t he media the enzy me is in can affect selectivity makes the presented mechanism from Figure 6 where the catechol intermediate is bound after forming is questionable. This suggests that in some

PAGE 28

23 cases the catechol intermediate is released before being made in to a quinone. Future work looking into enhancing monophenolase activity and decreasing diphenolase activity could possibly yield a mutant that is solely a monophenol oxidase which could provide important information for monophenolase ligand synthesis. A variation in the dicopper active site has been found in Ralstonia solanacearum that varies the monophenolase and diphenolase in vivo Two polyphenol oxidases have been identified in this species, one with a higher monophenolase to diphenolase activity rati o and another with almost entirely diphenolase activity as in catechol oxidases (Hernandez Romero, D. et al., 200 6 ). The supposed function of tyrosinases and/or catechol oxidases in bacteria is thought to be related to pathogenesis. Bacteria that typically have polyphenol oxidase activity are typically ones that interact with plants. Ralstonia specifically is a soil born plant pathogen that infects roots. The expression of a tyrosinase with an activity ratio of monophenolase/diphenolase greater than 1 and a catechol oxidase which has a monophenolase/diphenolase activity ratio of 0 probably means the two work together to produce melanin for the bacteria. The tyrosinases in these bacteria have two methionine residues near two of the histidines that complex wit h CuB. In class 1 copper enzymes, the presence of methionines close to the copper site alter the redox potential thus lowering the catalytic affectivity (Hernandez Romero, D. et al., 200 6 ). Site directed mutagenesis could be used to find if these residues are decreasing the CuB binding affinity which subsequently causes there to be more activity around CuA, which is thought to be more heavily involved in the monophenolase reaction. When comparing these bacterial polyphenolases to already known structures, t here is a relationship between a residue surrounding CuB and whether carboxylated or

PAGE 29

24 decarboxylated substrates are preferred. Some enzymes that have a seventh histidine that does not play a role in cop per binding near the entrance of the active site near C uB causes a greater affinity for carboxylated substrates. On the other hand, other enzymes have a leucine residue in the place of the histidine that creates a preference for decarboxylated substrates (Hernandez Romero, D. et al., 2005). Certain hemocyanins have low level diphenolase activity largely when disassociated into monomers with specificity varying on what is attached to the catechol molecule. Kinetic analysis shows however that plain catechol alone tends to have a generally quicker rate in Cancer pagurus (Idakieva, K. et al., 2013). When comparing hemocyanins from Limulus polyphemus and Cancer pagurus, it was found that there was diphenolase activity in the Cancer but not the Limulus This difference is thought to be due to access which can be speculated upon by looking at thermo stability. Cancer hemocyanin is less thermo stable which suggests that the protein is more malleable, and can therefore allow access to the active site (Idak ieva, K. et al., 2013). The treatment of hemocyanins with detergents has been shown to cause oxygenase activity by slightly al., 2011). As previously mentioned, SD S has been seen to increase tyrosinase activity (Goldfeder, M., Egozy, M., et al., 2013). In Cancer 4 mM SDS increases the slight diphenolase activity of hemocyanin by two fold (Idakieva, K. et al., 2013). The addition of SDS has also been found to induce activity in non enzymatic hemocyanins at concentrations above the critical micelle concentrations (Cong Y. et al., 2009 ). Depending on the hemocyanin, SDS increases or induces polyphenolase activity variably.

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25 Crustacean hemocyanins treated with SDS have an increase in activity for several minutes before active site integrity is thought to be lost. On the other hand Eurypelma californicu m hemocyanin remains stable for several hours after treatment with SDS (Idakieva K. et al., 2013 ). Increases o f catalytic activity caused by detergents a ffect certain substrates more than others that are probably dependant on how accessible the substrate is to the newly opened active site (Idakieva, K. et al., 2013). This theme of SDS increasing enzymatic activity is curious since it causes denaturation in most other proteins. The crystal structures depicting the conformational change that allows hemocyanin to execute mono or diphenolase activity has been found using the protein from the arthropod Pandinus imperato r commonly known as the king scorpion (Cong, Y. et al., 2009). The major change that is hypothesized to induce activity is a twist in domain I that further exposes the active site shown in Figure 1 1

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26 Figure 1 1 : A 3D variance map of Pandinus imperato r hemocyanin monomer. This hemocyanin is comprised of twelve subunits separated into three domains (I, II, and III). The oxygen binding site is located on domain II. The purple denotes differences in structure with arrows indicating areas with a greater co ncentration of changes. The dotted circle shows the area deemed domain I (Cong, Y. et al., 2009). The specific shift in each monomer in conjunction with quaternary changes in the hemocyanin (twenty four subunits split into two dodecamers each comprised of two hexamers; a 6x4mer hemocyanin) allows substrate access (Cong, Y. et al., 2009). A possible reason for improved or induced enzymatic activity is the conserved nature of the active site that is not compromised by the addition of detergents while surroun ding amino acids are affected. It has been established that hemocyanins have inducible in vitro phenoloxidase activity H owever, there are thought to be hemocyanins that are converted into tyrosinase and/or catechol oxidase homologues in vivo (Wright, J. et al., 2012). This process is thought to be related to sclerotization and immunity in some arthropods (Wright, J. et al., 2012). One hypothesis is that the excess of hemocyanin that is present in arthropods

