Copper-tyrosyl radical enzymes

Advances have been made since 2000 that contribute to our understanding of the biogenesis, structure and mechanism of copper-containing tyrosyl radical enzymes. Efforts to detail the biogenesis of galactose oxidase have produced the structure of the precursor enzyme, which provides a framework for emerging mechanistic studies. The role of the tyrosyl radical of cytochrome c oxidase is being defined in studies that aim to understand the His-Tyr crosslink, the location of the radical and, by direct attempts, to provide evidence for the radical during turnover.     

On the role of superoxide radical in the mechanism of action of galactose oxidase

The inhibition of galactose oxidase by superoxide dismutase is a function of the method of assay, nature of substrate, and composition of incubation and assay mixtures, as well as the concentration of dismutase. A reasonable level of inhibition is attained only when superoxide dismutase is present prior to the onset of catalysis although this effect is not observed under all conditions tried. Peroxidase activates galactose oxidase and blocks its interaction with either superoxide dismutase or catalase. These results further obscure the possible role of superoxide radical in the galactose oxidase reaction.     

Resonance Raman evidence for tyrosine involvement in the radical site of galactose oxidase

Resonance Raman data are reported for the redox-activated form of galactose oxidase from Dactylium dendroides. Excitation within the red (659 nm) and blue (457.9 nm) absorption bands leads to strong resonance enhancement of ligated tyrosine vibrational modes at 550, 1170, 1247, 1484, and 1595 cm-1. The ring mode frequencies are unusually low, indicating a decreased bond order in the ring. The spectra clearly differ in both frequencies and relative intensities from those characteristic of known aromatic pi-radicals. Enhancement of tyrosine ring modes on excitation within absorption bands previously associated with the presence of the radical in the active site suggests that the ligated tyrosine residue is present in the radical site and may stabilize this radical species through formation of a charge transfer complex. A dramatically different Raman spectrum is observed for the N3- adduct of galactose oxidase, exhibiting a single strong 1483 cm-1 feature. The intense visible-near IR absorption bands for galactose oxidase may derive from transitions within a charge transfer complex between an aromatic free radical and a tyrosine-copper complex.     

The radical chemistry of galactose oxidase

Galactose oxidase is a free radical metalloenzyme containing a novel metalloradical complex, comprised of a protein radical coordinated to a copper ion in the active site. The unusually stable protein radical is formed from the redox-active side chain of a cross-linked tyrosine residue (Tyr-Cys). Biochemical studies on galactose oxidase have revealed a new class of oxidation mechanisms based on this free radical coupled-copper catalytic motif, defining an emerging family of enzymes, the radical-copper oxidases. Isotope kinetics and substrate reaction profiling have provided insight into the elementary steps of substrate oxidation in these enzymes, complementing structural studies on their active site. Galactose oxidase is remarkable in the extent to which free radicals are involved in all aspects of the enzyme function: serving as a key feature of the active site structure, defining the characteristic reactivity of the complex, and directing the biogenesis of the Tyr-Cys cofactor during protein maturation.     

A tyrosine-derived free radical in apogalactose oxidase

Oxidation of apogalactose oxidase with ferricyanide leads to the formation of a stable free radical exhibiting distinctive optical absorption and EPR spectral features. The radical is associated with absorption in both near-UV and near-IR spectral regions, and its EPR spectrum is characteristic of an aromatic free radical with gav = 2.005. Reconstitution of both the apoenzyme and the free radical-containing form with copper substantially restores both the absorption spectra and the catalytic activity of the active enzyme, indicating that the preparation of the radical species does not significantly damage the protein. The absence of a free radical EPR signal in reconstituted and activated galactose oxidase containing nearly stoichiometric copper suggests the radical is an active site species relating to the free radical-coupled copper site previously proposed for this enzyme. Isotopic labeling experiments demonstrate that the radical derives from a tyrosine residue. The distinctive spectra associated with this radical indicate an environment which is different from that associated with the tyrosyl phenoxyl sites in other free radical enzymes.     

