The genetic control of the molybdoflavoproteins in Aspergillus nidulans. IV. A comparison between purine hydroxylase I and II.

The purine hydroxylases I and II of Aspergillus nidulans [previously called xanthine dehydrogenases I and II: Scazzocchio, Holl and Foguelman, Eur. J. Biochem. 36, 428--445 (1973)] have been studied in crude extracts. The two enzymes differ in their substrate specificities, purine hydroxylase II being able to accept nicotinate as a substrate and unable to hydroxylate xanthine. The kinetics of inhibition with allopurinol and oxypurinol are also different, the two analogues being pseudo-irreversible inhibitors of purine hydroxylase I, while allopurinol is a competitive inhibitor of purine hydroxylase II and oxypurinol shows anti-competitive inhibition. Differences in electro-phoretic mobility and molecular size are also shown. We have failed to show the formation of hybrid purine hydroxylase I/II molecules. While a common evolutionary origin of the purine hydroxylases could be postulated, the data reveal a considerable divergence.     

Evidence for a mu-oxo-bridged binuclear iron cluster in the hydroxylase component of methane monooxygenase. M?ssbauer and EPR studies

M?ssbauer and EPR studies of a highly active hydroxylase component of methane monooxygenase isolated from Methylosinus trichosporium OB3b are reported. The M?ssbauer spectra of the oxidized (as isolated) hydroxylase show iron in a diamagnetic cluster containing an even number of Fe3+ sites. The parameters are consistent with an antiferromagnetically coupled binuclear cluster similar to those of hemerythrin and purple acid phosphatases. Upon partial reduction of the hydroxylase, an S = 1/2 EPR spectrum with g values at 1.94, 1.86, and 1.75 (gav = 1.85) is observed. Such spectra are characteristic of oxo-bridged iron dimers in the mixed valent Fe(II).Fe(III) state. Further reduction leads to the appearance of a novel EPR resonance at g = 15. Comparison with an inorganic model compound for mu-oxo-bridged binuclear iron suggests that the g = 15 signal is characteristic of the doubly reduced state of the cluster in the protein. In this state, the M?ssbauer spectra exhibit two quadrupole doublets typical of high spin Fe2+, consistent with the Fe(II).Fe(II) form of the cluster. The spectral features of the iron center of the hydroxylase in three oxidation states are all similar to those reported for mu-oxo (or mu-hydroxo)-bridged binuclear iron clusters. Since no known monooxygenase contains such a cluster, a new oxygenase mechanism is suggested. Three different preparative methods yielded hydroxylases spanning a 9-fold range in specific activity, yet the same cluster concentration and spectral characteristics were observed. Thus, other parameters than those measured here have a major influence on the activity.     

Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.     

Properties of anthranilate hydroxylase (deaminating), a flavoprotein from Trichosporon cutaneum

Anthranilate hydroxylase was purified from the yeast Trichosporon cutaneum. This enzyme is a simple flavoprotein which apparently does not require any additional cofactor for the conversion of anthranilate to 2,3-dihydroxybenzoate. Anthranilate hydroxylase has Mr of approximately 95,000, with subunit Mr of 50,000; it contains 2 mol of FAD/mol of enzyme. A number of compounds in addition to anthranilate serve as substrates, or effectors, for this enzyme. Oxygen-labeling experiments show that the oxygen atom at the 3-position of the product, 2,3-dihydroxybenzoate, originates from O2, while that at the 2-position is derived from H2O. A mechanism is proposed involving imine formation and hydrolysis during the reaction with the flavin hydroperoxide formed from reduced enzyme flavin and molecular oxygen. This proposal is in accord with the mechanism postulated for other flavoprotein aromatic hydroxylases.     

Molybdopterin in carbon monoxide oxidase from carboxydotrophic bacteria.

