Polyphenol oxidases in plants

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.

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Copper Active Sites in Biology

Fruits constitute a commercially important and nutritionally indispensable food commodity. Being a part of a balanced diet, fruits play a vital role in human nutrition by supplying the necessary growth regulating factors essential for maintaining normal health. Fruits are widely distributed in nature. One of the limiting factors that influence their economic value is the relatively short ripening period and reduced post-harvest life. Fruit ripening is a highly coordinated, genetically programmed, and an irreversible phenomenon involving a series of physiological, biochemical, and organoleptic changes, that finally leads to the development of a soft edible ripe fruit with desirable quality attributes. Excessive textural softening during ripening leads to adverse effects/spoilage upon storage. Carbohydrates play a major role in the ripening process, by way of depolymerization leading to decreased molecular size with concomitant increase in the levels of ripening inducing specific enzymes, whose target differ from fruit to fruit. The major classes of cell wall polysaccharides that undergo modifications during ripening are starch, pectins, cellulose, and hemicelluloses. Pectins are the common and major components of primary cell wall and middle lamella, contributing to the texture and quality of fruits. Their degradation during ripening seems to be responsible for tissue softening of a number of fruits. Structurally pectins are a diverse group of heteropolysaccharides containing partially methylated D-galacturonic acid residues with side chain appendages of several neutral polysaccharides. The degree of polymerization/esterification and the proportion of neutral sugar residues/side chains are the principal factors contributing to their (micro-) heterogeneity. Pectin degrading enzymes such as polygalacturonase, pectin methyl esterase, lyase, and rhamnogalacturonase are the most implicated in fruit-tissue softening. Recent advances in molecular biology have provided a better understanding of the biochemistry of fruit ripening as well as providing a hand for genetic manipulation of the entire ripening process. It is desirable that significant breakthroughs in such related areas will come forth in the near future, leading to considerable societal benefits.

Fruit Ripening Phenomena–An Overview

Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.

Two classes of common phenolic plant pigments, the anthraquinones and the flavonols, contain many members mutagenic in the Salmonella/mammalian microsome assay. Several reports on the mutagenicity or other genetic or “carcinogenic” effects of these compounds have appeared in the literature or have been presented at various scientific meetings. The object of this review paper is to assess the present state of knowledge with respect to the genetic toxicity of these agents and their dietary intake and metabolic fate in man. Such information is necessary for formulating an assessment of genetic or carcinogenic risk to man posed by these agents.

A review of the genetic effects of naturally occurring flavonoids, anthraquinones and related compounds.

We identified methylenecyclopropylacetic acid, a known metabolite of hypoglycin A, in the urine of two patients with Jamaican vomiting sickness. Excretion of unusual dicarboxylic acids such as 2-ethylmalonic, 2-methylsuccinic, glutaric, adipic and dicarboxylic acids with eight and 10 carbon chains were also detected in both patients. The amounts of these dicarboxylic acids were 70 to 1000 times higher than normal. These metabolites have also been identified in urine of hypoglycin-treated rats. This evidence links hypoglycin A to Jamaican vomiting sickness as its causative agent. Urinary excretion of short-chain fatty acids was also increased up to 300 times higher than normal. These results indicate that, despite their clinical and histologic similarities, the cause and biochemical mechanisms of Jamaican vomiting sickness differ distinctly from those of Reye's syndrome in which these abnormal urinary metabolites are not appreciably increased.

Eccleston A. Kean

Papers

Jamaican vomiting sickness. Biochemical investigation of two cases.

Selective inhibition of acyl-CoA dehydrogenases by a metabolite of hypoglycin.

β-Galactosidase from Coffea Arabica and its role in fruit ripening

Identification of ethylmalonic acid in urine of two patients with the vomitting sickness of Jamaica

γ-Glutamyl transpeptidase of the ackee plant