Protein carbonylation: 2,4-dinitrophenylhydrazine reacts with both aldehydes/ketones and sulfenic acids

Most of the assays for detection of carbonylated proteins, the most general and widely used marker of severe protein oxidation, involve derivatization of the carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to formation of a stable dinitrophenyl hydrazone product. Here, by using a Cys-containing model peptide and high-resolution mass spectrometry, we demonstrate that DNPH is not exclusively selective for carbonyl groups, because it also reacts with sulfenic acids, forming a DNPH adduct, through the acid-catalyzed formation of a thioaldehyde intermediate that is further converted to an aldehyde. β-Mercaptoethanol prevents the formation of the DNPH derivative because it reacts with the oxidized Cys residue, forming the corresponding disulfide.     

Derivatization of gamma-glutamyl semialdehyde residues in oxidized proteins by fluoresceinamine

Oxidative modification of proteins is implicated in a number of physiologic and pathologic processes. Metal-catalyzed oxidative modification usually causes inactivation of enzymes and the appearance of carbonyl groups in amino acid side chains of the protein. We describe use of fluoresceinamine to label certain of those carbonyl groups. Fluoresceinamine reacted with those carbonyl groups to form a Schiff base which was reduced by cyanoborohydride to yield a stable chromophore on the oxidized residue. The high molar absorbtivity of the fluorescein moiety conferred high sensitivity upon the method. Labeled peptides were readily identified after tryptic digestion of oxidized glutamine synthetase. Further, acid hydrolysis of labeled glutamine synthetase allowed isolation of the derivatized, oxidized residue. The oxidized amino acid was identified as gamma-glutamyl semialdehyde. During metal-catalyzed oxidation, the inactivation of glutamine synthetase paralleled the appearance of gamma-glutamyl semialdehyde.     

Sequence of a peptide susceptible to mixed-function oxidation. Probable cation binding site in glutamine synthetase

Mixed-function oxidation of glutamine synthetase from Escherichia coli causes loss of catalytic activity. The inactivation correlates with the loss of 1 of 16 histidine residues/subunit (Levine, R.L. (1983) J. Biol. Chem. 258, 11823-11827). A cyanogen bromide peptide containing the oxidizable histidine has been isolated. Within the protein, the sequence is Met-His-Cys-His-Met. This hydrophilic sequence likely forms one of the divalent metal-binding sites of glutamine synthetase. Binding of Fe2+ to this site permits generation of an activated oxygen species which reacts with a nearby histidine residue. This site-specific free radical mechanism accounts for the specificity of the mixed-function oxidation.     

A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies

The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.     

Molecular characterization of NikD,a new flavoenzyme important in the biosynthesis of nikkomycin antibiotics

Nikkomycin antibiotics are potent inhibitors of chitin synthase, effective as therapeutic antifungal agents in humans and easily degradable insecticides in agriculture. NikD is a novel flavoprotein that catalyzes the oxidation of Delta(1)- or Delta(2)-piperideine-2-carboxylate, a key step in the biosynthesis of nikkomycin antibiotics. The resulting dihydropicolinate product may be further oxidized by nikD or converted to picolinate in a nonenzymic reaction. Saturated nitrogen heterocycles (L-pipecolate, L-proline) and 3,4-dehydro-L-proline act as alternate substrates. The ability of nikD to oxidize 3,4-dehydro-L-proline, but not 1-cyclohexenoate, suggests that the enzyme is specific for the oxidation of a carbon-nitrogen bond. An equivalent reaction is possible with the enamine (Delta(2)), but not the imine (Delta(1)), form of the natural piperideine-2-carboxylate substrate. Apparent steady-state kinetic parameters for the reaction of nikD with Delta(1)- or Delta(2)-piperideine-2-carboxylate (k(cat) = 64 min(-1); K(m) = 5.2 microM) or 3,4-dehydro-L-proline (k(cat) = 18 min(-1); K(m) = 13 mM) were determined in air-saturated buffer by measuring hydrogen peroxide formation in a coupled assay. NikD appears to be a new member of the monomeric sarcosine oxidase (MSOX) family of amine oxidizing enzymes. The enzyme contains 1 mol of flavin adenine dinucleotide (FAD) covalently linked to Cys321. The covalent flavin attachment site and two residues that bind substrate carboxylate in MSOX are conserved in nikD. NikD, however, exhibits an unusual long-wavelength absorption band, attributed to charge-transfer interaction between FAD and an ionizable (pK(a) = 7.3) active-site residue. Similar long-wavelength absorption bands have been observed for flavoproteins containing an active site cysteine or cysteine sulfenic acid. Interestingly, Cys273 in nikD aligns with an active-site histidine in MSOX (His269) that is, otherwise, a highly conserved residue within the MSOX family.     