The carbon monoxide oxidases (COXs) purified from the carboxydotrophic bacteria Pseudomonas carboxydohydrogena and Pseudomonas carboxydoflava were found to be molybdenum hydroxylases, identical in cofactor composition and spectral properties to the recently characterized enzyme from Pseudomonas carboxydovorans (O. Meyer, J. Biol. Chem. 257:1333-1341, 1982). All three enzymes exhibited a cofactor composition of two flavin adenine dinucleotides, two molybdenums, eight irons and eight labile sulfides per dimeric molecule, typical for molybdenum-containing iron-sulfur flavoproteins. The millimolar extinction coefficient of the COXs at 450 nm was 72 (per two flavin adenine dinucleotides), a value similar to that of milk xanthine oxidase and chicken liver xanthine dehydrogenase at 450 nm. That molybdopterin, the novel prosthetic group of the molybdenum cofactor of a variety of molybdoenzymes (J. Johnson and K. V. Rajagopalan, Proc. Natl. Acad. Sci. U.S.A. 79:6856-6860, 1982) is also a constituent of COXs from carboxydotrophic bacteria is indicated by the formation of identical fluorescent cofactor derivatives, by complementation of the nitrate reductase activity in extracts of Neurospora crassa nit-l, and by the presence of organic phosphate additional to flavin adenine dinucleotides. Molybdopterin is tightly but noncovalently bound to the protein. COX, sulfite oxidase, xanthine oxidase, and xanthine dehydrogenase each contains 2 mol of molybdopterin per mol of enzyme. The presence of a trichloroacetic acid-releasable, so-far-unidentified, phosphorous-containing moiety in COX is suggested by the results of phosphate analysis.     

Cofactor determination and spectroscopic characterization of the selenium-dependent purine hydroxylase from Clostridium purinolyticum

Purine hydroxylase (PH) from Clostridium purinolyticum contains a labile selenium cofactor and belongs to a class of enzymes known as the selenium-dependent molybdenum hydroxylases. The presence of approximately 1.1 mol of molybdenum, 0.87 mol of selenium, and 3.3 mol of iron per mol of PH was determined by atomic absorption spectroscopy. Enzyme preparations with lower than stoichiometric amounts of selenium exhibited correspondingly lower hydroxylase activities. Bound FAD, 1 mol per mol enzyme, was confirmed by UV-vis and fluorescence spectroscopy. CMP, released by acid hydrolysis, indicated the presence of a molybdopterin cytosine dinucleotide cofactor. The fully active PH utilized NADP(+) as an electron acceptor, and kinetic analysis revealed an optimal k(cat) of 412 s(-1) using hypoxanthine as the hydroxylase substrate. Xanthine, NAD(+), and NADPH had no significant effect on this reaction rate. A selenium-independent NADPH oxidase activity was exhibited by native PH. Electron paramagnetic resonance spectroscopy revealed the presence of a Mo(V) desulfo signal, FAD radical, and 2Fe-2S centers in hypoxanthine-reduced PH. No hyperfine coupling of selenium, using (77)Se isotope-enriched PH, was observed in any of the EPR active signals studied. The appearance of the desulfo signal suggests that the ligands of Mo in selenium-dependent molybdenum hydroxylases are different from the well-studied mammalian xanthine oxidoreductases (XOR) and aldehyde oxidoreductases (AOR) and suggests a unique role for Se in catalysis.     

The molybdenum iron-sulphur protein from Desulfovibrio gigas as a form of aldehyde oxidase.

The molybdenum iron-sulphur protein originally isolated from Desulfovibrio gigas by Moura, Xavier, Bruschi, Le Gall, Hall & Cammack [(1976) Biochem. Biophys. Res. Commun. 72, 782-789] has been further investigated by e.p.r. spectroscopy of molybdenum(V). The signal obtained on extended reduction of the protein with sodium dithionite has been shown, by studies at 9 and 35 HGz in 1H2O and 2H2O and computer simulations, to have parameters corresponding to those of the Slow signal from the inactive desulpho form of various molybdenum-containing hydroxylases. Another signal obtained on brief reduction of the protein with small amounts of dithionite was shown by e.p.r. difference techniques to be a Rapid type 2 signal, like that from the active form of such enzymes. In confirmation that the protein is a molybdenum-containing hydroxylase, activity measurements revealed that it had aldehyde:2,6-dichlorophenol-indophenol oxidoreductase activity. No such activity towards xanthine or purine was observed. Salicylaldehyde was a particularly good substrate, and treatment of the protein with it also gave rise to the Rapid signal. Molybdenum cofactor liberated from the protein was active in the nit-1 Neurospora crassa nitrate reductase assay. It is concluded that the protein is a form of an aldehyde oxidase or dehydrogenase. From the intensity of the e.p.r. signals and from enzyme activity measurements, 10-30% of the protein in the sample examined appeared to be in the functional form. The evolutionary significance of the protein, which may represent a primitive form of the enzyme rather than a degradation product, is discussed briefly.     