Streptomyces coelicolor oxidase (SCO2837p): a new free radical metalloenzyme secreted by Streptomyces coelicolor A3(2)

The SCO2837 open-reading frame is located within the conserved central core region of the Streptomyces coelicolor A3(2) genome, which contains genes required for essential cellular functions. SCO2837 protein (SCO2837p) expressed by Pichia pastoris is a copper metalloenzyme, catalyzing the oxidation of simple alcohols to aldehydes and reduction of dioxygen to hydrogen peroxide. Distinct optical absorption spectra are observed for oxidized and one-electron reduced holoenzyme, and a free radical EPR signal is present in the oxidized apoprotein, characteristic of the Tyr-Cys redox cofactor previously reported for fungal secretory radical copper oxidases, galactose oxidase and glyoxal oxidase, with which it shares weak sequence similarity. SCO2837p was detected in the growth medium of both S. coelicolor and a recombinant expression host (Streptomyces lividans TK64) by Western blotting, with the expression level dependent on the nature of the carbon source. This represents the first characterized example of a prokaryotic radical copper oxidase.     

Radiation inactivation of galactose oxidase,a monomeric enzyme with a stable free radical

To determine the radiation sensitivity of galactose oxidase, a 68 kDa monomeric enzyme containing a mononuclear copper ion coordinated with an unusually stable cysteinyl‐tyrosine (Cys‐Tyr) protein free radical. Both active enzyme and reversibly rendered inactive enzyme were irradiated in the frozen state with high‐energy electrons. Surviving polypeptides and surviving enzyme activity were analyzed by radiation target theory giving the radiation sensitive mass for each property. In both active and inactive forms, protein monomer integrity was lost with a single radiation interaction anywhere in the polypeptide, but enzymatic activity was more resistant, yielding target sizes considerably smaller than that of the monomer. These results suggest that the structure of galactose oxidase must make its catalytic activity unusually robust, permitting the enzymatic properties to survive in molecules following cleavage of the polymer chain. Radiation target size for loss of monomers yielded the mass of monomers indicating a polypeptide chain cleavage after a radiation interaction anywhere in the monomer. Loss of enzymatic activity yielded a much smaller mass indicating a robust structure in which catalytic activity could be expressed in cleaved polypeptides.     

The electronic structure of the Cys-Tyr(*) free radical in galactose oxidase determined by EPR spectroscopy

Galactose oxidase is a metalloenzyme containing a novel metalloradical complex in its active site, comprised of a mononuclear copper ion associated with a protein free radical. The free radical has been shown to be localized on an intrinsic redox cofactor, 3'-(S-cysteinyl)tyrosine (Cys-Tyr), formed by a posttranslational covalent coupling of tyrosine and cysteine side chains in a self-processing reaction. The role of the thioether linkage in the function of the Cys-Tyr cofactor is unresolved, and some computational studies have suggested that the thioether substituent has a negligible effect on the properties of the tyrosyl free radical. In order to address this question experimentally, we have incorporated site-selectively labeled tyrosine ((2)H, (13)C, (17)O) into galactose oxidase using an engineered tyrosine auxotroph strain of Pichia pastoris . (33)S was also incorporated into the protein. EPR spectra for the Cys-Tyr(*) free radical in each of these isotopic variants were analyzed to extract nuclear hyperfine parameters for comparison with theoretical predictions, and the unpaired spin distribution in the free radical was reconstructed from the hyperfine data. These labeling studies allow the first comprehensive experimental evaluation of the effect of the thioether linkage on the properties of Cys-Tyr(*) and indicate that previous calculations significantly underestimated the contribution of this feature to the electronic ground state of the free radical.     

o,o-Dityrosine in native and horseradish peroxidase-activated galactose oxidase

Treatment of galactose oxidase with catalytic amounts of horseradish peroxidase results in increases in both enzyme activity and Cu(II)-associated absorbance. This reaction requires O₂ and is reversed upon removal of O₂ or peroxidase. o,o-Dityrosine is detected in amino acid hydrolysates of peroxidase-treated galactose oxidase as a ninhydrin peak. Furthermore, even native enzyme contains this species as detected by fluorescence measurements. Peroxidase treatment increases the amount of dityrosine present. The dityrosine forms an intramolecular crosslink, the first such crosslink found in a nonstructural protein. The peroxidase-catalyzed formation of the dityrosine and putative precursor radical(s) is thought to involve a tyrosyl ligand to the Cu(II) in galactose oxidase. Such a radical may be involved in the activation observed.     