PAGE 32

27 causes some of the proteins to take on the role of polyphenol oxidase since some species do not have a tyrosinase or catechol oxidase identified (Decker, H. et al., 2007). Arthropods with no polyphenol oxidase activity in their hemocytes, such as chelicerates and isopods, are thought to be arthropods t hat use hemocyanin as a phenoloxidase (Terwilliger, N. B., 2007). This is possible since various bio available enzymes, such as serine protease s have been seen to activate hemocyanin in vitro without denaturing it (Cong, Y. et al., 2009). Other arthropods that do not use hemocyanin for oxygen transport have genetic sequences that are very similar in sequence that yield a tyrosinase zymogen that is activated upon the cleavage of an N terminal polypeptide (Burmester T., 2001 ). Melanin Production and Uses Both catechol oxidase and tyrosinase play a role in defense against pathogens and insects in fungi and plants (Mayer, A. M., 2006). It does this through the production of melanin (Figure 1 2 ) which encases exposed tissue (Klabunde, T. et al., 1998 ). The process of fruit browning, such as in the case of bananas, can be attributed to catechol oxidase or tyrosinase. When a sweet potato is cut in half and is left out, the exposed inside undergoes a physical alteration where it toughens. This is also d ue to catechol oxidase. It does this by releasing the enzyme from vesicles in response to injury and the presence of dioxygen. This causes the cascade resulting in quinone polymers that cross link surface proteins forming a cuticle (Jacobson, E. S., 2000). These melanoprotein assemblies are what cause the blackening or browning of exposed tissue. Work has been done to inhibit the effects of catechol oxidase and tyrosinase to preserve food. This has been studied in apples where the oxidation mediated by thes e polyphenol oxidases has negative effects on color, taste, flavor, and nutritional content (Holderbaum, D. F. et al.,

PAGE 33

28 2010). There is evidence however, that melanin and its intermediates have antioxidative, anti inflammatory, i mmune and anti tumor propert ies (Dutot, M. et al., 20120 Preventing the production of melanin may result in the loss healthy secondary metabolites. Figure 1 2 : Melanin biosynthesis pathway from Agaricus bisporus tyrosinase (Falguera, V. et al., 2010). This mechanism is typical of many catechol oxidases and tyrosinases. Using the oxidation of grated apple samples, a correlation between levels of polyphenols present and enzymatic browning has been hypothesized. Depending on the apple genotype, low polyphenol content correlates with slow enzymatic browning (Holderbaum, D. F et al., 2010). Tannin antioxidants often have monophenol or diphenol constituents that would act as substrates to tyrosinase or catechol oxidase in reaction

PAGE 34

29 mechanisms that would not lead to pigment molecules. Procyanidins in apples have been found to in hibit enzyme activity which would throw off the relationship between polyphenol concentration and enzyme activity (Mayer, A. M., 2006). Previously it has been mentioned that intermediates in the polyphenolase reaction mechanism have positive health benefit s making the removal of polyphenols result in the loss of nutritive value (Holderbaum, D. F. et al., 2010). The loss of polyphenols mediated by genetic modification may remove constituents that result in enzymatic browning, but it may also remove secondary metabolites that act as antioxidants. Further work should qualify the health benefits of various polyphenol concentrations in different apple cultivars. Fungi are also thought to use tyrosinase as a part of pathogen production to deter fungivores. Certain fungi have also been found to excrete tyrosinase in the presence of other fungi for an unknown reason (Jacobson, E. S., 2000). Most fungal melanins are derived from 1,8 dihydroxynaphthalene (DHN) as opposed to L tyrosine which results in biosynthetic poly S., 2000). Melanin polymerization is highly variable so pinpointing melanin structures is very difficult and very few have been structurally characterized. Since tyrosinase is what cata lyzes melanin formation and resultant skin pigmentation, the effective application of tyrosinase inhibitors to prevent skin darkening is of interest for several reasons. There are many dermatological disorders that could be slowed down through the usage of tyrosinase inhibitors (Park, J. et al., 2010). Specifically polyphenols found in a fermented soybean paste made in Korea have been very effective in inhibiting tyrosinase activity (Park, J. et al., 2010). The polyphenol inhibitors are

PAGE 35

30 antioxidant flavones (a class of tannin) that directly inhibit oxidation done by tyrosinase by occupying the active site (Park, J. et al., 2010). This is curious since aureusidin synthase produces aurones that are very structurally similar to flavones. Antioxidants are usuall y implemented as free radical scavengers However, tyrosinases do not normally release the phenoxyl radicals that are created as an intermediate in the enzyme reaction so antioxidant scavengers would not have an opportunity to act on the radical intermedia tes (Rolff, M., Schottenheim, J. et al., 2011; Dutot, M. et al., 2012). The mechanism behind this tannin inhibition of tyrosinase could yield information on tannin inhibitory effects since tannins are widely being implemented as radical scavengers while ot her effects are less understood. Melanin has also been found to be a functioning antioxidant in some species. Melanized Cryptococcus neoformans cells were ten times more likely to survive after the introduction of nitrogen and oxygen free radical species ( Jacobson, E. S., 2000). Human melanin plays a similar function so implementing tyrosinase inhibitors would likely cause a greater generation of free radical species cause by UV in the skin. Free radicals are known to cause DNA damage resulting in cancer. T yrosinase inhibitors are used in a variety of beauty products in East Asia (Park, J. et al., 2010). The dermatological disorders that would supposedly be prevented by tyrosinase inhibitors would be at the cost of a lessened ability of melanin to scavenge f ree radicals or absorb the UV itself which would make UV radiation from the sun more harmful. It is debated whether tyrosinase has a role in insect cuticular sclerotization since there is another enzyme that can enact the hydroxylation of tyrosine into DO PA (Anderson, S. O., 2010). Tyrosinase in insects is known to play a role in defense against harmful microorganisms and wound healing after its post translational activation due to