The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy.

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250-300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(II). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(II) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(II) at pH 7.2 was also found to affect its 1H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(II) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(II) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(III) (S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines.     

Deoxyhypusine hydroxylase is a Fe(II)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.     

X-ray absorption spectroscopic investigation of the resting ferrous and cosubstrate-bound active sites of phenylalanine hydroxylase

Previous studies of ferrous wild-type phenylalanine hydroxylase, [Fe(2+)]PAH(T)[], have shown the active site to be a six-coordinate distorted octahedral site. After the substrate and cofactor bind to the enzyme ([Fe(2+)]PAH(R)[L-Phe,5-deaza-6-MPH(4)]), the active site converts to a five-coordinate square pyramidal structure in which the identity of the missing ligand had not been previously determined. X-ray absorption spectroscopy (XAS) at the Fe K-edge further supports this coordination number change with the binding of both cosubstrates to the enzyme, and determines this to be due to the loss of a water ligand.     

The genetic control of molybdoflavoproteins in Aspergillus nidulans. A xanthine dehydrogenase I half-molecule in cnx- mutant strains of Aspergillus nidulans.

The cnx- group of mutants of Aspergillus nidulans lacks xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) and nitrate reductase (EC 1.6.6.3) activities and are thought to be defective in the synthesis of a molybdenum-containing cofactor, 'cnx', common to xanthine dehydrogenase and nitrate reductase [Pateman, J.A., Rever, B.M., Cove, D.J. and Roberts, D.B. (1964) Nature (Lond.) 201, 58-60]. The cnx cofactor has a role in maintaining the aggregated multimeric structure of nitrate reductase [MacDonald, D.W., Cove, D.J. and Coddington, A. (1974) Mol. Gen. Genet. 128, 187-199]. We report here that, in cnx- mutants grown under conditions inducing xanthine dehydrogenase I, a species cross-reacting with antisera to the native enzyme and of half its molecular weight is present, together with cross-reacting molecules of similar molecular weight to the native enzyme. This suggests that the cnx cofactor has a role in maintaining the aggregated structure of xanthine dehydrogenase I. Both cross-reacting species are capable of passing reducing equivalents from NADH to a tetrazolium salt, showing that the cnx cofactor is not necessary for enzymic activity towards NADH.     

Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii

A new nitrogenase from Azotobacter vinelandii has been isolated and characterized. It consists of two proteins, one of which is almost identical with the Fe protein (component 2) of the conventional enzyme. The second protein (Av1'), however, has now been isolated and shown to differ completely from conventional component 1, i.e., the MoFe protein. This new protein consists of two polypeptides with a total molecular weight of around 200,000. In place of Mo and Fe it contains V and Fe with a V:Fe ratio of 1:13 +/- 3. The ESR spectrum of Av1' also differs from conventional component 1 in that lacks the g = 3.6 resonance that arises from the FeMo cofactor but contains an axial signal with gav less than 2 as well as inflections in the g = 4-6 region possibly arising from an S = 3/2 state. This new enzyme can reduce dinitrogen, protons, and acetylene but is only able to utilize 10-15% of its electrons for the reduction of acetylene.     

