Photoactivation of pseudomonad L-tryptophan oxygenase by electron ejection from its substrate, L-tryptophan

Chemically oxidized, catalytically inactive, pseudomonad l-tryptophan-2,3-dioxygenase (EC 1.13.1.12) can be photoactivated aerobically as well as anaerobically by light of wavelength less than 360 nm. The substrate, l-tryptophan, must be present for photoactivation to proceed. In these studies, a CCl₄ filter was used to block light of wavelength less than 265 nm, preventing photolysis of water and the concomitant production of H₂O₂ (known reductant of tryptophan oxygenase). Photoactivation is not inhibited by superoxide dismutase or formate and is only slightly inhibited by catalase. Nonsubstrate analogues of l-tryptophan, 5-fluorotryptophan (binds to the catalytic site), and α-methyltryptophan (binds to the allosteric site), separately or in concert, do not mediate photoactivation, while another substrate, 6-fluorotryptophan, can. Saturation of the allosteric site with α-methyltryptophan increases the extent of photoactivation in the presence of a nonsaturating level of l-tryptophan, indicating that photoactivation is dependent on the extent of saturation of the catalytic site by l-tryptophan. During the time course of photoactivation, catalytic activity increases faster than does the formation of ferroheme enzyme, indicating that the fully reduced enzyme, (ferroheme)₂(Cu⁺)₂, is formed from the fully oxidized enzyme, (ferriheme)₂(Cu²⁺)₂, subsequent to photoactivation. A significant amount of the half-reduced, catalytically active enzyme, (ferriheme)₂(Cu⁺)₂, exists during the time course of photoactivation. We propose that the mechanism by which electrons enter tryptophan oxygenase is via “electron ejection” [T. R. Hopkins and R. Lumry (1972) Photochem. Photobiol.15, 555–566] from a photoexcited l-tryptophan bound at, the catalvtic site.     

Tryptophan 2,3-dioxygenase: a review of the roles of the heme and copper cofactors in catalysis.

L-Tryptophan, 2,3-dioxygenase (EC 1.13.11.11) has been purified to homogenity from L-tryptophan induced Pseudomonas acidovorans (ATCC 11299b) and from L-tryptophan and cortisone induced rat liver. The enzyme from both sources is composed of four subunits and contains two g-atoms copper and two moles heme per mole tetramer. The proteins from the two sources are not identical. Three oxidation states of tryptophan oxygenase have been isolated: (1) fully oxidized, [Cu(II)]2[Ferriheme]2; (2) half reduced, [Cu(i)]2[ferriheme]2; and (3) fully reduced, [Cu(I)]2[ferroheme]2. Catalytic activity is dependent solely on the presence of Cu(I) in the enzyme, the heme may be either ferro or ferri. The presence of Cu(II) in the enzyme results in a requirement for an exogenous reductant, such as ascorbate, in order to elicit enzymic activity. Ligands, such as cyanide and carbon monoxide, can inhibit catalysis by binding to either or to both the copper and heme moieties. Metal complexing agents, such as bathocuproinesulfonate and bathophenanthrolinesulfonate, can inhibit catalysis by binding to Cu(I) resent only in catalytically active enzyme molecules. During catalysis by the fully reduced form of the enzyme, molecular oxygen binds to the heme moieties, while during catalysis by the half reduced form of the enzyme it does not, presumably binding instead to the Cu(I) moieties. Enzymes that catalyze similar reactions have been purified from other sources. Indoleamine 2,3-dioxygenase appears to be a heme protein, but its copper content is unknown. Pyrrolooxygenases appear to be completely different enzymes, although they have not yet been purified to homegeneity.     

Radiocopper in L-tryptophan 2,3-dioxygenase isolated from Pseudomonas acidovorans grown in the presence of 64Cu (II).