PAGE 36

31 the removal of an N osinases have found that they do not play a role in sclerotization. Another enzyme group, laccases, have been found through RNAi to be crucial to the cuticular sclerotization process and the thickness and structure of the procuticle in several insects (And erson, S. O., 2010). Polyphenol oxidases (laccases, tyrosinases, or catechol oxidases) in insects have been found to have a positive feedback with the ecdysone hormone in the hemolymph which relates polyphenol oxidase expression with the molting process (W ang, M. et al., 2012). In crustaceans, tyrosinase is involved in defense against microorganisms as well as the sclerotization process after molting or wound healing (Terwilliger, N. B., 2007). The sclerotization process involves the formation of a hard sh ell from a pore using melanin derived products and catalyzed by a polyphenol oxidase (Burmester T., 2001 ). The difference in the function of tyrosinase in the process of sclerotization thus varies throughout arthropods. Furthermore some arthropods might u se hemocyanin as a mobile enzyme in the sclerotization process since there are some species that lack functional tyrosinases (Terwilliger, N. B., 2007). This variation of enzymatic functionality could be used to help trace phylogenic branches in the future Mollusk tyrosinases have proved to be elusive but several have been isolated. They have been found in the mantle and periostracum and are involved in the growth of shells in bivalves and gastropods (Zhang, C. et al., 2006). Since both mollusks and arthro pods have hemocyanin and tyrosinases that may or may not be involved in shell formation, figuring out the interplay of tyrosinases, laccases, and hemocyanins in cuticle hardening, sclerotization, and periostracum formation would be helpful in illustrating the process.

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32 Laccases are multi copper containing enzymes that contain type 1, 2, and 3 copper active sites. Type 3 active sites have been discussed in detail with the characteristic coupled dicopper center that is bridged by dioxygen. Type 1 blue copper centers have a tight coordination with a cysteine residue giving it an absorbance band of around ~600nm that gives the enzyme a blue color. Type 2 copper centers bind nitrogen or nitroso ligands and have characteristic EPR properties but no distinctive opt ical spectra absorbencies (Bertrand, T. et al., 2002). Since these enzymes have so many actives sites, they have relatively low amounts of substrate specificity. Multicopper oxidases can mediate the four step single electron reduction of dioxygen to water through the oxidation of various substrates such as ortho and para diphenols, catecholamines, and arylamines (Dalisay, D. S. et al., 2011; Bertrand, T. et al., 2002). Laccases reacting with ortho diphenols has caused some terminology mix ups resulting in confusion as to whether a process is mediated by one of the class 3 copper active site enzymes or a multicopper enzyme with a type 3 active site present. When a process is described, often the enzyme is classified by what it does and not what it looks like Because of this many enzymes have been classified under the umbrella term, polyphenol oxidases or PPOs. Laccases are found in plants, fungi, and certain bacteria or insects (Bertrand, T. et al., 2002). There are cases where a species expresses genes for both kinds of enzyme but what process each is doing is not distinguished. Laccases in some species carry out the biosynthesis of melanin as catechol oxidase or tyrosinase would (Dalisay, D. S. et al., 2011). Melanin has been shown to be used for defense in plants and animals but it is used offensively in fungi. Specifically Cryptococcus neoformans is a pathogenic fungus that can infect people and essentially turn neurotransmitters into melanin (Dalisay, D. S. et al.,

PAGE 38

33 2011). The use of inhibitors to stop this dangerous pathogenic mechanism relies on the knowledge that this fungus uses laccase and not tyrosinase in its biosynthesis of melanin. The two types of enzymes use different inhibitors, so trying to have an inhibi tor bind a PPO is non specific and potentially dangerous. Both laccases and tyrosinases are present at the same time in insects but their functions differ. Tyrosinases are found throughout ses are mainly evident on the cuticle (Anderson, S. O., 2010). As previously mentioned, RNAi experiments have determined that laccases are what catalyze cuticle formation in insects however PPO is a term still used to describe the enzyme involved with inse ct sclerotization. Biomimetic Ligand Introduction When attempting to synthesize biomimetic ligands for catechol oxidase, many considerations must be taken into account regarding specificity. The two copper ions in the active site of catechol oxidase in it s active form are 2.9 apart, have bound dioxygen, and only catalyze the oxidation of ortho catechols into ortho quinones. If a tyrosinase was the aim of the synthesis, the ligand must be able to catalyze to oxidation of a monophenols into an ortho diphen ol, and the ortho diphenol into an ortho quinone. Hemocyanin biomimetic ligands would aim to solely bind dioxygen without having any catalytic activity. The usage of model ligands is implemented to discover the coordination chemistry possibilities of the m uch larger protein binding sites in regards to the oxidative state of the copper atoms.