X-ray absorption spectroscopy structural investigation of early intermediates in the mechanism of DNA repair by human ABH2

Human ABH2 repairs DNA lesions by using an Fe(II)- and αKG-dependent oxidative demethylation mechanism. The structure of the active site features the facial triad of protein ligands consisting of the side chains of two histidine residues and one aspartate residue that is common to many non-heme Fe(II) oxygenases. X-ray absorption spectroscopy (XAS) of metallated (Fe and Ni) samples of ABH2 was used to investigate the mechanism of ABH2 and its inhibition by Ni(II) ions. The data are consistent with a sequential mechanism that features a five-coordinate metal center in the presence and absence of the α-ketoglutarate cofactor. This aspect is not altered in the Ni(II)-substituted enzyme, and both metals are shown to bind the cofactor. When the substrate is bound to the native Fe(II) complex with α-ketoglutarate bound, a five-coordinate Fe(II) center is retained that features an open coordination position for O(2) binding. However, in the case of the Ni(II)-substituted enzyme, the complex that forms in the presence of the cofactor and substrate is six-coordinate and, therefore, features no open coordination site for oxygen activation at the metal.     

Spectroscopic properties of the hydroxylase of methane monooxygenase

The hydroxylase component of methane monooxygenase (EC 1.14.13.25), which catalyzes the oxidation of methane to methanol, has been studied by visible, electron spin resonance and X-ray spectroscopies. The enzyme appears to possess a mu-oxo- or mu-hydroxo-bridged binuclear iron site, with no sulfur ligands to the cluster. Each Fe has 4-6 oxygen (or nitrogen) ligands, at an average distance of 1.92 +/- 0.03 A. The Fe-Fe distance is 3.05 +/- 0.05 A. Essentially all of the irons are in the Fe3+ state as the enzyme is prepared, but reduction with N-methylphenazonium methosulfate generates ESR-detectable states that appear to emanate from mixed-valence binuclear sites. One of these, with gav near 1.85, displays typical Curie law microwave saturation behavior, but the other, gav near 1.73, has a very potent method of spin-relaxation. Together they account for approximately 0.6 spins per molecule.     

Mechanism of oxygen activation by tyrosine hydroxylase

The mechanism by which the tetrahydropterin-requiring enzyme tyrosine hydroxylase (TH) activates dioxygen for substrate hydroxylation was explored. TH contains one ferrous iron per subunit and catalyzes the conversion of its tetrahydropterin cofactor to a 4a-carbinolamine concomitant with substrate hydroxylation. These results are in accord with shared mechanisms of oxygen activation by TH and the more commonly studied tetrahydropterin-dependent enzyme phenylalanine hydroxylase (PAH) and strongly suggest that a peroxytetrahydropterin is the hydroxylating species generated during TH turnover. In addition, TH can also utilize H2O2 as a cofactor for substrate hydroxylation, a result not previously established for PAH. A detailed mechanism for the reaction is proposed. While the overall pattern of tetrahydropterin-dependent oxygen activation by TH and PAH is similar, the H2O2-dependent hydroxylation performed by TH provides an indication that subtle differences in the Fe ligand field exist between the two enzymes. The mechanistic ramifications of these results are briefly discussed.     

Occurrence of molybdenum in the nicotinic acid hydroxylase from Clostridium barkeri

Molybdenum, assayed by atomic absorption spectrometry, copurifies with the selenium-containing nicotinic acid hydroxylase from Clostridium barkeri. Fluorescence spectral studies on the enzyme indicate the presence, along with flavin, of another component. The fluorescence spectra of this component obtained after the aerobic denaturation of the nicotinic acid hydroxylase are similar to the fluorescence properties reported for the “pterin-like” cofactor from xanthine oxidase and several other molybdoproteins. Nicotinic acid hydroxylase from C. barkeri contains molybdenum, selenium, iron, acid-labile sulfur, and flavin with the occurrence of a “pterin-like” cofactor also a likely component.     

