Dioxygenases without requirement for cofactors and their chemical model reaction: compulsory order ternary complex mechanism of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase involving general base catalysis by histidine 251 and single-electron oxidation of the substrate dianion

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to the alpha/beta hydrolase fold family, catalyzing the cleavage of 1H-3-hydroxy-4-oxoquinaldine (I) and 1H-3-hydroxy-4-oxoquinoline (II) to N-acetyl- and N-formylanthranilate, respectively, and carbon monoxide. Bisubstrate steady-state kinetics and product inhibition patterns of HodC, the C69A protein variant of Hod, suggested a compulsory-order ternary-complex mechanism, in which binding of the organic substrate precedes dioxygen binding, and carbon monoxide is released first. The specificity constants, k(cat)/K(m,A) and k(cat)/K(m,O)()2, were 1.4 x 10(8) and 3.0 x 10(5) M(-1) s(-1) with I and 1.2 x 10(5) and 0.41 x 10(5) M(-1) s(-1) with II, respectively. Whereas HodC catalyzes formation of the dianion of its organic substrate prior to dioxygen binding, HodC-H251A does not, suggesting that H251, which aligns with the histidine of the catalytic triad of the alpha/beta hydrolases, acts as general base in catalysis. Investigation of base-catalyzed dioxygenolysis of I by electron paramagnetic resonance (EPR) spectroscopy revealed formation of a resonance-stabilized radical upon exposure to dioxygen. Since in D(2)O spectral properties are not affected, exchangeable protons are not involved, confirming that the dianion is the reactive intermediate that undergoes single-electron oxidation. We suggest that in the ternary complex of the enzyme, direct single-electron transfer from the substrate dianion to dioxygen may occur, resulting in a radical pair. Based on the estimated spin distribution within the radical anion (observed in the model reaction of I), radical recombination may produce a C4- or C2-hydroperoxy(di)anion. Subsequent intramolecular attack would result in the 2,4-endoperoxy (di)anion that may collapse to the reaction products.     

Penetration of dioxygen into proteins studied by quenching of phosphorescence and fluorescence

Experiments were done to measure the ability of dioxygen to collisionally quench the phosphorescent and fluorescent tryptophans in alcohol dehydrogenase and alkaline phosphatase. In all cases, luminescence is quenched with rate constants close to 1 x 10(9) M-1 s-1. The rate of reaching the buried tryptophans is little affected by solvent viscosity due to added glycerol. Quenching by dioxygen is not due to a protein-opening reaction. It appears to be rate limited by internal protein diffusion rather than at the entry step. Dioxygen appears to enter the proteins directly, as in liquidlike diffusion, rather than through transiently forming channels that are only present a small fraction of the time. A high-pressure oxygen system is described that considerably facilitates fluorescence quenching experiments.     

Determination of the dioxygen quenching constant for protein and model indole triplets

The decay of the indole triplet of single tryptophan-containing proteins and model compounds can be readily determined at room temperature in solution by monitoring the triplet absorption or emission following an exciting laser pulse. The dioxygen triplet quenching constants, can be measured for all these molecules and compared to the analogous singlet values determined by fluorescence methods. The dioxygen triplet quenching constant (tkq) ranged from a high of 5.1.10(9) M-1.s-1 for the exposed indole of corticotropin to a low of 0.1.10(9) M-1.s-1 for the buried indole of asparaginase. The ratio of these values with their respective dioxygen singlet quenching constants (skq), tkq/skq, ranged from 0.3 to 0.6 for aqueous exposed polypeptide indoles. For globular proteins the tkq/skq value is observed to be 0.2 +/- 0.1. This lower value for protein indoles is not attributable to 'bulk' environmental or hydrogen bonding effects, since the magnitude of tkq/skq (= 0.5 +/- 0.1) for model indoles was independent of solvent dielectric constant, polarity, and proticity. Temperature-dependence studies were done to test whether tkq could be used to characterize the nature of the protein matrix. The activation energy (Ea) for tkq was found to be 11 +/- 2 kcal/mol for most proteins. This Ea was independent of whether the indole side-chain was solvent exposed or buried in the non-aqueous protein interior. Large Ea values were also obtained for model indoles, naphthalene and nalidixic acid, dissolved in water, whereas the same compounds dissolved in 95% ethanol exhibited much smaller Ea values. These data, in combination with the observation that the tkq of model indoles is insensitive to changes in solvent viscosity, indicate that dioxygen quenching at the triplet level can not be easily used to characterize the dynamics of proteins.     

