Vincent Massey

The production of superoxide anion radicals in the reaction of reduced flavins and flavoproteins with molecular oxygen

Abstract The generation of O2•− in the reaction of reduced flavins and several flavoproteins has been shown. This has been demonstrated by the ability to catalyse the aerobic reduction of cytochrome c and the inhibition of this reduction by erythrocuprein. In contrast to the results with the intact enzyme, the reduction of cytochrome c by deflavoxanthine oxidase is not inhibited by erythrocuprein, indicating that the site of production of O2•− in the native enzyme is flavin, rather than non-heme iron.

Introduction: flavoprotein structure and mechanism.

UNDERSTANDING OF THE vERsATILE chemistry of flavins and the mechanisms of action of flavoprotein enzymes has progressed enormously in the last 10 years, especially since the X-ray crystal structures of many flavoenzymes have become available and meaningful site-directed mutagenesis of putative active site residues has been possible. This issue of The FASEB Journal contains the first in a series of articles that will attempt to review and summarize the present state of knowledge of representatives of the various classes of flavoenzymes. In this series, wherever possible, information regarding the catalytic mechanism obtained from solution studies will be correlated with structural information obtained by Xray crystallographic techniques. It has been apparent for many years that flavoenzymes can be grouped into a relatively small number of classes, where members within the same class share many common properties differing from those of other classes, including the types of reactions catalyzed, the ability to use molecular oxygen as acceptor, and the nature of auxiliary redox centers. In this series, examples of most of these classes will be considered. The basis for classification and details of common properties have been reviewed previously (1) but for convenience will be reiterated here.

Direct demonstration of superoxide anion production during the oxidation of reduced flavin and of its catalytic decomposition by erythrocuprein

The oxidation of reduced flavins by molecular oxygen at neutral to alkaline pH produces substantial yields of the superoxide anion, O2•. This species is rapidly destroyed by catalytic quantities of the copper protein, erythrocuprein, and by stoichiometric quantities of ferricytochrome c.

An optomechanical transducer in the blue light receptor phototropin from Avena sativa

The PHOT1 (NPH1) gene from Avena sativa specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine/threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of A. sativa phototropin with various 13C/15N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by 1H and 31P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain conducive to autophosphorylation of NPH1.

[73] Determination of nonheme iron, total iron, and copper

Publisher Summary This chapter describes the methods for the determination of nonheme iron, total iron, and copper. Nonheme iron may be extracted quantitatively from most materials with trichloroacetic acid, example NADH dehydrogenase, succinie dehydrogenase, and the liberated iron. For the determination of total iron in biological samples, the material for analysis is most commonly subjected to an acid digestion prior to carrying out the color reaction. A number of different methods for digesting the sample and developing the color are described (methods A, B, and C). A method is also described for the determination of total iron using an extraction procedure in place of the acid digestion (method D). There are a number of reagents, which form more or less specific colored complexes with copper, so that interference can usually be overcome by selection from the methods of determination available. This is especially so for biological samples which are generally free from interfering elements, and preliminary separations are seldom required. As for iron determinations, the color reaction with copper is usually preceded by digestion of the sample (methods A and B). An extraction method has also been described, and has been used to determine the valency state of the copper in the sample.

L-lactate oxidase and L-lactate monooxygenase: Mechanistic variations on a common structural theme

Properties of L-lactate oxidase from Aerococcus viridans are described. The gene encoding the enzyme has been isolated. From its cDNA sequence the amino acid sequence has been derived and shown to have high similarity with those of other enzymes catalyzing oxidation of L-alpha-hydroxy acids, including flavocytochrome b2, lactate monooxygenase, glycolate oxidase, mandelate dehydrogenases and a long chain alpha-hydroxy acid oxidase. The enzyme is expressed in Escherichia coli, and is a flavoprotein containing FMN as prosthetic group. It shares many properties of other alpha-hydroxy acid oxidizing enzymes, eg stabilization of the anionic semiquinone form of the flavin, facile formation of flavin-N(5)-sulfite adducts and a set of conserved amino acid residues around the bound flavin. Steady-state and rapid reaction kinetics of the enzyme have been studied and found to share many characteristics with those of L-lactate monooxygenase, but to differ from the latter in quantitative aspects. It is these quantitative differences between the two enzymes which account for the differences in the overall reactions catalyzed. These differences arise from different stabilities of a common intermediate of reduced flavin enzyme and pyruvate. In the case of the monooxygenase this complex is very stable and is the form that reacts with O2 to give a complex in which the oxidative decarboxylation occurs, yielding the products, acetate, CO2, and H2O (Lockridge O, Massey V, Sullivan PA (1972) J Biol Chem 247, 8097-8106). With lactate oxidase, the complex dissociates rapidly, with the result that it is the free reduced flavin form of the enzyme that reacts with O2, to give the observed products, pyruvate and H2O2.