Functional annotation and characterization of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1

The genome of Rhodococcus jostii RHA1 contains an unusually large number of oxygenase encoding genes. Many of these genes have yet an unknown function, implying that a notable part of the biochemical and catabolic biodiversity of this Gram-positive soil actinomycete is still elusive. Here we present a multiple sequence alignment and phylogenetic analysis of putative R. jostii RHA1 flavoprotein hydroxylases. Out of 18 candidate sequences, three hydroxylases are absent in other available Rhodococcus genomes. In addition, we report the biochemical characterization of 3-hydroxybenzoate 6-hydroxylase (3HB6H), a gentisate-producing enzyme originally mis-annotated as salicylate hydroxylase. R. jostii RHA1 3HB6H expressed in Escherichia coli is a homodimer with each 47kDa subunit containing a non-covalently bound FAD cofactor. The enzyme has a pH optimum around pH 8.3 and prefers NADH as external electron donor. 3HB6H is active with a series of 3-hydroxybenzoate analogues, bearing substituents in ortho- or meta-position of the aromatic ring. Gentisate, the physiological product, is a non-substrate effector of 3HB6H. This compound is not hydroxylated but strongly stimulates the NADH oxidase activity of the enzyme.     

NAD(P)+-independent aldehyde dehydrogenase from Pseudomonas testosteroni. A novel type of molybdenum-containing hydroxylase

Aldehyde dehydrogenase from Pseudomonas testosteroni was purified to homogeneity. The enzyme has a pH optimum of 8.2, uses a wide range of aldehydes as substrates and cationic dyes (Wurster's blue, phenazine methosulphate and thionine), but not anionic dyes (ferricyanide and 2.6-dichloroindophenol), NAD(P)+ or O2, as electron acceptors. Haem c and pyrroloquinoline quinone appeared to be absent but the common cofactors of molybdenum hydroxylases were present. Xanthine was not a substrate and allopurinol was not an inhibitor. Alcohols were inhibitors only when turnover of the enzyme occurred in aldehyde conversion. The enzyme has a relative molecular mass of 186,000, consists of two subunits of equal size (Mr 92,000), and 1 enzyme molecule contains 1 FAD, 1 molybdopterin cofactor, 4 Fe and 4 S. It is a novel type of NAD(P)+-independent aldehyde dehydrogenase since its catalytic and physicochemical properties are quite different from those reported for already known aldehyde-converting enzymes like haemoprotein aldehyde dehydrogenase (EC 1.2.99.3), quino-protein alcohol dehydrogenases (EC 1.1.99.8) and molybdenum hydroxylases.     

Further characterization of two different,reversible aldehyde oxidoreductases from Clostridium formicoaceticum,one containing tungsten and the other molybdenum

The tungsten- and the molybdenum-containing aldehyde oxidoreductases from Clostridium formicoaceticum show, for aldehydes, K _m values<30 M and K _i values of millimolar concentrations. The tungsten-containing aldehyde oxidoreductase is inactivated to 50% by 3 mM KCN within 1 min, by 1 mM ferricyanide within 5 min, and by 0.05 mM chloralhydrate within 30 s. The molybdenum-containing AOR shows 50% inactivation within 1 min only with 70 mM KCN. The tungsten-containing enzyme is very sensitive to oxygen, especially in the reduced state, whereas the molybdenum-containing enzyme exhibits only moderate oxygen sensitivity without being markedly influenced by the redox state of the enzyme. The tungsten in the aldehyde oxidoreductase is bound to a pterin cofactor (Wco) of the mononucleotide form that is known for molybdopterin cofactor (Moco). The nature of the molybdenum cofactor in the molybdenum-containing aldehyde oxidoreductase is still unclear. The UV/VIS spectrum of the tungsten-containing aldehyde oxidoreductase shows a broad absorption in the range of 400 nm with a millimolar absorption coefficient of 18.1 (reduced form) and 24.8 (dehydrogenated form) at 396 nm. The epr spectrum exhibits two different W(V) signals with the following g values for signal A: 2.035, 1.959, 1.899 and signal B: 2.028, 2.017, 2.002. Dithionite-reduced enzyme shows signals of 4Fe–4S or 2Fe–2S clusters. Initial rate studies with different substrates for the carboxylate reduction led to a Bi Uni Uni Bi mechanism.Abbreviations AOR aldehyde oxidoreductase - NH ₂ CO-MV 1,1-carbamoylmethylviologen - MV methylviologen - TMV 1,1,2,2-tetramethylviologen