Effect of galactose on free radical reactions of polymorphonuclear leukocytes.

To account for impaired bactericidal activity of polymorphonuclear leukocytes during galactosemia, we have investigated the effects of galactose upon free radical reactions associated with the oxygen-dependent killing mechanism of guinea pig polymorphonuclear leukocytes. Millimolar levels of galactose, which are encountered in the circulation of infants with galactosemia, did not affect the chemical reaction of Superoxide anion with either ferricytochrome c or nitroblue tetrazolium. Galactose did, however, significantly inhibit the reaction of hydroxyl radical with methional, and we propose that such hydroxyl radical removal would be deleterious to normal bactericidal activity. Additional experiments demonstrated that polymorphonuclear leukocytes incubated in the presence of galactose reduced less ferricytochrome c than polymorphonuclear leukocytes incubated without galactose. These results imply that galactose impairs the cellular release of Superoxide anion, which would further disable bactericidal activity.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Cyclodecapeptides to mimic the radical site of tyrosyl-containing proteins.

Tyrosyl radicals are involved in many biologically important processes. The development of model compounds to mimic radical enzyme active sites, such as galactose oxidase (GO), has widely contributed to an enhanced understanding of their spectral properties, structural attributes and even reactivity. An emerging approach towards the synthesis of such active site mimetics is the use of peptidic ligands. The potential of cyclodecapeptides to bear phenoxyl radicals has been evaluated through three compounds. LH(4) (2+) is a cyclodecapetide containing two histidine residues (mimicking His(496) and His(581) of GO) and two tyrosine residues (mimicking Tyr(495) and the Tyr(272)* radical of GO). L(tBu)H(4) (2+) and L(OMe)H(4) (2+) incorporate 2,4,6-protected phenols in place of each tyrosine in LH(4) (2+). The deprotonation constants of each peptide determined by potentiometric titrations showed that there are some interactions between the acido-basic residues. Cyclic voltammetric studies revealed that only the peptides incorporating 2,4,6-protected phenolates exhibit reversible redox couples and are thus precursors of radicals stable enough to persist in solution. These studies also showed L(OMe2-) to possess the lower oxidation potential, indicating that this peptide, in its radical form, is the most stabilized. The electrochemically generated radical species have been characterized by EPR spectroscopy.     

Inactivation of cysteine proteases by (acyloxy)methyl ketones using S'-P' interactions

We have synthesized (acyloxy)methyl ketone inactivators of papain, cathepsin B, and interleukin-1beta conversion enzyme (ICE) that interact with both the S and S' subsites. The value of k(inact)/K(i) for these inactivators is strongly dependent on the leaving group. For example, Z-Phe-Gly-CH(2)-X is a poor inactivator of papain when X is OCOCH(3) (k(inact)/K(i) = 2.5 M(-)(1) s(-)(1)) but becomes a potent inactivator when X is OCO-L-Leu-Z (k(inact)/K(i) = 11 000 M(-)(1) s(-)(1)). Since these leaving groups have similar chemical reactivities, the difference in potency must be attributed to interactions with the S' sites. The potency of the leaving group correlates with the P' specificity of papain. Similar results are also observed for the inactivation of cathepsin B by these compounds. A series of inactivators with the general structure Fmoc-L-Asp-CH(2)-X were designed to inactivate ICE. No inhibition was observed when X was OCOCH(3). In contrast, ICE is inactivated when X is OCO-D-Pro-Z (k(inact)/K(i) = 131 M(-)(1) s(-)(1)). These results demonstrate that S'-P' interactions can be utilized to increase the efficacy and selectivity of (acyloxy)methyl ketone inactivators.     