L-Tryptophan 2,3-dioxygenase (EC 1.13.11.11), isolated from L-tryptophan-induced Pseudomonas acidovorans, ATCC 11299b, which has been grown in a medium containing 64Cu(NO3)2, has been shown to contain radiocopper. At several stages of purification of the enzyme samples were taken, and these were subjected to disc acrylamide gel electrophoresis in the presence of 10 mM L-tryptophan. After electrophoresis the position of the yellow heme band, corresponding to tryptophan oxygenase, was visually located, and the gels were sliced and counted. A large peak of radioactivity was seen to occur at the location on the gel of tryptophan oxygenase no matter what the stage of purification. Treatment of each sample before electrophoresis for 30 min at 37 degrees with gamma-globulins prepared from rabbits sensitized to homogeneous pseudomonad tryptophan oxygenase greatly reduced this peak of radioactivity, whereas treatment of each sample with rabbit preimmune gamma-globulin did not. This direct demonstration of the presence of coper in pseudomonad tryptophan oxygenase, using 64-Cu, avoided the problems and artifacts inherent in the usual techniques of copper analysis and unequivocally refutes the recent contention of Ishimura and Hayaishi ((1973) J. Biol.Chem. 248, 8610-8612) "that copper is not an essential component of L-tryptophan 2,3-dioxygenase of Pseudomonas." The presence of copper in pseudomonad and rat liver tryptophan oxygenases, previously reported by us (Brady, F. O., Monaco, M. E., Forman, H. J., Schutz, G., and Feigelson, P. (P. (1972) J. Biol. Chem. 247, 7915-7922), is reaffirmed by the experiments reported herein.     

A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase

A quantitative yield of half-reduced (ferrous-ferric) cytochrome c peroxidase from Pseudomonas aeruginosa has been obtained by using either ascorbate or NADH as reductant of the resting (ferric-ferric) enzyme along with phenazine methosulfate as mediator. The formation of Compounds I and II from the half-reduced enzyme and hydrogen peroxide has been studied at 25 degrees C using rapid-scan spectrometry and stopped-flow measurements. The spectra of Compound I in the Soret and visible regions were recorded within 5 ms after mixing the half-reduced enzyme with H2O2. The spectrum of the primary compound at the Soret region had a maximum at 414 nm, and in the visible region at 528 and 556 nm. The spectrum of Compound I showed no bands in the 650-nm region, excluding the possibility of a pi-cation radical being part of the catalytic mechanism. Compound I was stable for at least 12 s when no reducing equivalents were present. In the presence of reduced azurin, half-reduced enzyme reacted with H2O2 to form Compound II within 50 ms. The spectrum of Compound II had a Soret maximum at 411 nm. In the visible region the Compound II spectrum was close to that of the totally oxidized, resting enzyme form. In the presence of excess azurin, Compound II was converted rapidly to the half-reduced enzyme form. The kinetics of Compound I formation was also followed with peracetic acid, ethylhydroperoxide, and m-chloroperbenzoic acid as electron acceptors. The rate constants of these reactions are diminished compared to that of hydrogen peroxide, indicating a closed structure for the heme pocket of the enzyme.     

Spectroscopic and equilibrium properties of the indoleamine 2,3-dioxygenase-tryptophan-O2 ternary complex and of analogous enzyme derivatives. Tryptophan binding to ferrous enzyme adducts with dioxygen, nitric oxide, and carbon monoxide

The dioxygen adduct of the heme protein indoleamine 2,3-dioxygenase has been generated at -30 degrees C in mixed solvents, and spectroscopic and equilibrium studies of its L-tryptophan (substrate) binding properties have been carried out for the first time. Comparative studies have also been performed with the NO and CO adducts of the ferrous enzyme. Under the conditions employed (-30 degrees C), both autoxidation and turnover (L-tryptophan + O2----formylkynurenine) of the ternary complex are effectively suppressed. Structural identification of the ternary complex is based on the 1:1 molar stoichiometry for the substrate-oxygenated enzyme adduct formation (Kd approximately 10(-4) M), the time-dependent linear product formation (turnover) at -20 degrees C, and the quantitative conversion of the complex to the ferrous CO derivative by bubbling with CO. Binding of L-tryptophan to the oxygenated enzyme leads to decreases in the intensities of its major absorption bands (lambda max 415, 541, 576 nm) and to a blue shift of its Soret peak. Interestingly, among the ferrous enzyme derivatives examined, only the substrate-bound oxygenated enzyme exhibits solvent-dependent Soret absorption peak positions, e.g., lambda max 411.5 and 413.5 nm in 65% (v/v) aqueous glycerol and ethylene glycol, respectively. In addition, indole binds to the oxygenated enzyme, causing a red shift of its Soret peak in these solvents only in the presence of substrate (411.5----414 nm and 413.5----414.5 nm, respectively), while similar effects of indole are independent of tryptophan for the other ferrous enzyme derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)     

The non-fluorescence of 4-fluorotryptophan.