PAGE 39

34 In testing mono and diphenol oxidase biomimetic ligands, the various electron states copper can be in may result in one Cu II or two Cu I ions complexing up with the inte nded active site (Arnold, A et al., 2012). Since the enzyme itself requires the complexing of O 2 to the copper coordination site with two Cu I atoms to alternate from the inactive deoxy to the active oxy form, synthetic ligands that complex a single Cu II at om with O 2 are not desirable however will still be discussed. The difference in counter ion structure can determine the different copper uptake by the ligand (Arnold, A et al., 2012). Depending on the structure of the ligands synthesized, different geometr ies have been achieved. There have been various 2 2 bis oxo, and 1,2 peroxo adducts present in syntheses making it less likely for a ligand to execute the same binding chemistry as the proteins however, the various adducts have all been seen to function as either a monophenolase, diphenolase, or both (Rolff, M., Schottenhe im, J. et al., 2011). Unidentate imidazole compounds when placed in aqueous conditions with peroxide and Cu I have been found to self assemble at 125 C. These ligands yield a complex that resembles the active site of the three type 3 copper enzymes. The c omplex under the same conditions under which it was made also demonstrated monophenol hydroxylation. This auto assembly and tyrosinase functionality suggest that the active site is innately thermodynamically stable which allowed it to be effectively conser ved and ubiquitous (Citek C. et al., 2012 ). Multiple uni dentate 1,2 d imethylimidazole ligands assemble dicopper peroxo cores at low temperatures. However if tris (2 pyridyl) methylamine is present, which is known to form a peroxo bridged dicopper II complex the peroxide copper core will transfer to the tridentate ligand showing favorability to higher dentate ligands (Sanyal, I. et al., 1991). The ability of the core to transfer once assembled

PAGE 40

35 shows how dynamic the self assembly is even at low temperatures. A problem with unidentate imidazole ligands is the coordination with copper is a end on 1,2 peroxo complex with dioxygen (Sanyal, I. et al., 1993). Although adducts formed by these small ligands are not the desired 2 2 configuration, the exhibition o f activity is promising. There is an increase in catechol oxidase activity by low weight ligands there are anionic compounds in solution (Bakshi, R. et al., 2011). The process of self assembly and improvements due to anionic media give some insight into th e rudimentary processes of diphenol oxidation. Phenoxyl radicals are often formed by mono oxygenase ligands upon binding the monophenol (Rolff, M. et al., 2011). The reaction is thought to begin as the substrate hydrogen is adopted by the dioxygen in the active site. Radical species created as intermediate are prevented from disassociating by the oxy radical binding to the copper core (Rolff, M. et al., 2011). This ability to stop radical formation gives copper containing ligands various possible applicati ons. Currently synthetic Cu II ligands are being implemented as radical scavengers in vivo mice models with promising anti diabetic and anti carcinogenic effects (Starha, P. et al., 2009). Tyrosinase chelate ligands with two donor groups have been found to have greater activity than ligands with three donor gr oups. With two donor groups the copper center is more electrophilic, it arranges into the most efficient geometry with more ease, and facilitates the conversion of ortho catechol to ortho quinone due t o a higher redox potential (Rolff, M., Schottenheim, J. et al., 2011). Since tyrosinases have three donor groups the fact that synthetic two donor group ligands have more activity is unexpected

PAGE 41

36 (Rolff, M., Schottenheim, J. et al., 2011). The redox potentia l of in vivo tyrosinases is most likely increased by the flexibility of the histidines around the coppers, the surrounding amino acids, and the environment the enzyme is in. Synthesized Ligands Bispidine ligand complexes have been found to exhibit diphenol oxidase abilities as well as irreversibly binding peroxide as it oxidizes the copper atoms depending on whether the ligand has end on or side on structures (Comba, P. et al., 2012). Figure 1 3 : Variou s bispidine ligand systems with variations in structure to find adduct differences (Comba, P. et al., 2012). Previous catalytic ligands create a side on adduct while end on structures have no activity. All of these bispidine ligands except L 1 (Figure 1 3 ) have catalytic diphenolase activity and form end on adducts that are unusually stable. There is a side reaction that occurs with the excess of catechol where the end on adduct having longer intermolecular distances between the copper atoms allows for two s ubstrates to bind a copper a piece (Figure 1 4 ) resulting in no product (Comba, P. et al., 2012).

PAGE 42

37 Figure 1 4 : Bispidine side reaction lowering the reaction turnover (Comba, P. et al., 2012). The addition of a methylpyridine has a positive effect on the r ate only with L 5 (Figure 13 ). The n=1 comparable ligand L 4 (Figure 13 ) has a much slower rate meaning the length of the linker has an effect on the influence of a nitrogen containing functional group. On the other hand L 2 (Figure 13 ) has a much faster rate than L 3 (Figure 13 ) showing the polarity of the functional group influences the effect of the length of the linker. Active site flexibility has been seen to be an important factor in the type 3 copper protein coordination motifs. Macrocyclic nitrogen con taining groups have been found to bind inorganics with a considerable degree of flexibility (McKie R. et al., 2007 ). Macrocyclic ligands of this type have been found to mimic catechol oxidase activity but this style of ligand does not provide the constraints that are found in the enzymes since it is accessible from both directions (Banu, K. S. et al., 2008). However, the affect of solvent and the steric properties of these ligands provide information on how the ligand compares with the enzyme.

