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.     

Phenylalanine hydroxylase: absolute configuration and source of oxygen of the 4a-hydroxytetrahydropterin species

The formation of tyrosine from phenylalanine catalyzed by rat liver phenylalanine hydroxylase is coupled to the generation of a 4a-hydroxy adduct from the requisite tetrahydropterin cofactor. As indicated by its circular dichroism (CD) spectrum, the optical activity of the adduct generated from racemic 6-methyltetrahydropterin requires stereoselectivity of the oxygenation. The absolute configuration of this new stereocenter is 4a(S)-hydroxy-6(RS)-methyltetrahydropterin by analogy to the CD spectrum of one of the four stereoisomers of 5-deaza-4a-hydroxy-6-methyltetrahydropterin. The source of the 4a-hydroxy oxygen is O2, as demonstrated by the observation of a 18O-induced 13C shift in the 13C NMR spectrum of the adduct when generated from [4a-13C]-6-methyltetrahydropterin and 18O2.     

Interaction of metal ions with nucleic acids. Interaction of copper(II) with guanosine and its derivatives.

Interaction of copper(II) with guanosine, 2'-deoxyguanosine, 1-methylguanosine, 7-methylguanosine and GMP was studied withe use of spectroscopic and magneto-chemical methods. The main site of copper(II) binding in guanosine is nitrogen N-7; participation of N-1 is not excluded. The involvement of carbonyl oxygen in copper binding or copper chelation to N-7 and 0-6 is rather unlikely. A crystalline complex of copper(II) with GMP [Cu(C10H12O8N5P) .(H2O)3] was obtained, and it was demonstrated that copper(II) is bound with N-7 and the phosphate group.     

Affinity labeling of the active site and the reactive sulfhydryl associated with activation of rat liver phenylalanine hydroxylase

A pterin analogue, 5-[(3-azido-6-nitrobenzylidene)amino]-2,6-diamino-4-pyrimidinone (ANBADP), was synthesized as a probe of the pterin binding site of phenylalanine hydroxylase. The photoaffinity label has been found to be a competitive inhibitor of the enzyme with respect to 6,7-dimethyltetrahydropterin, having a Ki of 8.8 +/- 1.1 microM. The irreversible labeling of phenylalanine hydroxylase by the photoaffinity label upon irradiation is both concentration and time dependent. Phenylalanine hydroxylase is covalently labeled with a stoichiometry of 0.87 +/- 0.08 mol of label/enzyme subunit. 5-Deaza-6-methyltetrahydropterin protects against inactivation and both 5-deaza-6-methyltetrahydropterin and 6-methyltetrahydropterin protect against covalent labeling, indicating that labeling occurs at the pterin binding site. Three tryptic peptides were isolated from [3H]ANBADP-photolabeled enzyme and sequenced. All peptides indicated the sequence Thr-Leu-Lys-Ala-Leu-Tyr-Lys (residues 192-198). The residues labeled with [3H]ANBADP were Lys198 and Lys194, with the majority of the radioactivity being associated with Lys198. The reactive sulfhydryl of phenylalanine hydroxylase associated with activation of the enzyme was also identified by labeling with the chromophoric label 5-(iodoacetamido)fluorescein [Parniak, M. A., & Kaufman, S. (1981) J. Biol. Chem. 256, 6876]. Labeling of the enzyme resulted in 1 mol of fluorescein bound per phenylalanine hydroxylase subunit and a concomitant activation of phenylalanine hydroxylase to 82% of the activity found with phenylalanine-activated enzyme. Tryptic and chymotryptic peptides were isolated from fluorescein-labeled enzyme and sequenced. The modified residue was identified as Cys236.     