The derivative 4-fluorotryptophan was confirmed to have negligible fluorescence at 25 degrees C and 285 nm (tryptophan/4-fluorotryptophan quantum-yield ratio greater than 100:1). However, photolysis experiments on tryptophan and 4-fluorotryptophan, in which loss of starting material was measured by reverse-phase h.p.l.c., demonstrated that 4-fluorotryptophan was significantly more photochemically active than the parent tryptophan, with the 4-fluorotryptophan photolysis quantum yield being 7 times larger than that of tryptophan at 25 degrees C and 285 nm. In addition, at 77 K and 275 nm 4-fluorotryptophan displayed strong fluorescence and phosphorescence, with emission quantum yields comparable with those of tryptophan at 77 K and 275 nm.     

Multiple molecular forms of diarylpropane oxygenase, an H2O2-requiring, lignin-degrading enzyme from Phanerochaete chrysosporium

Three different molecular forms of the H2O2-requiring heme enzyme, diarylpropane oxygenase, were isolated from the extracellular medium of Na-acetate buffered, agitated cultures of Phanerochaete chrysosporium. Forms I, II, and III were separated by DEAE-Sepharose and further purified on Sephadex G-100. Absorption maxima of the native, reduced, and a variety of ligand complexes of the three enzyme forms are essentially identical, indicating similar heme environments. All forms also have similar, but not identical, reactivity. The homogeneous proteins oxidized a diarylpropane, an olefin, a beta-aryl ether dimer, a phenylpropane, phenylpropane diols, and veratryl alcohol. Identical products were produced from each form. However, the specific activities of the three homogeneous enzymes for veratryl alcohol oxidation were 18.75, 11.80, and 8.48 mumol min-1 mg-1. In the presence of one equivalent of H2O2 the Soret maximum of diarylpropane oxygenase II shifted from 408 to 418 nm, and two additional maxima appeared at 526 and 553 nm, indicating the presence of an Fe(IV)-oxo species equivalent to horseradish peroxidase II. This oxidized species could be reduced back to the native form by veratryl alcohol and several reducing agents (e.g., Na2S2O4, NH2NH2, thiourea, or NADH). The molecular weights of diarylpropane oxygenases I, II, and III were approximately 39,000, 41,000, and 43,000, respectively. The major form (II) (85% of the activity) contained approximately 6% neutral carbohydrate. The affinity of the forms for concanavalin A-agarose suggests that they all are glycoenzymes.     

Studies of the copper and heme cofactors of pseudomonad L-tryptophan-2,3-dioxygenase by electron paramagnetic resonance spectroscopy

l-Tryptophan-2,3-dioxygenase, (EC 1.13.1.12) purified from Pseudomonas acidovorans, is inactivated on aerobic aging or on treatment with K₃Fe(CN)₆, but regains activity in the presence of reducing agents such as sodium ascorbate. Examination of oxidized, inactive enzyme by electron paramagnetic resonance (epr) spectroscopy has revealed the presence of high spin ferriheme (g = 6.2) and of Cu(II) (g_⊥ = 2.065, g_∥ = 2.265) in the enzyme.The epr signal of Cu(II) in inactive tryptophan oxygenase is attenuated on the addition of ascorbate, whereas the high spin ferriheme signal is unaffected, indicating that the site of action of reducing agents in activating the enzyme is the enzymic copper. Quantitation of the Cu(II) signal in inactive tryptophan oxygenase by double integration accounts for 45% of the total copper.Addition of l-tryptophan to either inactive or active enzyme produces a decrease of 44 ± 5% of the epr signal of high spin ferriheme and the emergence of the epr signal of a low spin ferriheme (g_{1, 2, 3} = 2.66, 2.20, 1.81). Disappearance of the high spin ferriheme is hyperbolic (Hill coefficient, n = 1.02) with respect to l-tryptophan concentration, while the appearance of the low spin ferriheme is sigmoidal (Hill coefficient, n = 1.33) with respect to l-tryptophan concentration. The characteristics of the epr signal of this low spin ferriheme are intermediate between those of the signals of the hydroxides of hemoglobin and myoglobin and those in which two histidines are ligated to the ferriheme of hemoglobin. This may be the first example of the observation by epr of an allosteric parameter of an enzyme.     

pH dependence of the cooperative interactions and conformation of tryptophan oxygenase.