Oxygenation of carbon monoxide by bovine heart cytochrome c oxidase

Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1), as the terminal enzyme of the mammalian mitochondrial electron transport chain, has long been known to catalyze the reduction of dioxygen to water. We have found that when reductively activated in the presence of dioxygen, the enzyme will also catalyze the oxidation of carbon monoxide to its dioxide. Two moles of carbon dioxide is produced per mole of dioxygen, and similar rates of production are observed for 1- and 2-electron-reduced enzyme. If 13CO and O2 are used to initiate the reaction, then only 13CO2 is detected as a product. With 18O2 and 12CO, only unlabeled and singly labeled carbon dioxide are found. No direct evidence was obtained for a water-gas reaction (CO + H2O----CO2 + H2) of the oxidase with CO. The CO oxygenase activity is inhibited by cyanide, azide, and formate and is not due to the presence of bacteria. Studies with scavengers of partially reduced dioxygen show that catalase decreases the rate of CO oxygenation.     

Chemical and spectroscopic evidence for the formation of a ferryl Fea3 intermediate during turnover of cytochrome c oxidase

When partially reduced cytochrome c oxidase samples are reoxidized with dioxygen, an EPR-silent dioxygen intermediate, which is at the three-electron level of dioxygen reduction, is trapped at the dioxygen reduction site. The intermediate has novel spectral features at 580 and 537 nm. Combined optical and EPR results reveal that this intermediate reacts rapidly with CO at 277-298 K causing the abolition of the 580/537 mm features and the appearance of a rhombic CuB EPR signal. A ferryl Fea3, or an intermediate at the same formal level of oxidation, is proposed to oxidize CO to CO2 producing an EPR-detectable CuB adjacent to a low-spin ferrous Fea3-dioxygen (or carbon monoxide) adduct.     

Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-D-glucamine dithiocarbamate

Iron(II)-dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)-dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N-(dithiocarboxy)sarcosine (dtcs) and N-methyl-d-glucamine dithiocarbamate (mgd) are 56 and -25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)-dtcs and iron(II)-mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)-dtcs and -mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6-3.2) x 10(4) and (0.7-1.1) x 10(5) M(-1) s(-1), respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)-dtcs and iron(II)-mgd by hydrogen peroxide is significantly slower (ca. 10-15 M(-1) s(-1)). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)-dtcs and iron(III)-mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M(-1) s(-1), respectively.     

Kinetic studies on the reaction between Trametes villosa laccase and dioxygen

A laccase from the fungus Trametes villosa (TviL) was investigated in order to elucidate the reaction mechanism of the reduction of dioxygen to water performed by this blue multi-copper oxidase. The ability of TviL to activate dioxygen was studied by stopped-flow experiments and under steady-state conditions. In the stopped-flow experiments TviL was reduced with a small excess of 4-hydroxyphenylacetic acid and afterwards the re-oxidation process was monitored by stopped-flow techniques by mixing with excess dioxygen. The reaction between reduced TviL and dioxygen was studied in the temperature range 10-35 degrees Celsius and with the concentration of dioxygen between 30 and 240microM. The observed rate constant k(obs) is found to be linear dependent on the dioxygen concentration and the observed second-order rate constant for the re-oxidation of reduced TviL is, at 25 degrees Celsius, determined to be 1.14x10(6)M(-1)s(-1). The activation energy, E(a), is from the same data determined to be 22kJmol(-1). Oxidation of different phenols (4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, guaiacolsulfonic acid and hydroquinone) under steady state conditions was investigated at concentrations of dioxygen ranging from 60 to 250microM. This line of experiments showed that TviL follows a ping-pong mechanism, and an observed second-order rate constant for the reaction with dioxygen of 7.1x10(5)M(-1)s(-1) at 25 degrees Celsius was found with 4-hydroxyphenylacetic acid as reducing substrate. The two kinetic methods resulted in observed rate constants of equal magnitudes for the reaction with dioxygen, which suggests that the rate limiting step(s) is (are) included in both the reactions studied by the two different techniques.     

Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-d-glucamine dithiocarbamate

Iron(II)-dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)-dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N-(dithiocarboxy)sarcosine (dtcs) and N-methyl-d-glucamine dithiocarbamate (mgd) are 56 and -25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)-dtcs and iron(II)-mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)-dtcs and -mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6-3.2) x 10(4) and (0.7-1.1) x 10(5) M(-1) s(-1), respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)-dtcs and iron(II)-mgd by hydrogen peroxide is significantly slower (ca. 10-15 M(-1) s(-1)). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)-dtcs and iron(III)-mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M(-1) s(-1), respectively.     

Nature of oxygen activation in glucose oxidase from Aspergillus niger: the importance of electrostatic stabilization in superoxide formation.

Glucose oxidase catalyzes the oxidation of glucose by molecular dioxygen, forming gluconolactone and hydrogen peroxide. A series of probes have been applied to investigate the activation of dioxygen in the oxidative half-reaction, including pH dependence, viscosity effects, 18O isotope effects, and solvent isotope effects on the kinetic parameter Vmax/Km(O2). The pH profile of Vmax/Km(O2) exhibits a pKa of 7.9 +/- 0.1, with the protonated enzyme form more reactive by 2 orders of magnitude. The effect of viscosogen on Vmax/Km(O2) reveals the surprising fact that the faster reaction at low pH (1.6 x 10(6) M-1 s-1) is actually less diffusion-controlled than the slow reaction at high pH (1.4 x 10(4) M-1 s-1); dioxygen reduction is almost fully diffusion-controlled at pH 9.8, while the extent of diffusion control decreases to 88% at pH 9.0 and 32% at pH 5.0, suggesting a transition of the first irreversible step from dioxygen binding at high pH to a later step at low pH. The puzzle is resolved by 18O isotope effects. 18(Vmax/Km) has been determined to be 1.028 +/- 0.002 at pH 5.0 and 1.027 +/- 0.001 at pH 9.0, indicating that a significant O-O bond order decrease accompanies the steps from dioxygen binding up to the first irreversible step at either pH. The results at high pH lead to an unequivocal mechanism; the rate-limiting step in Vmax/Km(O2) for the deprotonated enzyme is the first electron transfer from the reduced flavin to dioxygen, and this step accompanies binding of molecular dioxygen to the active site. In combination with the published structural data, a model is presented in which a protonated active site histidine at low pH accelerates the second-order rate constant for one electron transfer to dioxygen through electrostatic stabilization of the superoxide anion intermediate. Consistent with the proposed mechanisms for both high and low pH, solvent isotope effects indicate that proton transfer steps occur after the rate-limiting step(s). Kinetic simulations show that the model that is presented, although apparently in conflict with previous models for glucose oxidase, is in good agreement with previously published kinetic data for glucose oxidase. A role for electrostatic stabilization of the superoxide anion intermediate, as a general catalytic strategy in dioxygen-utilizing enzymes, is discussed.     

Direct detection of a dioxygen adduct of cytochrome a3 in the mixed valence cytochrome oxidase/dioxygen reaction

Time-resolved resonance Raman spectra have been recorded during the reaction of mixed valence (a3+ a2+(3)) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 10 microseconds subsequent to carbon monoxide photolysis, a mode is observed at 572 cm-1 that shifts to 548 cm-1 when the experiment is repeated with 18O2. The appearance of this mode is dependent upon the laser intensity used and disappears at higher incident energies. The high frequency data in conjunction with the mid-frequency data allow us to assign the 572 cm-1 mode to the Fe-O stretching vibration of the low-spin O2 adduct that forms in the mixed valence cytochrome oxidase/dioxygen reaction. The 572 cm-1 v(Fe2(+)-O2) frequency in the mixed valence enzyme/O2 adduct is essentially identical to the 571 cm-1 frequency we measured for this mode during the reduction of O2 by the fully reduced enzyme (Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1989) J. Am. Chem. Soc. 111, 6439-6440; Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1990) J. Am. Chem. Soc. 112, 1297), which indicates that the O2-bound cytochrome a3 site is independent of the redox state of the cytochrome a/CuA pair. The photolabile oxy intermediate is replaced by photostable low- or intermediate-spin cytochrome a3+(3), with t1/2 congruent to 200 microseconds.     