THE REACTION OF REDUCED XANTHINE DEHYDROGENASE WITH MOLECULAR OXYGEN REACTION KINETICS AND MEASUREMENT OF SUPEROXIDE RADICAL

Xanthine dehydrogenase (XDH) from bovine milk contains significant activity in xanthine/oxygen turnover assays. The oxidative half-reaction of XDH with molecular oxygen has been studied in detail, at 25 °C, pH 7.5, to determine the basis of the preference of XDH for NAD over oxygen as oxidizing substrate. Spectral changes of XDH accompanying oxidation were followed by stopped-flow spectrophotometry. The amount of superoxide radicals formed during oxidation was investigated to assess the ability of XDH to catalyze production of oxygen radicals. Reduced XDH reacts with oxygen in at least 4 bi-molecular steps, with 1.7-1.9 mol of superoxide per mol of XDH formed from the last 2 electrons oxidized. A model is discussed in which the flavin hydroquinone transfers electrons to oxygen to produce hydrogen peroxide at a rate constant of at least 72,000 M−1 s−1, whereas flavin semiquinone reduces oxygen to form superoxide as slow as 16 M−1 s−1. Steady-state kinetics of xanthine/oxygen and NADH/oxygen turnover of XDH were determined to have kcat values of 2.1 ± 0.1 and 2.5 ± 0.9 s−1, respectively, at 25 °C, pH 7.5. XDH is therefore capable of catalyzing the formation of reduced oxygen species at one-third the rate of xanthine/NAD turnover, 6.3 s−1 (Hunt, J., and Massey, V. (1992) J. Biol. Chem. 267, 21479-21485), in the absence of NAD. As XDH contains a significant and intrinsic xanthine oxidase activity, estimates of relative amounts of XO and XDH based solely upon turnover assays must be made with caution. Initial-rate assays containing varying amounts of xanthine, NAD, and oxygen indicate that at 100% oxygen saturation, NADH formation is only inhibited at concentrations of xanthine and NAD below Km for each substrate.

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide. These conflicting requirements are met by the coordination of multiple movements involving the protein, the two cofactors, and the substrate. Here, using the R220Q mutant form of para-hydroxybenzoate hydroxylase, we show that in the absence of substrate, the large βαβ domain (residues 1–180) and the smaller sheet domain (residues 180–270) separate slightly, and the flavin swings out to a more exposed position to open an aqueous channel from the solvent to the protein interior. Substrate entry occurs by first binding at a surface site and then sliding into the protein interior. In our study of this mutant, the structure of the complex with pyridine nucleotide was obtained. This cofactor binds in an extended conformation at the enzyme surface in a groove that crosses the binding site of FAD. We postulate that for stereospecific reduction, the flavin swings to an out position and NADPH assumes a folded conformation that brings its nicotinamide moiety into close contact with the isoalloxazine moiety of the flavin. This work clearly shows how complex dynamics can play a central role in catalysis by enzymes.

Tight binding inhibitors of xanthine oxidase.

The enzyme is well-suited to carry out the catalysis of these oxidative reactions, contain- ing as it does four redox-active sites: molybdenum, flavin adenine dinucleotide (FAD) and a pair of iron-sulfur centers of the ferredoxin type (Fe2S2). The purine substrates interact with the enzyme at the molybdenum site of xanthine oxidase and oxygen at the flavin site. The two metabolic reactions above Constitute the final steps of purine catab- olism in primates and as such play an important role in determining the in vivo steady state levels of the various purines, particularly that of uric acid. The introduction of

Purification and characterization of glycolic acid oxidase from pig liver.

1. 1. A procedure for the isolation of glycolic acid oxidase (glycolate:O2 oxidoreductase, EC 1.1.3.1) from pig liver is described. The enzyme has been crystallized and the amino acid composition has been determined. 2. 2. Glycolic acid oxidase binds a variety of anions. Sulfate, sulfite, chloride, heptanoate, oxalate and phosphate each cause characteristic changes in the visible absorption spectrum of the enzyme. Studies on these effects have suggested that the FMN prosthetic group may be near one or more positively charged groups and also a hydrophobic region of the protein. 3. 3. The pK for the ionization of the 3-imino nitrogen in free FMN (pK = 10.3) is shifted to about pH 8 in FMN bound to glycolic acid oxidase. The observed pK is increased (to pH 9.3) when the enzyme titration is performed in the presence of oxalate. 4. 4. Glycolic acid oxidase contains a second chromophore which has not yet been identified. FMN can be selectively removed from the protein to give a flavin-free protein which is green in color. This green material has absorption maxima at 328,425, and 600 mμ and a fluorescence emission maximum at about 450 mμ. 5. 5. The visible absorption of glycolic acid oxidase is bleached in an autogenous reaction. This reaction, which is spectrally similar to the bleaching that occurs when sulfite is added to the enzyme, has been partly characterized. 6. 6. The unusual absorption spectrum of pig liver glycolic acid oxidase and possibly other glycolic acid oxidases is due, at least in part, to anion binding, the presence of the unidentified green chromophore, and autogenous bleaching.