Allosteric interactions in the cupro-heme enzyme tryptophan oxygenase (EC 1.13.11.11) of Pseudomonas acidovorans are shown to be pH-dependent. Increasing the assay pH from 6.0 to 8.0 progressively desensitizes the enzyme from both homotropic and heterotropic ligand interactions. This pH-dependent reversible transition has a pK of 6.2. Hill coefficients for the substrate L-tryptophan of 2.0 and 1.4 were measured at pH 6.0 and pH 7.0, respectively. In attempting to identify the enzymatic residue (or residues) responsible for these pH-dependent effects, the enzyme was observed to be irreversibly inactivated by photoinduced oxidation in the presence of the sensitizer, methylene blue. The photoinactivated enzyme showed a loss of one-half its Soret (405 nm) absorption which accompanied the loss of one-half its heme and histidine contents. This first order photoinduced inactivation was pH-dependent and corresponded to a requirement for a protonated species with a pK of 6.2. These results suggest that histidine residues may be involved in the catalytic function and in mediating cooperative interactions of tryptophan oxygenase. Absolute and difference sedimentation velocity analyses indicate that the molecule undergoes a conformational transition when the pH is decreased from pH 8.0 to pH 6.0. This conformational alteration, measured as a 3.9% increase in S20, w can be regarded as an equivalent decrease in the frictional coefficient. If, a more or less spherical shape to the molecule is assumed, then, the 3.9% decrease in the frictional coefficient between pH 8.0 and 6.0 corresponds to a 12% decrease in apparent hydrodynamic volume of the enzyme. Thus, protonation of an enzymatic moiety, possibly histidine, determines both the conformational and functional interactions between enzymatic sites.     

Tryptophan catabolism in Bacillus megaterium.

Bacillus megaterium grows in a medium containing L-tryptophan as the sole carbon, nitrogen, and energy source. Kynurenine, anthranilic acid, and catechol are metabolic intermediates, suggesting that this organism used the anthranilic acid pathway for tryptophan degradation. Cells that grow on L-tryptophan oxidize kynurenine, alanine, and anthranilic acid and the presence of tryptophan oxygenase (EC 1.13.1.12), kynureninase (EC 3.7.1.3), and catechol oxygenase (EC 1.13.1.1) in cell extracts provide additional evidence for the degradative pathway in B. megaterium. Tryptophan oxygenase is inhibited by sodium azide, potassium cyanide, and hydroxylamine, indicating that the enzyme has a functional heme group. D-Tryptophan is not a substrate for tryptophan oxygenase, and the D-isomer does not inhibit this enzyme. Formamidase (EC 3.5.1.9) and anthranilate hydroxylase are not detectable in extracts. Tryptophan catabolism is inducible in B megaterium and is subject to catabolite repression by glucose and glutamate. Arginine does not cause repression, and kynurenine induces both tryptophan oxygenase and kynureninase.     

Resonance Raman spectroscopic characterization of compound III of lignin peroxidase

Resonance Raman (RR) spectra of several compounds III of lignin peroxidase (LiP) have been measured at 90 K with Soret and visible excitation wavelengths. The samples include LiPIIIa (or oxyLiP) prepared by oxygenation of the ferrous enzyme, LiPIIIb generated by reaction of the native ferric enzyme with superoxide, LiPIIIc prepared from native LiP plus H2O2 followed by removal of excess peroxide with catalase, and LiPIII* made by addition of excess H2O2 to the native enzyme. The RR spectra of these four products appear to be similar and, thus, indicate that the environments of these hexacoordinate, low-spin ferriheme species must also be very similar. Nonetheless, the Soret absorption band of LiPIII* is red-shifted by 5 nm from the 414-nm maximum common to LiPIIIa, -b, and -c [Wariishi, H., & Gold, M.H. (1990) J. Biol. Chem. 265, 2070-2077]. Analysis of the iron-porphyrin vibrational frequencies indicates that the electronic structures for the various compounds III are consistent with an FeIIIO2.-formulation. The spectral changes observed between the oxygenated complex and the ferrous heme of lignin peroxidase are similar to those between oxymyoglobin and deoxymyoglobin. The contraction in the core sizes in compound III relative to the native peroxidase is analyzed and compared with that of other heme systems. EPR spectra confirm that the high-spin ferric form of the native enzyme, with an apparent g = 5.83, is converted into the EPR-silent LiPIII* upon addition of excess H2O2. Its magnetic behavior may be explained by anti-ferromagnetic coupling between the low-spin FeIII and the superoxide ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reaction of hemin with H2O2