Effect of cocaine on the tyrosine hydroxylase of rat hypothalamus]

The influence of cocaine on tyrosine hydroxilase of rat brain hypothalamus was investigated in vivo (0.5 mg/kg) and in vitro (10(--6)--10(--5)M). Cocaine was used as a substance with a known adrenergic type of action. It was shown that under standard conditions cocaine in vitro increased the enzyme activity and decreased the Km for DMPH4 cofactor without changing Vmax of the reaction analyzed by the membrane enzyme. Cocaine in vitro decreased the tyrosine hydroxylase activity, especially that of the membrane enzyme. In this case there occurred a decrease of Km for DMPH4 and a decrease of Vmax of the reaction. The decrease of Vmax is considered to be the result of the secondary effect of cocaine.     

Carcinine-β-cyclodextrin derivatives as scavenger entities of OH radicals and SOD-like properties of their copper(II) complexes

6-Deoxy-6-{4-[N-(2-aminoethyl)propaneamide]imidazolyl}cyclohepta amylose (CDcarc) and 6-{3-amine-N-[2-(imidazol-4-yl)ethyl]propaneamide}-6-deoxycyclohepta amylose (CDcrac) were synthesized with the aim to obtain copper(II) complexes able to scavenge superoxide radical. The copper(II) complexes were studied by means of UV-Vis, ESR, CD, ESI-MS spectroscopies to gain information about the species present in solution as function of the pH. The antioxidant activity was assayed against superoxide enzymatically generated and compared with that obtained from copper(II) complex with underivatized carcinine. The hydroxyl radical scavenging ability of these new ligands was also tested.     

Synthesis and biological activity of N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid

2-Deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a potent inhibitor of thymidylate synthase. Its analogue, N(alpha)-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-propargylamino]phenylacetyl]-L-glutamic acid, containing p-aminophenylacetic acid residue substituting p-aminobenzoic acid residue, was synthesized. The new analogue exhibited a moderately potent thymidylate synthase inhibition, of linear mixed type vs. the cofactor, N(5,10)-methylenetetrahydrofolate. The Ki value of 0.34 microM, determined with a purified recombinant rat hepatoma enzyme, was about 30-fold higher than that reported for inhibition of thymidylate synthase from mouse leukemia L1210 cells by ICI 198583 (Hughes et al., 1990, J. Med. Chem. 33, 3060). Growth of mouse leukemia L5178Y cells was inhibited by the analogue (IC50 = 1.26 mM) 180-fold weaker than by ICI 198583 (IC50 = 6.9 microM).     

The synthesis, structure and SOD-like behaviors of a μ-imidazolato-dicopper(II) complex with a binucleating hexaazamacrocycle

The synthesis, crystal structure and magnetic properties of the imidazolate-bridged dinuclear copper(II) complex [LCu₂(Im)(DMSO)₂](ClO₄)₃(DMSO), where ImH is imidazole, and L is 3, 6, 9, 17, 20, 23-hexaazatricyclo[23.3.1.1^11,13]triaconta-1(29), 11(30), 12, 14, 25(26), 27-hexaene, have been studied. Single crystal X-ray diffraction determination reveals the distorted tetragonal pyramid geometries of the imidazolate bridged dicopper(II) center are incorporated within the binucleating macrocycle. Both copper atoms are five-coordinated by four basal nitrogen atoms (three from the macrocycle and one from the imidazolate) and one oxygen atom from the DMSO molecule. Two coordinated DMSO molecules are situated at the same side of the macrocycle. The Cu–Cu separation in the complex is 5.93 Å. Magnetic measurements and ESR spectroscopy studies exibit an antiferromagnetic exchange interaction with a coupling constant of J= − 26.94 cm⁻¹. Investigations on the pH-dependent ESR of the title compound in frozen 50% aqueous DMSO solution confirm the existence of the bridged cation [LCu₂(Im)]³⁺ as a major species in solution mainly in the range 5.2pH11.5. The imidazolate bridge is broken in the pH range of 5.2–3.3, which is close to that for the native enzyme.     