PAGE 43

38 Figure 15 : Five Robson type macrocyclic ligands that form dinuclear Cu II complexes and exhibit diphenolase activity (Banu, K. S et al., 2008). Using 3,5 di tert butylcatechol as a substrate these ligan ds (Figure 15 ) after copper had been introduced, either formed stable Cu II substrate adducts or had diphenolase activity depending on the solvent. In m ethanol only ligand 2 (Figure 15 ) formed a stable adduct

PAGE 44

39 while the rest had activity. In acetonitrile lig ands 2, 3, and 4 (Figure 15 ) all formed stable adducts while the other two have activity with the substrate (Banu, K. S. et al., 2008). Despite more active site/substrate adducts f orming with acetonitrile, the substrate adduct found in the presence of methanol is the most stable. The ligand with the most catalytic ability after the copper is introduced is ligand complex 1 (Figure 15 ) which forms a 6 membered chelate ring where its c ounterpart ligand complex 2 forms a 5 membered chelate ring. Ligand complex 5 (Figure 15 ) has the next best catalytic activity since the al kyl groups in 3 and 4 (Figure 15 ) causes steric hindrance towards the substrate (Banu, K. S. et al., 2008). Monodenta te ligand binding is thought to occur in these macrocyclic ligands which follow the binding motif associated with diphenolase activity. The solvent effect on activity and the various differences in binding affinity show how various reaction and coordinatio n environments can alter how the substrate interacts with the copper center. These ligands do not bind dioxygen but rather utilize the alcohol groups present in the ligand. Having no available oxygen atoms to donate prevent this type of ligand from executi ng monophenolase activity. Substrate access blockage is thought to be a major reason most hemocyanins have little to no enzymatic activity in its native form so not being able to release bound peroxide has no biological counterpart (Idakieva, K. et al., 20 13). These ligands have no blockage and the ability for a substrate or dioxygen to complex up irreversibly however, can give insight into the specifics of what makes the complex viable (Comba, P. et al., 2012). Trinuclear copper containing ligands have also been employed to carry out catechol oxidase activity. Some of t hese complexes have been found to have

PAGE 45

40 enantioselectivity and result in a single enantiomer having greater affinity for the ligand active site (Mutti, F. G. et al., 2009). Figure 1 6 : Tr inuclear copper ligand with enantiomer specific reactions with certain substrates (Mutti, F. G. et al., 2009). This ligand binds diphenols using the two A metal centers as designated on the above Figure 1 6 Two substrates were found to have enantiomer sele ctivity: DOPA OMe and norepinephrine. Both substrates have more binding affinity to the D compared to the L form. Since laccases have spectator copper atoms near the type 3 copper active site, this kind of ligand could be used to characterize multi copper containing enzymes. One of the earliest ligands (Figure 1 7 ) with both mono and diphenolase activity is bis{bis[2 (1 methyl 2 benzimidazolyl) ethyl]amino} m xylene (L66) (Casella, L. et al., 1996). At temperatures of its ability to carry out mono and diphenolase activity.

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41 Figure 1 7 bis{bis[2 (1 methyl 2 benzimidazolyl) ethyl]amino} m xylene with r eactions with mono (methyl 4 hydroxybenzoate) and diphenols ( 2,4 di tert butyl phenolate ) (Rolff, M., Schottenheim, J. et al., 2011). The complex did not retain its functionality at higher temperature due to the Michael addition of the reactants with the p roduct intermediates. This reaction integrity loss at room temperature does not stop the quinone product from being produced but rather, ~40% of the yield is lost to the side reaction (Casella, L. et al., 1996).

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42 Figure 1 8 : Reaction product due to Michael addition to the quinone intermediate (Casella, L. et al., 1996). Desp ite this set back, this ligand was the first small molecule model of tyrosinase to bind dioxygen and have both mono and diphenolase activity (Rolff, M., Schottenheim, J. et al., 2011). The ligand also assemble d a 2 2 adduct when the copper atoms complex with peroxide making it biomimetic in the sense that it follow ed the motif of class 3 copper proteins. Karlin and coworkers were one of the first gr oups of people to explore ligands that mimic the side on dicopper peroxo moiety (Rolff, M., Schottenheim, J. et al., 2011). Early on they discovered a ligand that executed monophenol hydroxylation in a manner similar to tyrosinase with the same dicopper pe roxo core adduct as the enzyme. The ligand use d two pyridyl alkylamines that are xylyl bridged which effectively comple d with two Cu I atoms.

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43 Figure 1 9 : The general synthesis reactions for ligands created (Karlin, K. D. et al., 2012). Upon the introduct ion of dioxygen, the ligand rotates creating the 2 2 adduct as depicted in figure 20

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44 Figure 20 : Model system with 2 2 side on adducts forming with the central arene being hydroxylated (Karlin, K. D. et al., 2012). These ligands using 4 pyridal s ubstituents have recently been synthesized and used to study copper dioxygen kinetics.

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45 Figure 2 1 : 4 Pyridal ligands used to measure the rate of dioxygen binding with different pyridal R groups illustrating three distinct oxidation states (1, 2, and 3) (Karlin, K. D. et al., 2012). These ligands (1a 1d) (Figure 2 1 ) at low temperatures in dichloromethane complex with dioxygen (2a 2d) (Figure 2 1 ) reversibly (k 1 for forward reaction, k 1 for reverse reaction) (Figure 20 ) which will subsequently hydroxylate the arene ring (k 2 (Figure 19) ; 3a 3d) (Figure 2 1 ). At low temperatures (~ rapidly (Figure 20 ) (k 1 >>>k 1 ) forming the side on dicopper peroxo core which then undergoes a unimolecular decay from 2 to 3 (F igure 20 ). At higher temperatures, 2 is only partially formed which means k 1 must be taken into account. Dioxygen has been hypothesized to bind to one of the copper atoms creating an undetectable superoxo