The kinetics of formation of the dominant intermediate (CII) formed between hemin and H2O2 has been studied by the stopped-flow method. CII is preceded by a precursor (CI) for which a steady state is established at an early stage of the reaction. The formation of CI from hemin and H2O2 causes only a marginal change in the optical absorbance (A). The transition CI----CII is accompanied by a substantial decrease of A in the Soret region. Relevant rate constants (or combinations of them) and the molar absorption coefficients of the intermediates at 400 nm have been determined. The absorption spectrum of CII in the Soret region has been evaluated. Aspects of the catalysis of decomposition of H2O2 by hemin in relation to the Fe3+ ion and catalase are discussed.     

The influence of some methodological factors on measurement of tryptophan oxygenase activities in crude homogenates of rat liver.

1. With two different methods for assaying the tryptophan oxygenase activity in rat liver homogenates, the effects of some methodological factors on the activity of the enzyme were studied. 2. In fed, but not in starved, rats a compound(s) absorbing at 365 nm, interfering with the reading of kynurenine absorbance, disappeared gradually during incubation. 3. A correction for this tryptophan-independent reaction was necessary in order to determine correct tryptophan oxygenase activity. 4. Blood remaining in liver tissue post mortem can serve as a source of cofactor haem for tryptophan oxygenase, causing spuriously high values for the activity of the holoenzyme form of tryptophan oxygenase. 5. A rapid and progressive activation of tryptophan oxygenase post mortem occurs in undisrupted liver tissue, and this activation is temperature-dependent.     

Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.     

Nitric oxide-generated P420 nitric oxide synthase: characterization and roles for tetrahydrobiopterin and substrate in protecting against or reversing the P420 conversion

The neuronal NO synthase (nNOS) heme binds self-generated NO, and this negatively regulates NO synthesis. Here we utilized the nNOS oxygenase domain and full-length nNOS along with various spectroscopic methods to (1) study formation of the six-coordinate ferrous NO complex and its conversion to a five-coordinate NO complex and (2) investigate the spectral and catalytic properties of the five-coordinate NO complex following its air oxidation to a ferric enzyme. NO bound quickly to ferrous nNOS oxygenase to form a six-coordinate NO complex (kon and koff values of 1.25 x 10(-)3 mM-1 s-1 and 128 s-1 at 10 degreesC, respectively) that was stable in the presence of L-arginine or tetrahydrobiopterin (BH4) but was converted to a five-coordinate NO complex in a biphasic process (k = 0.1 and 0.01 s-1 at 10 degreesC) in the absence of these molecules. Air oxidation of the ferrous six-coordinate NO complex generated an enzyme with full activity and ferrous-CO Soret absorbance at 444 nm. In contrast, oxidation of the five-coordinate NO complex generated an inactive dimer with ferrous-CO Soret absorbance at 420 nm, indicating nNOS was converted to a ferric P420 form. Incubation of ferric P420 nNOS with BH4 alone or BH4 and L-arginine resulted in time-dependent reactivation of catalysis and associated recovery of P450 character. Thus, nNOS is a heme-thiolate protein that can undergo a reversible P450-P420 conversion. BH4 has important roles in preventing P420 formation during NO synthesis, and in rescuing P420 nNOS.     

Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism

We have examined the interaction of tryptophan synthase and tryptophanase with the tryptophan analogues oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Since these analogues have tetrahedral geometry at carbon 3 of the heterocyclic ring, they are structurally similar to the indolenine tautomer of L-tryptophan, a proposed intermediate in reactions of L-tryptophan. Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 microM) 10-100-fold lower than the corresponding Km or KI values for L-tryptophan. Addition of oxindolyl-L-alanine or 2,3-dihydro-L-tryptophan to solutions of the alpha 2 beta 2 complex of tryptophan synthase results in new absorption bands at 480 or 494 nm, respectively, which are ascribed to a quinonoid or alpha-carbanion intermediate. Spectrophotometric titration data give half-saturation values of 5 and 25 microM, which are comparable to the KI values obtained in kinetic experiments. Our finding that both enzymes catalyze incorporation of tritium from 3H2O into oxindolyl-L-alanine is evidence that both enzymes form alpha-carbanion intermediates with oxindolyl-L-alanine. These results support the proposal that the indolenine tautomer of L-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase. In addition, we have found that oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent adduct.     