Amino acids in segment IVS6 and beta-subunit interaction support distinct conformational changes during Ca(v)2.1 inactivation

Ca(v)2.1 mediates voltage-gated Ca2+ entry into neurons and the release of neurotransmitters at synapses of the central nervous system. An inactivation process that is modulated by the auxiliary beta-subunits regulates Ca2+ entry through Ca(v)2.1. However, the molecular mechanism of this alpha1-beta-subunit interaction remains unknown. Herein we report the identification of new determinants within segment IVS6 of the alpha(1)2.1-subunit that markedly influence channel inactivation. Systematic substitution of residues within IVS6 with amino acids of different size, charge, and polarity resulted in mutant channels with rates of fast inactivation (k(inact)) ranging from a 1.5-fold slowing in V1818I (k(inact) = 0.98 +/- 0.09 s(-1) compared with wild type alpha(1)2.1/alpha2-delta/beta1a k(inact) = 1.35 +/- 0.25 s(-1) to a 75-fold acceleration in mutant M1811Q (k(inact) = 102 +/- 3 s(-1). Coexpression of mutant alpha(1)2.1-subunits with beta(2a) resulted in two different phenotypes of current inactivation: 1) a pronounced reduction in the rate of channel inactivation or 2) an attenuation of a slow component in I(Ba) inactivation. Simulations revealed that these two distinct inactivation phenotypes arise from a beta2a-subunit-induced destabilization of the fast-inactivated state. The IVS6- and beta2a-subunit-mediated effects on Ca(v)2.1 inactivation are likely to occur via independent mechanisms.     

Evidence against transition metal-independent hydroxyl radical generation by xanthine oxidase

It is shown by the use of EPR spectroscopy that formation of the hydroxyl radical adduct with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the xanthine-xanthine oxidase system is hydrogen peroxide-independent. Production of the DMPO-hydroxyl radical adduct is inhibited by superoxide dismutase but is unaffected by purified grades of catalase. Hydroxyl radicals are a secondary product of the decomposition of the DMPO-superoxide radical adduct and are also formed as a result of trace metals such as iron present in the buffer. These results are in contrast with a recent report (Kuppusamy, P., and Zweier, J. W. (1989) J. Biol. Chem. 264, 9880-9884) in which the assertion is made that the hydroxyl radical adduct arises from the trapping of hydroxyl radicals generated via the direct reduction of hydrogen peroxide by xanthine oxidase. It is demonstrated here that treatment of phosphate buffer with the chelator deferoxamine mesylate is not in itself sufficient to suppress the effect of contaminating adventitious metal ions in xanthine-xanthine oxidase incubations.     

Stereoselective hydrogen abstraction by galactose oxidase

The fungal enzyme galactose oxidase is a radical copper oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on galactose oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state for substrate oxidation. In this work, these detailed mechanistic studies have been extended using a series of stereospecifically monodeuterated substrates, including 1-O-methyl-alpha-D-galactose as well as unsubstituted benzyl alcohol and 3- and 4-methoxy and 4-nitrobenzyl derivatives. Synthesis of all of these substrates was based on oxidation of the alpha,alpha'-dideuterated alcohol to the corresponding (2)H-labeled aldehyde, followed by asymmetric hydroboration using alpha-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by mass spectrometry to quantitatively evaluate the substrate dependence of the stereoselectivity of the catalytic reaction. For all of these substrates, the selectivity for pro-S hydrogen abstraction was at least 95%. This selectivity appears to be a direct consequence of constraints imposed by the enzyme on the orientation of substrates bearing a branched beta-carbon. Steady state analysis of kinetic isotope effects on V/K has resolved individual contributions from primary and alpha-secondary kinetic isotope effects in the reaction, providing a test for the involvement of an electron transfer redox equilibrium in the oxidation process. Multiple isotope effect measurements utilizing simultaneous labeling of the substrate and solvent have contributed to refinement of the relation between proton transfer and hydrogen atom transfer steps in substrate oxidation.     

A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase.

Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.     

Kinetic comparison of reduction and intramolecular electron transfer in milk xanthine oxidase and chicken liver xanthine dehydrogenase by laser flash photolysis