A kinetic study on the binding of monomeric and polymeric derivatives of NAD+ to yeast alcohol dehydrogenase

The binding to yeast alcohol dehydrogenase of NAD+ and its five derivatives (N6-[2-[N-[2-[N-(2-methacrylamidoethyl)carbamoyl]ethyl] carbamoyl]ethyl]-NAD (I), N6-[N-[2-[N-(2-methacrylamidoethyl) carbamoyl]ethyl]carbamoylmethyl]-NAD (II), copolymer of I with acrylamide (PA-I), copolymer of II with acrylamide (PA-II), and copolymer of I with N,N-dimethylacrylamide (PDMA-I] were studied statically and kinetically by the stopped-flow method by using the quenching of the enzyme fluorescence in the presence of pyrazole. Apparent dissociation constants and apparent rate constants were determined therefrom. It was concluded that (1) the N6-CH2CH2CO group (of I) is effective in making the derivative bind more strongly as well as faster than NAD+, while the N6-CH2CO group (of II) is not; and (2) the binding of the polymer derivatives of NAD+ to the enzyme is not essentially weaker and slower than that of native NAD+, but is even faster in some cases. The coenzymic activities of the above compounds were also determined with yeast alcohol dehydrogenase, pig heart malate dehydrogenase, and rabbit muscle lactate dehydrogenase.     

Structural correlation of catecholase-like activities of oxy-bridged dinuclear copper(II) complexes

Eight oxy-bridged dinuclear copper(II) complexes with catecholase-like sites, [Cu(L1)X]2 (HL1 = 1-diethylaminopropan-2-ol, X=N3- 1, NCO- 2, and NO2- 3), [Cu(L2)X]2 (HL2=N-ethylsalicylaldimine, X=NO3- 4, Cl- 5, N3- 6, NCS- 7), and [Cu(L3)]2(ClO4)2, 8 (HL3=N-(salicylidene)-N'-(2-pyridylaldene)propanediamine) have been prepared and characterized. The single crystal X-ray analysis show that the structures of complexes 6 and 8 are dimeric with two adjacent copper(II) atoms bridged by pairs of micro-oxy atoms from the L2 and L3 ligands. Magnetic susceptibility measurements in the temperature range 4-300 K indicate significant antiferromagnetic coupling for 4, 5 and 7 and ferromagnetic coupling for 6 between the copper(II) atoms. The catecholase activity of complexes for the oxidation of 3,5-di-tert-butylcatechol by O2 was studied and it was found that the complexes with the bond distance of Cu(II)...Cu(II) located at 2.9-3.0 A show higher catecholase activity.     

Acid-base properties and copper(II) complexes of dipeptides containing histidine and additional chelating bis(imidazol-2-yl) residues

Copper(II) complexes of dipeptides of histidine containing additional chelating bis(imidazol-2-yl) agent at the C-termini (PheHis-BIMA [N-phenylalanyl-histidyl-bis(imidazol-2-yl)methylamine] and HisPhe-BIMA [N-histidyl-phenylalanyl-bis(imidazol-2-yl)methylamine]) were studied by potentiometric, UV-Visible and Electron Paramagnetic Resonance (EPR) techniques. The imidazole nitrogen donor atoms of the bis(imidazol-2-yl)methyl group are described as the primary metal binding sites forming stable mono- and bis(ligand) complexes at acidic pH. The formation of a ligand-bridged dinuclear complex [Cu2L2]4+ is detected in equimolar solutions of copper(II) and HisPhe-BIMA. The coordination isomers of the dinuclear complex are described via the metal binding of the bis(imidazol-2-yl)methyl, amino-carbonyl and amino-imidazole(His) functions. In the case of the copper(II)-PheHis-BIMA system the [NH2, N-(amide), N(Im)] tridentate coordination of the ligand is favoured and results in the formation of di- and trinuclear complexes [Cu2H(-1)L]3+ and [Cu3H(-2)L2]4+ in equimolar solutions. The presence of these coordination modes shifts the formation of "tripeptide-like" ([NH2, N-, N-, N(Im)]-coordinated) [CuH(-2)L] complexes into alkaline pH range as compared to other dipeptide derivatives of bis(imidazol-2-yl) ligands. Although there are different types of imidazoles in these ligands, the deprotonation and coordination of the pyrrole-type N(1)H groups does not occur below pH 10.     