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46 intermediate before creating the side on adduct. T he rate of dioxygen binding (k 1 ) was found to increase from 1a to 1b to 1c which shows the more electron donating capacity the ligand has, the more favorable the dioxygen binding is. The unsymmetrical ligand 1d however, has a lower k 1 than its symmetrical counterpart 1. This is seen as evidence that the formation of a superoxo intermediate is not rate determining since the coordination environment change would not affect superoxo binding rate. There are steric constraints that decrease the bond strength of the dicopper peroxo core giving 2d a higher k 1 The asymmetrical nature of 1d makes the arene hydroxylation step (k 2 ) much less favorable at low temperatures. At higher temperatures however, it loses stability which promotes the decay from 2d to 2c (Karli n, K. D. et al., 2012). Some bidentate alkylamine xylyl based ligands have been found to form end on adducts and do not hydroxylate the arene ring (Mandal, S. et al., 2012). The ligands synthesized by Karlin and co workers determined that the bis oxo Cu III 2 equilibrium adduct (Figure 4 ) does not occur. These ligands (Figure 22 ) however do form that adduct.

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47 Figure 22 : Ligands synthesized with four novel complexes generated: L H,H with MeCN and PPh 3 (1 and 2), and L Me,Me with MeCN and PPh 3 (3 and 4) (Mandal, S. et al., 2012). Ligands 1 and 3 (Figure 2 2 ) result in arene ring hydroxylation but rather uniquely, 3 does this despite forming the bis oxo adduct at low temperatures which has not been re, this adduct was found to have both mono and diphenolase activity. Figure 2 3 : Reaction of bis oxo (Figure3) adduct from ligand 3 (Figure 2 2 ) with 2,4 di tert

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48 At room temperature this ligand had both mono and diphenolase and could create stoichiometric amounts of product in the absence of dioxygen meaning the active site would remain reduced after it reacts. Figure 2 4 : The various reaction pathways with the ar ene hydroxylated ligand and the 2 2 dicopper peroxo adduct (Mandal, S. et al., 2012). This ligand was found to have strongly antiferromagnetically oxidized copper atoms which is indicative of class 3 copper proteins. The active site of class 3 copper proteins varies depending on the protein but in its active form is strongly antiferromagnetically coupled, and there is a distance of around

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49 ~4 between the two copper atoms. Ligands with larger Cu Cu bond lengths (~7.5 ) have no steric capability of both binding a diphenol but can still maintain diphenolase activity. Also EPR spectra have shown that even at this longer distance, the two copper atoms are sometimes still coupled (Martinez, A. et al., 2012). Computational a nalysis using density functional theory on ligands with known rate values have been used to help understand the theoretical step by step substrate reaction mechanisms with thermodynamically viable energy values (Martinez, A. et al., 2012). Figure 2 5 : Li gands (A, B, and C) previously synthesized with known rate values when reacting with catechol (Martinez, A. et al., 2012). Experimentally, ligand A (Figure 2 5 ) has been found to have a Cu Cu distance of 7.34 while ligand B (Figure 2 5 ) has 7.52 between the two atoms. Ligand C (Figure 2 5 ) is used as a comparison between a lone copper versus two coupled coppers and the effects on the reaction energies.

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50 Figure 2 6 : The binding energies of ligand A and B with catechol or its anion (Martinez, A. et al., 201 2). The above computational scheme (Figure 2 6 ) shows that theoretically the catecholate anion is likely to form before the substrate binds since the reaction scheme is significantly more favorable in the third row compared to the first row Also having a m etal bound hydroxide anion in the active site coordination environment is less favorable. Since the step shown is a water displacement step, the hydroxide anion is less willing to be moved thus increasing the amount of energy the step takes by having a gre ater electron density. Ligand B has a higher electron accepting ability than A in its structure this making it more energetically efficient.

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51 Figure 2 7 : The binding energies of the mononuclear copper ligand C (Martinez, A. et al., 2012). The mononuclear copper ligand (Figure 2 7 ) shows the energies required for catechol or catecholate anion to displace water. When generally comparing the energies of the dinuclear and mononuclear ligands, it is clear that the dinuclear reaction pathway is more favorable. Th e coupled copper atoms do not both bind the ligand but one increases the electron acceptor capacity of the other (Martinez, A. et al., 2012). Tetradentate N 4 donor ligands have been found to have diphenolase activity by using both mononuclear Cu II and di nuclear Cu I containing copper centers (Figure 2 8 ).

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52 Figure 2 8 : Four ligands exhibiting diphenolase activity (Ramadan, A. E. M. et al., 2012). These ligands showed that the disassociation constant of the X group in the case of (1) and (2) (Figure 2 8 ) largely affects the rate of the disassociation which was found to be less with the hydroxyl group than the chloride (Ramadan, A. E. M. et al., 2012). This is in agreement with the theoretical analysis done by Martinez where the amount of energy it takes to displace what is around the copper atom directly affects the rate of catalysis. Also similar to the results of Martinez, complex (4) reacts at a greater rate than the other three. The rate measured for these ligands was for the oxidation of 3,5 ditertbuty lcatechol to 3,5 ditertbutylquinone.

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53 Figure 2 9 : Reaction mechanism using a mononuclear Cu II ligand (Figure 27) (Ramadan, A. E. M. et al., 2012). The coordination around the Cu II ligands is depicted (Figure 2 9 ) showing an intermediate radical that is formed before the product. This usage of single copper ligands is interesting because they catalyze a reaction that single copper enzymes do not. By altering ligands with enzymatic activities, the components can be modified to cause slight changes in the reaction mechanism to see what environmental factors play a role. Using a bis benzimidazolyl diamide ligand (Figure 30 ) and nitrate anions, Bakshi found two separate reaction mechanisms depending on the spectator ion present (Figures 30 and 31) (Bakshi, R. et al., 2011).

