Based on its generally accessible I/II redox couple and bioavailability, copper plays a wide variety of roles in nature that mostly involve electron transfer (ET), O2 binding, activation and reduction, NO2− and N2O reduction and substrate activation. Copper sites that perform ET are the mononuclear blue Cu site that has a highly covalent CuII-S(Cys) bond and the binuclear CuA site that has a Cu2S(Cys)2 core with a Cu-Cu bond that keeps the site delocalized (Cu(1.5)2) in its oxidized state. In contrast to inorganic Cu complexes, these metalloprotein sites transfer electrons rapidly often over long distances, as has been previously reviewed.1–4 Blue Cu and CuA sites will only be considered here in their relation to intramolecular ET in multi-center enzymes. The focus of this review is on the Cu enzymes (Figure 1). Many are involved in O2 activation and reduction, which has mostly been thought to involve at least two electrons to overcome spin forbiddenness and the low potential of the one electron reduction to superoxide (Figure 2).5,6 Since the Cu(III) redox state has not been observed in biology, this requires either more than one Cu center or one copper and an additional redox active organic cofactor. The latter is formed in a biogenesis reaction of a residue (Tyr) that is also Cu catalyzed in the first turnover of the protein. Recently, however, there have been a number of enzymes suggested to utilize one Cu to activate O2 by 1e− reduction to form a Cu(II)-O2•− intermediate (an innersphere redox process) and it is important to understand the active site requirements to drive this reaction. The oxidases that catalyze the 4e−reduction of O2 to H2O are unique in that they effectively perform this reaction in one step indicating that the free energy barrier for the second two-electron reduction of the peroxide product of the first two-electron step is very low. In nature this requires either a trinuclear Cu cluster (in the multicopper oxidases) or a Cu/Tyr/Heme Fe cluster (in the cytochrome oxidases). The former accomplishes this with almost no overpotential maximizing its ability to oxidize substrates and its utility in biofuel cells, while the latter class of enzymes uses the excess energy to pump protons for ATP synthesis. In bacterial denitrification, a mononuclear Cu center catalyzes the 1e- reduction of nitrite to NO while a unique µ4S2−Cu4 cluster catalyzes the reduction of N2O to N2 and H2O, a 2e− process yet requiring 4Cu’s. Finally there are now several classes of enzymes that utilize an oxidized Cu(II) center to activate a covalently bound substrate to react with O2.



Figure 1

Copper active sites in biology.





Figure 2

Latimer Diagram for Oxygen Reduction at pH = 7.0 Adapted from References 5 and 6.



This review presents in depth discussions of all these classes of Cu enzymes and the correlations within and among these classes. For each class we review our present understanding of the enzymology, kinetics, geometric structures, electronic structures and the reaction mechanisms these have elucidated. While the emphasis here is on the enzymology, model studies have significantly contributed to our understanding of O2 activation by a number of Cu enzymes and are included in appropriate subsections of this review. In general we will consider how the covalency of a Cu(II)–substrate bond can activate the substrate for its spin forbidden reaction with O2, how in binuclear Cu enzymes the exchange coupling between Cu’s overcomes the spin forbiddenness of O2 binding and controls electron transfer to O2 to direct catalysis either to perform two e− electrophilic aromatic substitution or 1e− H-atom abstraction, the type of oxygen intermediate that is required for H-atom abstraction from the strong C-H bond of methane (104 kcal/mol) and how the trinuclear Cu cluster and the Cu/Tyr/Heme Fe cluster achieve their very low barriers for the reductive cleavage of the O-O bond.

Much of the insight available into these mechanisms in Cu biochemistry has come from the application of a wide range of spectroscopies and the correlation of spectroscopic results to electronic structure calculations. Thus we start with a tutorial on the different spectroscopic methods utilized to study mononuclear and multinuclear Cu enzymes and their correlations to different levels of electronic structure calculations.

/pdf/copper-active-sites-in-biology-lhhabv47if.pdf

Copper Active Sites in Biology

Four new quercetin-derived oxidation products (1-4) and lunularin-4-O-beta-D-glucoside (5) were isolated from a water extract of onion (Allium cepa) skin, together with 17 other known compounds. Antibacterial assays for the isolated compounds showed that 2-(3,4-dihydroxyphenyl)-4,6-dihydroxy-2-methoxybenzofuran-3-one (1) presented selective activity against Helicobacter pylori strains and 3-(quercetin-8-yl)-2,3-epoxyflavanone (4) showed antibacterial activity against MRSA and H. pylori strains at the same time that it increased susceptibility of MRSA to beta-lactams. Evaluation of antioxidant activity against DPPH for the isolated compounds showed that the new derivative compounds (1-4) and 2,5,7,3',4'-pentahydroxy-3,4-flavandione (6) are more active than quercetin.