Inhibition of postcardiac arrest brain protein oxidation by acetyl-l-carnitine

Free radical mediated, site-specific protein oxidation has been implicated in the pathophysiology of ischemia/reperfusion brain injury. The purpose of this study was to determine whether this form of molecular damage could be detected in a clinically relevant model employing 10-min cardiac arrest in dogs followed by restoration of spontaneous circulation for up to 24 h. The effects of postichemic acetyl-L-carnitine administration of protein oxidation were also tested due to its previously reported improvement of brain energy metabolism and neurological outcome in this model. Following the experimental period, soluble proteints were extracted froma sample of frontal cortex and reacted with dinitrophenylhydrazine for spectrophotometric measurement of protein carbonyl groups. The most importance results of this study were that brain protein carbonyl groups were significantly elevated following 2 and 24 h of reperfusion compared to nonischemic controls, and that postichemic IV administration of acetyl-L-carnitine eliminated the increase in carbonyl groups observed at the 24-h period. These results indicate that brain protein oxidation does occur in a clinically relevant model of complete global cerebral ischemia and reperfusion, and that oxidation is inhibited under treatment conditions that improve neurological outcome.     

Inactivation of glutathione peroxidase by superoxide radical

The selenium-containing glutathione peroxidase, when in its active reduced form, was inactivated during exposure to the xanthine oxidase reaction. Superoxide dismutase completely prevented this inactivation, whereas catalase, hydroxyl radical scavengers, or chelators did not, indicating that O2 was the responsible agent. Conversion of GSH peroxidase to its oxidized form, by exposure to hydroperoxides, rendered it insensitive toward O2. The oxidized enzyme regained susceptibility toward inactivation by O2 when reduced with GSH. The inactivation by O2 could be reversed by GSH; however, sequential exposure to O2 and then hydroperoxides caused irreversible inactivation. Reactivity toward CN- has been used as a measure of the oxidized form of GSH peroxidase, whereas reactivity toward iodoacetate has been taken as an indicator of the reduced form. By these criteria both O2 and hydroperoxides convert the reduced form to oxidized forms. A mechanism involving oxidation of the selenocysteine residue at the active site has been proposed to account for these observations.     

Conjugation of glutathione to oxidized tyrosine residues in peptides and proteins

Tyrosine residues are sensitive to oxidation and can be converted to hydroperoxides either by superoxide reacting with the Tyr radical or by singlet oxygen. These hydroperoxides rearrange to bicyclic derivatives that are readily reduced to more stable hydroxides. The aromatic character of tyrosine is lost, but the product contains an α-β unsaturated carbonyl group and is, therefore, an electrophile. We have generated hydroxide derivatives of several Tyr-containing peptides and shown using liquid chromatography/mass spectrometry that they undergo Michael addition with GSH. For Tyr-Gly, rate constants of 9.2 and 11.8 m(-1)min(-1) were measured for the two chromatographically distinct isomers. Unusual for GSH addition to an electrophile, the reaction is reversible, with a half-life of many hours for the reverse reaction. These kinetics indicate that with a typical cellular concentration of 5 mm GSH, >95% Tyr-Gly hydroxide would become conjugated with a half-life of ～15 min. Sperm whale myoglobin forms a hydroperoxide on Tyr-151 in a hydrogen peroxide/superoxide-dependent reaction. We show that its hydroxide derivative reacts with GSH to form a conjugate. Detection of the conjugate required stabilization by reduction; otherwise, the reverse reaction occurred during tryptic digestion and analysis. Our findings represent a novel mechanism for peptide or protein glutathionylation involving a carbon-sulfur cross-link between oxidized Tyr and Cys. As with other electrophiles, the oxidized Tyr should undergo a similar reaction with Cys residues in proteins to give intramolecular or intermolecular protein cross-links. This mechanism could give rise to protein cross-linking in conditions of oxidative stress.     

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.     

Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions

A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation.     

Metal-catalyzed oxidation of Escherichia coli glutamine synthetase: correlation of structural and functional changes

Metal-catalyzed oxidation of proteins has been implicated in a variety of biological processes, particularly in the marking of proteins for subsequent proteolytic degradation. The metal-catalyzed oxidation of bacterial glutamine synthetase causes conformational, covalent, and functional alterations in the protein. To understand the structural basis of the functional changes, the time course of oxidative modification of glutamine synthetase was studied utilizing a nonenzymic model oxidation system consisting of ascorbate, oxygen, and iron. The structural modifications induced included: decreased thermal stability; weakening of subunit interactions; decrease in isoelectric point; introduction of carbonyl groups into amino acid side chains; and loss of two histidine residues. These changes did not denature the protein, but instead induced relatively subtle changes. Indeed, even the most extensively modified protein had a sedimentation velocity which was identical to that of the native enzyme. Comparison of the time courses of the structural and functional changes established that: (i) Loss of the metal binding site and of catalytic activity occurred with loss of one histidine per subunit; (ii) increased susceptibility to proteolysis occurred with loss of two histidine residues per subunit. Thus, oxidation at one site suffices to inactivate the enzyme, but two sites must be modified to induce susceptibility to proteolysis. The limited and specific changes induced by metal-catalyzed oxidation are consistent with a site-specific free radical mechanism.     

Evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A lyase arginine-41 as a catalytic residue: use of acetyldithio-coenzyme A to monitor product enolization

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to produce acetyl-CoA and acetoacetate. Arginine-41 is an invariant residue in HMG-CoA lyases. Mutation of this residue (R41Q) correlates with human HMG-CoA lyase deficiency. To evaluate the functional importance of arginine-41, R41Q and R41M recombinant mutant human HMG-CoA lyase proteins have been constructed, expressed, and purified. These mutant proteins retain structural integrity based on Mn(2+) binding and affinity labeling stoichiometry. R41Q exhibits a 10(5)-fold decrease in V(max); R41M activity is >or=10-fold lower than the activity of R41Q. Acetyldithio-CoA, an analogue of the reaction product, acetyl-CoA, has been employed to test the function of arginine-41, as well as other residues (e.g., aspartate-42 and histidine-233) implicated in catalysis. Acetyldithio-CoA supports enzyme-catalyzed exchange of the methyl protons of the acetyl group with solvent; exchange is dependent on the presence of Mg(2+) and acetoacetate. In comparison with wild-type human enzyme, D42A and H233A mutant enzymes exhibit 4-fold and 10-fold decreases, respectively, in the proton exchange rate. In contrast, R41Q and R41M mutants do not catalyze any substantial enzyme-dependent proton exchange. These results suggest a role for arginine-41 in deprotonation or enolization of acetyldithio-CoA and implicate this residue in the HMG-CoA cleavage reaction chemistry that leads to acetyl-CoA product formation. Assignment of arginine-41 as an active site residue is also supported by a homology model for HMG-CoA lyase based on the structure of 4-hydroxy-2-ketovalerate aldolase. This model suggests the proximity of arginine-41 to other amino acids (aspartate-42, glutamate-72, histidine-235) implicated as active site residues based on their function as ligands to the activator cation.     

Inactivation of Bacillus subtilis glutamine synthetase by metal-catalyzed oxidation.

Instability of Bacillus subtilis glutamine synthetase in crude extracts was attributed to site-specific oxidation by a mixed-function oxidation, and not to limited proteolysis by intracellular serine proteases (ISP). The crude extract from B. subtilis KN2, which is deficient in three intracellular proteases, inactivated glutamine synthetase similarly to the wild-type strain extract. To understand the structural basis of the functional change, oxidative modification of B. subtilis glutamine synthetase was studied utilizing a model system consisting of ascorbate, oxygen, and iron salts. The inactivation reaction appeared to be first order with respect to the concentration of unmodified enzyme. The loss of catalytic activity was proportional to the weakening of subunit interactions. B. subtilis glutamine synthetase was protected from oxidative modification by either 5 mM Mn2+ or 5 mM Mn2+ plus 5 mM ATP, but not by Mg2+. The CD-spectra and electron microscopic data showed that oxidative modification induced relatively subtle changes in the dodecameric enzyme molecules, but did not denature the protein. These limited changes are consistent with a site-specific free radical mechanism occurring at the metal binding site of the enzyme. Analytical data of the inactivated enzyme showed that loss of catalytic activity occurred faster than the appearance of carbonyl groups in amino acid side chains of the protein. In B. subtilis glutamine synthetase, the catalytic activity was highly sensitive to minute deviations of conformation in the dodecameric molecules and these subtle changes in the molecules could be regarded as markers for susceptibility to proteolysis.     

Identification of cysteine-10 of protein S18 as part of the mRNA-binding site of Escherichia coli ribosomes by affinity-labeling studies with a chemically reactive A-U-G analog.

The reaction of a bromoacetamidophenyl derivative of the initiation codon A-U-G (A-U-G) with tight couples of Escherichia coli ribosomes leads to an exclusive crosslinking of label to protein S18. This crosslinking inhibits A-U-G-directed fMet-tRNAfMet binding into the puromycin-sensitive site of ribosomes and stimulates elongation-factor-dependent binding of Met-tRNAmMet. It is, therefore, concluded that protein S18 is located at or near the aminoacyl-tRNA binding site of E. coli ribosomes. Peptide as well as amino acid analysis shows that the reaction between A-U-G and ribosomes took place at cysteine-10 of protein S18. A-U-G could not be crosslinked to ribosomal proteins of the temperature-sensitive E. coli strain 258ts, where arginine-11 of protein S18 is replaced by a cysteine residue.     