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54 Figure 30 : Benzimidazolyl ligand (Bakshi, R. et al., 2011). The substrate used, ditertbutylcatechol (DBTC), is converted to ditertbutylquinone (DBTQ) by the synthetic ligand in a water and meth anol solution saturated with dioxygen. Figure 31 : Mechanism with spectator phosphate anions in solution (Bakshi, R. et al., 2011).

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55 This reaction mechanism mirrors the diphenolase mechanism closely however its reaction is thought to be carried forward by the hydroxyl bound met state and not the oxy state. Figure 3 2 : Reaction carried out in the presence of acetate anions (Bakshi, R. et al., 2011). This reaction mechanism uses a DTBC radical as an intermediate. It also carries the reaction forward with a reduced copper atom. In both cases, the addition of a picolyl group near a nitrogen that complexes with a copper atom enhanced the rate of reaction. It is thought to do this because the addition of a basic group stabilizes the pH by acting as a proton s ponge (Bakshi, R. et al., 2011). By simply changing a spectator ion, the reaction mechanism changes drastically. This could also have major implications when looking at the enzyme activity in vivo Specifically, the tyrosinase monophenolase reaction mechanism is thought to have a radical intermediate that it does not release. Maybe this reaction intermediate is allowed due to acetate ions around the active site. Many

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56 tyrosinases and catechol oxidas es reside on the cell membrane. This means there are many phosphate groups around it in the form of the phospholipid bilayer. Perhaps in vivo the enzyme active site is near the phospha te head groups fortifies the substrate bound to the single Cu II in the d iphenolase reaction step. Some ligands that coordinate with a single copper atom can carry out the same reaction as many dinuclear copper containing class 3 copper proteins. By studying the chemistry behind what allows this, insight into some possible fact ors that affect the reaction in vivo can be deduced. Bis Tris Imidazole Ligand Proposal Since the active site of type 3 copper complex proteins are comprised entirely of histidine residues, the usage of imidazole functional units closely mirrors the biolog ical counterpart to the biomimetic mettaloenzyme ligands. A variety of tris and polydentate tris imidazole ligands have been recently synthesized with copper complex structures being noted (Volkman, J. and Nicholas, K. M., 2012). The creation of tris imida zole tripodal ligands has been accomplished and used in Fe III complexing. Tripodal N donor ligands have been found to coordinate Cu I in a way that is very similar to type 3 copper centers (Kujime, M. and Fujii, H., 2005). A viable but untested biomimetic ligand would be to attach two tripodal imidazole subunits to have one ligand unit bind two Cu I atoms. How to make a tripodal imidazole ligand is outlined in Volkman, 2004. Kujime outlines a synthesis that makes the same ligand as Volkman but includes varia tions with functional groups attached.

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57 Figure 3 3 : Metal binding ligand with three imidazole subunits (Kujime, M. and Fujii, H., 2005). This ligand can be attached to another one via a linker chain of any number of carbons long in a process outlined by McKie in the supplemental information (in this case a propyl chain is used). Figure 3 4 : Two imidazoles linked through the addition of 1, 3 bromopropane (McKie, R. et al., 2007). The process described by McKie uses only imidazole so alterations must be made to ensure the Kujime and Volkman ligand will react properly with the 1,3 bromopropane. This requires the alcohol group in the tripodal i midazole ligand to be protected since a cation in that position would be relatively stable which could cause the hydroxyl group to react with the 1,3 bromopropane or any of the other reactant in the synthesis process. By the addition of methanol in acidic solution, the OH would be replaced with a OHCH 3 which would allow the tripodal ligands to link.

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58 Figure 3 5 : Desired bis tris imidazole ligand. To keep the bromopropane from connecting chains of tripodal ligands, it would have to be added at a much lowe r concentration than trisimidazole present. These ligands would closely resemble the self assemblies seen at low temperatures while being tridentate rather than monodentate. The ligand is innately flexible which has been seen to be an important component o f ligand catalytic ability however the propyl linker may prevent the two sides from complexing with copper near each other. A longer chain may allow the two imidazole tripods with attached copper to be nearer to each other and thus display a more biomimeti c structure. Conclusion Class 3 copper proteins use a strongly biologically conserved and entropically favorable active site to bind dioxygen to two coupled copper atoms and by doing so, carry out varied and crucial biological functions. These functions range from carrying oxygen throughout the body, producing various pigments, creating novel bioavailable compounds, producing melanin for protection against sun and pathogens, to use as

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59 pathogens, or provide hardening and color to exoskeletons Biomimetic l igands are synthesized to help characterize the active site and provide subtle details about substrate specificity and kinetic activity. The importance of this copper active site motif is clear due to their many functions. The fact that six histidines and two reduced copper atoms will self assemble at low temperatures shows how energetically favorable the active site is, and probably has implications as to why primordial life adopted it for its use in oxidizing compounds. The study of ligands and protein st ructures will eventually yield information on how to oxidize compounds that will yield commercially useful products. The study of active sites can show how to inhibit the activity of pathogenic tyrosinases or prevent melanization in skin. Being able to con trol the biosynthesis of pigments can alter the coloration and secondary metabolite content. As more enzyme structures are elucidated, more proteins are discovered, or functions are uncovered, the understanding of this ubiquitous copper center, its coordin ation site chemistry, and evolutionary phylogeny, will give insight into overall metalloprotein structure and importance.