/pdf/antibacterial-and-antioxidant-activities-of-quercetin-1g6wicbhyz.pdf

Antibacterial and antioxidant activities of quercetin oxidation products from yellow onion (Allium cepa) skin.

https://dspace.library.uu.nl/bitstream/handle/1874/14262/3.Fusetti2002%20pp259-68.pdf;sequence=1

Crystal Structure of the Copper-Containing Quercetin 2,3-Dioxygenase from Aspergillus japonicus

Ring-cleaving dioxygenases catalyze key reactions in the aerobic microbial degradation of aromatic compounds. Many pathways converge to catecholic intermediates, which are subject to ortho or meta cleavage by intradiol or extradiol dioxygenases, respectively. However, a number of degradation pathways proceed via noncatecholic hydroxy-substituted aromatic carboxylic acids like gentisate, salicylate, 1-hydroxy-2-naphthoate, or aminohydroxybenzoates. The ring-cleaving dioxygenases active toward these compounds belong to the cupin superfamily, which is characterized by a six-stranded β-barrel fold and conserved amino acid motifs that provide the 3His or 2- or 3His-1Glu ligand environment of a divalent metal ion. Most cupin-type ring cleavage dioxygenases use an FeII center for catalysis, and the proposed mechanism is very similar to that of the canonical (type I) extradiol dioxygenases. The metal ion is presumed to act as an electron conduit for single electron transfer from the metal-bound substrate anion to O2, resulting in activation of both substrates to radical species. The family of cupin-type dioxygenases also involves quercetinase (flavonol 2,4-dioxygenase), which opens up two C-C bonds of the heterocyclic ring of quercetin, a wide-spread plant flavonol. Remarkably, bacterial quercetinases are capable of using different divalent metal ions for catalysis, suggesting that the redox properties of the metal are relatively unimportant for the catalytic reaction. The major role of the active-site metal ion could be to correctly position the substrate and to stabilize transition states and intermediates rather than to mediate electron transfer. The tentative hypothesis that quercetinase catalysis involves direct electron transfer from metal-bound flavonolate to O2 is supported by model chemistry.

Ring-cleaving dioxygenases with a cupin fold.

Quercetin 2,3-dioxygenase (2,3QD) is the only firmly established copper dioxygenase known so far. Depending solely on a mononuclear Cu center, it catalyzes the breakage of the O-heterocycle of flavonols, producing more easily degradable phenolic carboxylic acid ester derivatives. In the enzymatic process, two C—C bonds are broken and concomitantly carbon monoxide is released. The x-ray structures of Aspergillus japonicus 2,3QD anaerobically complexed with the substrate kaempferol and the natural substrate quercetin have been determined at 1.90- and 1.75-A resolution, respectively. Flavonols coordinate to the copper ion as monodentate ligands through their 3OH group. They occupy a shallow and overall hydrophobic cavity proximal to the metal center. As a result of a van der Waals contact between the most outward flavonol A-ring and Pro164, a flexible loop in front of the active site becomes partly ordered. Interestingly, flavonols bound to 2,3QD are bent at the C2 atom, which is pyramidalized. The increased local sp3 character at this atom may stabilize a carbon-centered radical activated for dioxygen attack. Glu73 coordinates the copper through its Oɛ1 atom. The short distance of about 2.55 A between its Oɛ2 atom and the flavonol O3 atom suggests that a hydrogen bond exists between the two atoms, indicating that Glu73 can act as a base in flavonol deprotonation and that it retains the proton. Structure-based geometric considerations indicate O2 binding to the flavonol C2 atom as the preferred route for flavonol dioxygenation.

/pdf/anaerobic-enzyme-substrate-structures-provide-insight-into-345j9fhjtq.pdf

Anaerobic Enzyme.Substrate Structures Provide Insight Into the Reaction Mechanism of the Copper- Dependent Quercetin 2,3-Dioxygenase.

The dioxygenase produced by Aspergillus flavus that oxidizes quercetin contains 2 moles of copper per mole of enzyme and binds at least 2 moles of substrate. The enzyme is not inhibited by sulfhydr...

Degradation of rutin by Aspergillus flavus. Studies on specificity, inhibition, and possible reaction mechanism of quercetinase

Evidence is presented that a single enzyme, quercetinase, is responsible for the degradation of quercetin by Aspergillus flavus to yield carbon monoxide and a depside, 2-protocatechuoylphloroglucinol carboxylic acid. A procedure for the isolation of the dioxygenase as a homogeneous protein is described. The most purified preparation degraded 10 800 μmoles of quercetin/h mg protein and was homogeneous as judged by ultracentrifugation and by electrophoresis. The molecular weight was determined as 111 000 + 4000. Km values for quercetin and oxygen as substrates were 5.2 × 10−6 M and 1.2 × 10−4 M respectively. The enzyme is a glycoprotein containing 27.5% carbohydrate and the amino acid composition is presented.

Degradation of rutin by Aspergillus flavus. Purification of the dioxygenase, querecentinase.

Aspergillus flavus produces an extracellular esterase that hydrolyses phenolic carboxylic acid acyl esters. An assay based upon the measurement of the rate of release of phloroglucinol on hydrolysi...

T. Oka

Papers

Degradation of rutin by Aspergillus flavus. Studies on specificity, inhibition, and possible reaction mechanism of quercetinase

Degradation of rutin by Aspergillus flavus. Purification of the dioxygenase, querecentinase.

Purification and properties of a phenol carboxylic acid acyl esterase from Aspergillus flavus.