Nitrite and nitric oxide treatment of Helix pomatia hemocyanin: single and double oxidation of the active site.

The reaction of nitrite and nitric oxide with Helix pomatia hemocyanin has been studied. One or both of the two copper ions in the active site can be oxidized, depending upon reaction conditions. The single oxidation of the oxygen binding site can be reversed by reduction with hydroxylamine, and the oxygen binding properties of the protein are simultaneously restored. The experiments, including electron paramagnetic resonance, indicate that nitric oxide is not a ligand of copper in the singly oxidized active site and that the oxidized copper ions is coupled to at least two nitrogen atoms of amino acid residues. The doubly oxidized protein can be reduced to a singly oxidized one with ascorbic acid or hydroxylamine; the latter reagent is again able to reduce the singly oxidized state and to restore the oxygen binding properties.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Influence of DNA binding on the formation and reactions of tryptophan and tyrosine radicals in peptides and proteins

The rate constant of the one-electron oxidation of the tryptophan (Trp) or tyrosine (Tyr) residues by Br- X 2 radical anions is strongly decreased when the peptides are bound to DNA. Oxidation by N X 3 is much less affected by binding. These results can be explained by electrostatic repulsion between the charged polyphosphate backbone and the Br- X 2 radicals. Once oxidized, the interacting aromatic residues react with the DNA in a first order process with a rate constant of the order 10(3) s-1. These results have been extended to the single strand binding protein: the product of gene 32 of phage T4 (gp 32). The pulse radiolysis study suggests that one Trp residue of the protein oxidized by the Br- X 2 radicals reacts with the DNA in the complex while one Tyr residue is buried upon association. It is also shown that the exposure of Trp and Tyr residues to radical attack depends on whether the T4 SSB protein is bound to native or heat-denatured DNA.     

Structure and function of corticosteroid-binding globulin: Role of carbohydrates

To study the site-specificity of human corticosteroid-binding globulin (CBG) glycosylation and the functional significance of individual carbohydrate chains in its molecule, a panel of recombinant CBG mutants containing each of the six potential glycosylation sites alone and in various combinations has been expressed in Chinese hamster ovary (CHO) cells. Analyses of these mutant glycoproteins showed that three of the glycosylation sites are only partially utilized, and this may contribute to the production of glycoforms with distinct physiological functions. Processing of individual carbohydrate chains (branching and fucosylation) is site-specific and may, thus, account for the formation of structural determinants essential for the recognition of CBG by cell membranes. Glycosylation at the only phylogenetically conserved consensus site, Asn²³⁸-Gly²³⁹-Thr²⁴⁰, is essential for the biosynthesis of CBG with steroid-binding activity. Evidence has been obtained to support the hypothesis that transient carbohydrate-polypeptide interactions between Trp²⁶⁶ and the maturing carbohydrate chain at Asn²³⁸ occur during early stages of the CBG biosynthesis which affect protein folding and formation of the steroid-binding site. Another tryptophan residue, Trp³⁷¹, has been found to be critical for CBG-steroid interactions and is likely located in the steroid-binding site.     

A kinetic study of the endogenous reduction of the oxidized sites in the primary cytochrome c peroxidase-hydrogen peroxide compound.

The primatry compound formed in the reaction between H2O2 and cytochrome c peroxidase is oxidized two equivalents above the native enzyme. The two oxidized sites are thought to be an Fe(IV) and an amino acid radical. In the absence of oxidizable substrate, the Fe(IV) and radical sites decay by apparent first-order processes but at different rates. It is likely that the decay involves both intra- and intermolecular electron-transfer reactions. The reduction of the Fe(IV) site depends upon the pH with a minimum reduction rate of 2.9-10(-5)s(-1) at pH 6. At pH 4 and 6, the reduction of the Fe(IV) site is facilitated by prior oxidation of amino acid residues in the protein.     

Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation

The mechanism of self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation has been studied by the methods of elastic and dynamic light-scattering and viscosimetry. Fibrin obtained from oxidized fibrinogen exhibits higher average fiber mass/length ratio compared with native fibrin. Fibrinogen ozonation sharply reduced the latent period preceding aggregation of protein molecules; however, the mechanism of self-assembly of ozonated and non-ozonated fibrinogen cluster was identical. In both cases flexible polymers are formed and reaching a certain critical length they form densely packed structures and aggregate. Using infrared spectroscopy, it has been shown that free radical oxidation of amino acid residues of fibrinogen polypeptide chains catalyzed by ozone results in formation of carbonyl, hydroxyl, and ether groups. It is concluded that fibrinogen peripheral D-domains are the most sensitive to ozonation, which causes local conformational changes in them. On one hand, these changes inhibit the reaction of longitudinal polymerization of monomeric fibrin molecules; on the other hand, they expose reaction centers responsible for self-assembly of fibrinogen clusters.