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.     

Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases

Phenylalanine hydroxylase (PAH) is a pterin-dependent non-heme metalloenzyme that catalyzes the oxidation of phenylalanine to tyrosine, which is the rate-limiting step in the catabolism of Phe. Chromobacterium violaceum phenylalanine hydroxylase (cPAH) has been prepared and its steady-state mechanism has been investigated. The enzyme requires iron for maximal activity. Initial rate measurements, done in the presence of the 6,7-dimethyl-5,6,7,8-tetrahydropterin (DMPH(4)) cofactor, yielded an average apparent k(cat) of 36+/-1 s(-1). The apparent K(M) values measured for the substrates DMPH(4), L-Phe, and O(2) are 44+/-7, 59+/-10, and 76+/-7 microM, respectively. Steady-state kinetic analyses using double-reciprocal plots revealed line patterns consistent with a sequential ter-bi mechanism in which L-Phe is the middle substrate in the order of binding. The occurrence of a line intersection on the double-reciprocal plot abscissa when either pterin or O(2) is saturated suggests that, prior to O(2) binding, DMPH(4) and L-Phe are in associative pre-equilibrium with cPAH. Together with an inhibition study using the oxidized cofactor, 7,8-dimethyl-6,7-dihydropterin, it is conclusive that the mechanism is fully ordered, with DMPH(4) binding the active site first, L-Phe second, and O(2) last. This represents the first conclusive steady-state mechanism for a PAH enzyme.     

(6R)-Tetrahydrobiopterin Increases the Activity of Tryptophan Hydroxylase in Rat Raphe Slices

The effects of (6R)- and (6S)-tetrahydrobiopterin (BPH4), tetrahydroneopterin, and 6-methyltetrahydropterin on the activity of tryptophan hydroxylase were investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measurement of 5-hydroxytryptophan (5-HTP) formation under inhibition of aromatic L-amino acid decarboxylase with use of HPLC-fluorometric detection. (6R)-BPH4 (the naturally occurring form) at 42 microM, tetrahydroneopterin at 50 microM, and 6-methyltetrahydropterin at 100 microM increased tryptophan hydroxylase activity to 350, 145, and 146% of control values, respectively. (6S)-BPH4, however, had no significant effects on tryptophan hydroxylase activity. These results suggest that tryptophan hydroxylase is subsaturating in vivo for the naturally occurring cofactor, (6R)-BPH4, and that the concentration of (6R)-BPH4 may play an important role for the regulation of tryptophan hydroxylase activity in vivo.     

Antagonists of calcium fluxes and calmodulin block activation of the p21-activated protein kinases in neutrophils

Neutrophils stimulated with fMLP or a variety of other chemoattractants that bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecular masses of approximately 63 and 69 kDa (gamma- and alpha-Pak). Previous studies have shown that products of phosphatidylinositol 3-kinase and tyrosine kinases are required for the activation of Paks. We now report that a variety of structurally distinct compounds which interrupt different stages in calcium/calmodulin (CaM) signaling block activation of the 63- and 69-kDa Paks in fMLP-stimulated neutrophils. These antagonists included selective inhibitors of phospholipase C (1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), the intracellular Ca(2+) channel (8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate), CaM (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), and CaM-activated protein kinases (N-[2-(N-(chlorocinnamyl)-N:-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide). This inhibition was dose-dependent with IC(50) values very similar to those that interrupt CaM-dependent reactions in vitro. In contrast, less active analogues of these compounds (1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(4-aminobutyl)-1-naphthalenesulfonamide; promethazine; 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine]) did not affect activation of Paks in these cells. CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), but not their less-active analogues (N-(6-aminohexyl)-1-naphthalenesulfonamide; promethazine), were also found to block activation of the small GTPases Ras and Rac in stimulated neutrophils along with the extracellular signal-regulated kinases. These data strongly suggest that the Ca(2+)/CaM complex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases.     