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63 Karlin, K. D., Zhang, C. X., Rheingold, A. L., Galliker, B., Kaderli, S., & Zuberbuehler, A. D. (2012). Reversible dioxygen binding and arene hydroxylation reactions: Kinetic and thermodynamic studies involving ligand electronic and structural variati ons. Inorganica Chimica Acta, 389 138 150. Klabunde, T., Eicken, C., Sacchettini, J., & Krebs, B. (1998). Crystal structure of a plant catechol oxidase containing a dicopper center. Nature Structural Biology, 5 (12), 1084 1090. Kujime, M., & Fujii, H. (2 005). Synthesis of sterically hindered tris(4 imidazolyl)carbinol ligands and their copper(I) complexes related to metalloenzymes. Tetrahedron Letters, 46 (16) Mandal, S., Mukherjee, J., Lloret, F., & Mukherjee, R. (2012). Modeling tyrosinase and catechola se activity using new m xylyl based ligands with bidentate alkylamine terminal coordination. Inorganic Chemistry, 51 (24), 13148 13161. Martin, A. G., Depoix, F., Stohr, M., Meissner, U., Hagner Holler, S., Hammouti, K., et al. (2007). Limulus polyphemus h emocyanin: 10 cryo EM structure, sequence analysis, molecular modelling and rigid body fitting reveal the interfaces between the eight hexamers. Journal of Molecular Biology, 366 (4), 1332 1350. Martinez, A., Membrillo, I., Ugalde Saldivar, V. M., & Gasqu e, L. (2012). Dinuclear copper complexes with imidazole derivative ligands: A theoretical study related to catechol oxidase activity. Journal of Physical Chemistry B, 116 (28), 8038 8044. Matoba, Y., Kumagai, T., Yamamoto, A., Yoshitsu, H., & Sugiyama, M. (2006). Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis. Journal of Biological Chemistry, 281 (13), 8981 8990. Mayer, A. M. (2006). Polyphenol oxidases in plants and fungi: Going places? A review. Phyto chemistry, 67 (21), 2318 2331. Mckie, R., Murphy, J. A., Park, S. R., Spicer, M. D., & Zhou, S. (2007). Homoleptic crown n heterocyclic carbene complexes. Angewandte Chemie International Edition, 46 (34), 6525 6528. Mutti, F. G., Zoppellaro, G., Gullotti, M., Santagostini, L., Pagliarin, R., Andersson, K. K., et al. (2009). Biomimetic modelling of copper enzymes: Synthesis, characterization, EPR analysis and enantioselective catalytic oxidations by a new chiral trinuclear copper(II) complex. European Journa l of Inorganic Chemistry, (4), 554 566.

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65 S anyal I., S trange R., B lackburn N., & K arlin K. (1991). Formation of a cop per dioxygen complex (Cu2 O2) using simple imidazole ligands. Journal of the American Chemical Society, 113 (12), 4692 4693. Sendovski, M., Kanteev, M., Ben Yosef, V. S., Adir, N., & Fishman, A. (2010). Crystallization and preliminary X ray crystallographi c analysis of a bacterial tyrosinase from bacillus megaterium Acta Crystallographica Section F Structural Biology and Crystallization Communications, 66 1101 1103. Sendovski, M., Kanteev, M., Ben Yosef, V. S., Adir, N., & Fishman, A. (2011). First structures of an active bacterial tyrosinase reveal copper plasticity. Journal of Molecular Biology, 405 (1), 227 237. Starha, P., Travnicek, Z., Herchel, R., Popa, I., Suchy, P., & Vanco, J. (2009). Dinuclear copper(II) complexes containing 6 (benz ylamino)purines as bridging ligands: Synthesis, characterization, and in vitro and in vivo antioxidant activities. Journal of Inorganic Biochemistry, 103 (3), 432 440. Suzuki, H., Furusho, Y., Higashi, T., Ohnishi, Y., & Horinouchi, S. (2006). A novel o am inophenol oxidase responsible for formation of the phenoxazinone chromophore of grixazone. Journal of Biological Chemistry, 281 (2), 824 833. Terwilliger, N. B. (2007). Hemocyanins and the immune response: Defense against the dark arts. Integrative and Com parative Biology, 47 (4), 662 665. Velkova, L., Dimitrov, I., Schwarz, H., Stevanovic, S., Voelter, W., Salvato, B., et al. (2010). Structure of hemocyanin from garden snail helix lucorum Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 157 (1), 16 25. Virador, V. M., Reyes Grajeda, J. P., Blanco Labra, A., Mendiola Olaya, E., Smith, G. M., Moreno, A., et al. (2010). Cloning, sequencing, purification, and crystal structure of grenache ( vitis vinifera ) polyphenol oxidase. Journal of Agricultural and Food Chemistry, 58 (2), 1189 1201. Volkman, J., & Nicholas, K. M. (2004). Efficient synthesis of tris(4 imidazolyl)methanol derivatives. Organic Letters, 6 (23) Volkman, J., & Nicholas, K. M. (2012). A synthetic quest for tris(imidazolyl) carboxylates and their metal complexes: Active site models for quercetin 2,3 dioxygenases and other non heme redox metalloenzymes. Tetrahedron, 68 (16), 3368 3376.

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