Stereospecificity of hepatic L-tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase [L-tryptophan--oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.     

Single-turnover of nitric-oxide synthase in the presence of 4-amino-tetrahydrobiopterin: proposed role for tetrahydrobiopterin as a proton donor

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric-oxide synthase (NOS) that serves as a one-electron donor to the oxyferrous.heme complex. 4-Aminotetrahydrobiopterin (4-amino-BH4) is a potent inhibitor of NO synthesis, although it mimics all allosteric and structural effects of BH4 and exhibits comparable redox properties. We studied the reaction of reduced endothelial NOS oxygenase domain with O2 in the presence of 4-amino-BH4 at -30 degrees C by optical and electron paramagnetic resonance (EPR) spectroscopy. With Arg as the substrate, we observed a trihydropteridine radical with a corresponding heme species that was oxyferrous, with a Soret maximum at 428 nm and no EPR signal. With NG-hydroxy-l-arginine (NHA) no pterin radical appeared, whereas an axial ferrous heme.NO complex was formed. The corresponding optical spectra, with Soret bands at 417/423 nm, suggest that the proximal sulfur ligand is protonated. Accordingly, 4-amino-BH4 serves as a one-electron donor to Fe(II).O2 with both Arg and NHA, but the reaction cycle cannot be completed with either substrate. We propose that protonation of Fe(II)O2- is inhibited in the presence of 4-amino-BH4. With Arg, dissociation of O2- and binding of O2 yields Fe(II).O2 and a pteridine radical; with NHA, reaction of the substrate with heme-bound O2- eventually yields Fe(II).NO and reduced 4-amino-BH4. These results suggest that BH4 donates a proton to Fe(II).O2- during catalysis and that inhibition by 4-amino-BH4 may be due to its inability to support this essential protonation step.     

Oxygen activation by a mixed-valent, diiron(II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase

myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to d-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by M?ssbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O(2). Both observations implicate the mixed-valent, diiron(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III).MI] with limiting O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III).MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O(2) stoichiometry in the reaction, 0.8 +/- 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O(2). The DG/O(2) yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III).MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O(2) from the II/II manifold.     

pH dependence of the reduction of dioxygen to water by cytochrome c oxidase. 1. The P(R) state is a pH-dependent mixture of three intermediates,A, P,and F

Recent studies on cytochrome oxidase have indicated that the putative "peroxy" intermediate in the catalytic cycle (P(R)) is a mixture of intermediates, including P and F [Sucheta, A., et al. (1998) Biochemistry 37, 17905-17914], and the bench-made P and F forms appear to have the same redox state (Fe(a3)(4+)=O(2-)), but a different protonation state [Fabian, M., and Palmer, G. (2001) Biochemistry 40, 1867-1874]. To explore the possibility that the putative P(R) state is a pH-dependent mixture of intermediates, we investigated the reduction of dioxygen to water by the fully reduced cytochrome oxidase at pH 6.2, 7.5, and 8.5 in the visible and Soret regions (350-800 nm) using the CO flow-flash technique. Singular value decomposition and global exponential fitting of the time-resolved absorption difference spectra resolved five apparent lifetimes. The fastest three (1.5, 13, and 34 micros) were independent of pH, while the two slowest rates (80-240 micros and 1.1-2.4 ms) decreased by a factor of 2-3 as the pH increased. When the time-resolved spectra were analyzed using a unidirectional sequential model, the spectra of the reduced enzyme and the dioxygen-bound intermediate, compound A, were found to be pH-independent. However, the putative P(R) intermediate was best represented by a pH-dependent mixture of compound A, P, and F. The ferryl form was favored at low pH. The subsequent intermediate is a ferryl with a pH-dependent electron transfer equilibrium between heme a and Cu(A), the reduced heme a being favored at low pH. These results suggest a pH-dependent reaction mechanism of the reduction of dioxygen to water by the fully reduced enzyme that is more complex than previously proposed.