Purification and state of activation of rat kidney phenylalanine hydroxylase

Phenylalanine hydroxylase, the enzyme that catalyzes the irreversible hydroxylation of phenylalanine to tyrosine, was purified from rat kidney with the use of phenyl-Sepharose, DEAE-Sephacel, and gel permeation high pressure liquid chromatography. Our most highly purified fractions had a specific activity in the presence of 6-methyltetrahydropterin, of 1.5 mumol of tyrosine formed/min/mg of protein, which is higher than has been reported hitherto. For the rat kidney enzyme, the ratio of specific activity in the presence of 6-methyltetrahydropterin to the specific activity in the presence of tetrahydrobiopterin (BH4) is 5. By contrast, this ratio for the unactivated rat liver hydroxylase is 80. These results indicate that the kidney enzyme is in a highly activated state. The rat kidney hydroxylase could not be further activated by any of the methods that stimulate the BH4-dependent activity of the rat liver enzyme. In addition, the kidney enzyme binds to phenyl-Sepharose without prior activation with phenylalanine. The phenylalanine saturation pattern with BH4 as a cofactor is hyperbolic with substrate inhibition at greater than 0.5 mM phenylalanine, a pattern that is characteristic of the activated liver hydroxylase. The molecular weight of the rat kidney enzyme as determined by gel permeation chromatography is 110,000, suggesting that the enzyme might be an activated dimer. We conclude, therefore, that phenylalanine hydroxylases from rat kidney and liver are in different states of activation and may be regulated in different ways.     

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.     

Synthesis and anti-inflammatory activities of N4,N5-disubstituted-3-methyl- H-pyrazolo[3,4-c]pyridazines

The synthesis and anti-inflammatory activity of 4,5-dihydroxy-3-methyl-1H-pyrazolo[3,4-c]pyridazine (4), 4,5-dichloro-3-methyl-1H-pyrazolo[3,4-c]pyridazine (5), 4,-benzoyloxy-3-methyl-1-benzoyl-1H-pyrazolo[3,4-c]pyridazin-5yl benzoate (6), 3-methyl-N4,N5-bis(4-methylphenyl)-1H-pyrazolo[3,4-c]pyridazine-4,5-diamine (7), 4[[5-(4-carboxyanilino)-3-methyl-1H-pyrazolo[3,4-c]pyridazin-4yl]amino]benzoic acid (8), N-[5-(benzoylamino)-3-methyl-1H-pyrazolo[3,4-c]pyridazin-4-yl]benzamide (9) and 3-methyl-N4,N5-bis[4-(1H-benzimidazol-2yl)phenyl]-1H-pyrazolo[3,4-c]pyridazine-4,5-diamine (10) are being reported.     

Structural studies of copper catalyzed hydroxylation reactions

The reaction of 2,6-diacetylpyridine, CuCl₂, and semicarbazide hydrochloride was studied as a function of pH to yield three different products: aqua(2-acetylsemicarbazone-3-hydroxo-6-acetylpyridine) chlorocopper(II) hemihydrate, 1, bis(2,6-diacetylpyridinedisemicarbazone)copper(I) chloride dimer, 2, and bis(2,6-diacetylpyridinedisemicarbazone)copper(II) chloride dimer, 3. The reaction of 1 with a mol of semicarbazide hydrochloride gave chloro(3-hydroxo-2,6-diacetylpyridinedisemicarbazone)copper(II) chloride hydrate, 4, and with a mol of semicarbazide hydrochloride and cupric chloride gave aquachlorocopper(II)(μ-chloro)(μ′-[2-acetylsemicarbazone-O,N-3-hydroxy-6-acetylsemicarbazonepyridine-O,N,N])aquacopper(II) trihydrate, 5. The crystal structures of compounds 1-5 were determined by X-ray diffraction. Some observations on the course of the reactions are given.     

Phenylalanine hydroxylase from Chromobacterium violaceum is a copper-containing monooxygenase. Kinetics of the reductive activation of the enzyme

Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.