A comparative study using laser flash photolysis of the kinetics of reduction and intramolecular electron transfer among the redox centers of chicken liver xanthine dehydrogenase and of bovine milk xanthine oxidase is described. The photogenerated reductant, 5-deazariboflavin semiquinone, reacts with the dehydrogenase (presumably at the Mo center) in a second-order manner, with a rate constant (k = 6 x 10(7) M-1 s-1) similar to that observed with the oxidase [k = 3 x 10(7) M-1 s-1; Bhattacharyya et al. (1983) Biochemistry 22, 5270-5279]. In the case of the dehydrogenase, neutral FAD radical formation is found to occur by intramolecular electron transfer (kobs = 1600 s-1), presumably from the Mo center, whereas with the oxidase the flavin radical forms via a bimolecular process involving direct reduction by the deazaflavin semiquinone (k = 2 x 10(8) M-1 s-1). Biphasic rates of Fe/S center reduction are observed with both enzymes, which are due to intramolecular electron transfer (kobs approximately 100 s-1 and kobs = 8-11 s-1). Intramolecular oxidation of the FAD radical in each enzyme occurs with a rate constant comparable to that of the rapid phase of Fe/S center reduction. The methylviologen radical, generated by the reaction of the oxidized viologen with 5-deazariboflavin semiquinone, reacts with both the dehydrogenase and the oxidase in a second-order manner (k = 7 x 10(5) M-1 s-1 and 4 x 10(6) M-1 s-1, respectively). Alkylation of the FAD centers results in substantial alterations in the kinetics of the reaction of the viologen radical with the oxidase but not with the dehydrogenase. These results suggest that the viologen radical reacts directly with the FAD center in the oxidase but not in the dehydrogenase, as is the case with the deazaflavin radical. The data support the conclusion that the environments of the FAD centers differ in the two enzymes, which is in accord with other studies addressing this problem from a different perspective [Massey et al. (1989) J. Biol. Chem. 264, 10567-10573]. In contrast, the rate constants for intramolecular electron transfer among the Mo, FAD, and Fe/S centers in the two enzymes (where they can be determined) are quite similar.     

Effect of zinc on superoxide-dependent hydroxyl radical production in vitro

Trace elements play an important role in oxygen metabolism and therefore in the formation of free radicals. Whereas iron and copper are usually the main enhancers of free radical formation, other trace elements, such as zinc and selenium, protect against the harmful effects of these radicals. To investigate the different protective mechanisms of zinc on radical formation, we examined the effects of added zinc and copper on superoxide dismutase activity. We also studied the effects of copper and iron on xanthine oxidase activity and on the Haber-Weiss cycle (iron, superoxide, and hydrogen peroxide), which generates hydroxyl radicals in vitro. The hypoxanthine/xanthine oxidase radical generating system contained a variety of different physiological ligands for binding the iron. This study confirmed the inhibitory effect of copper on xanthine oxidase activity. Moreover, it demonstrated that zinc inhibited hydroxyl radical formation when this formation was catalyzed by a citrate-iron complex in the hypoxanthine/xanthine oxidase reaction. Finally, human blood plasma inhibited citrate-iron-dependent hydroxyl radical formation under the same conditions. Although trace elements seemed responsible for this antioxidant activity of plasma, it is likely that zinc played no role as a plasma antioxidant. Indeed, calcium appeared to be responsible for most of this effect under our experimental conditions.     

Reaction of bovine cytochrome c oxidase with hydrogen peroxide produces a tryptophan cation radical and a porphyrin cation radical

Oxidized bovine cytochrome c oxidase reacts with hydrogen peroxide to generate two electron paramagnetic resonance (EPR) free radical signals (Fabian, M., and Palmer, G. (1995) Biochemistry 34, 13802-13810). These radicals are associated with the binuclear center and give rise to two overlapped EPR signals, one signal being narrower in line width (DeltaHptp = 12 G) than the other (DeltaHptp = 45 G). We have used electron nuclear double resonance (ENDOR) spectrometry to identify the two different chemical species giving rise to these two EPR signals. Comparison of the ENDOR spectrum associated with the narrow signal with that of compound I of horseradish peroxidase (formed by reaction of that enzyme with hydrogen peroxide) demonstrates that the two species are virtually identical. The chemical species giving rise to the narrow signal is therefore identified as an exchange-coupled porphyrin cation radical similar to that formed in horseradish peroxidase compound I. Comparison of the ENDOR spectrum of compound ES (formed by the reaction of hydrogen peroxide with cytochrome c peroxidase) with that of the broad signal indicates that the chemical species giving rise to the broad EPR signal in cytochrome c oxidase is probably an exchange coupled tryptophan cation radical. This is substantiated using H(2)O/D(2)O solvent exchange experiments where the ENDOR difference spectrum of the broad EPR signal of cytochrome c oxidase shows a feature consistent with hyperfine coupling to the exchangeable N(1) proton of a tryptophan cation radical.