Proton activities for three states of cytochrome P-450cam.

Changes in proton concentration during the binding of dioxygen, carbon monoxide, and for the exchange of dioxygen by carbon monoxide, at ferrous-cytochrome P-450cam were measured by direct titration. Insufficient proton release was observed to support protonation-deprotonation of an axial cysteinyl sulfur donor as a mechanism for generation of hyper spectra in only the carbonylated ferrous state. Measurement of the $\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value for CO binding as a function of pH (the carbon monoxide Bohr effect) confirms the direct titration data.     

Carbon monoxide oxygenase activity of cytochrome cd1

Cytochrome cd1 from the denitrifying bacterium Pseudomonas aeruginosa catalyzes the oxygenation of carbon monoxide by dioxygen. A minimum estimate of the turnover number for this activity is 7 mol of carbon dioxide produced per hour per mole of cytochrome subunit at 30 degrees C and pH 7. The reaction is 98% inhibited by 2.5 mM cyanide, but catalase has no effect. The reaction accounts for the unusual reduction of ferric cytochrome in the presence of carbon monoxide, but no additional reducing agent. The reaction is independent of the steady-state oxidation level of the cytochrome during turnover. Under anaerobic conditions, ferricyanide plus water may substitute for dioxygen as the source of oxidizing equivalents and atomic oxygen.     

Photoacoustic characterization of protein dynamics following CO photodetachment from fully reduced bovine cytochrome c oxidase

We report a protein conformational change following carbon monoxide photodetachment from fully reduced bovine cytochrome c oxidase that is hypothesized to be associated with changes in ligand mobility through a dioxygen access channel in the protein. Although not resolved by earlier photoacoustic or optical studies on this adduct, utilization of slightly lower temperatures revealed a process with a kinetic lifetime of about 70 ns at 10 degrees C. We measure an enthalpy change of about 8 kcal/mol in 0.050 M HEPES buffer that becomes less endothermic (DeltaH approximately 2 kcal/mol) at higher ionic strength. The volume contraction of about -0.7 mL/mol associated with the process almost doubles in higher ionic strength buffer systems. Measurements of samples in phosphate buffer systems are similar and appear to display the same subtle ionic strength dependence. Both the isolation of this photoacoustic signal component and the possible dependence on ionic strength of the thermodynamic parameters derived from its analysis appear analogous to and consistent with prior photoacoustic results monitoring CO photodetachment from the camphor complex of cytochrome P-450. Accordingly, we consider a similar model in which a conformational change results in movement of an exposed charged group or groups towards the interior of the protein, out of contact with solvent, as in the closing of a salt bridge.     

Kinetic and equilibrium studies of the reactions of heme-substituted horse heart myoglobins with oxygen and carbon monoxide.

In order to study the effects of chemical modifications of the vinyl groups of heme on oxygen and carbon monoxide binding to myoglobin, apomyoglobins from horse heart were reconstituted with six different hemins with various side chains. Laser flash photolysis experiments of these reconstituted myoglobins showed that the combination rate constants for oxygen (k') and carbon monoxide (l') were closely related to the electron-attractive properties of the side chains. The k' values obtained in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees were 0.83 (meso-), 2.4 (deutero-), 1.1 (reconstituted proto-), 1.2 (native proto-), 1.5 (2-formyl-4-vinyl-), 1.9 (2-vinyl-4-formyl-), and 2.7 X 10(7) M-1 S-1 (2,4-diformylmyoglobins), and the corresponding l' values were 2.8, 18, 4.8, 5.1, 7.1, 15, and 35 X 10(5) M-1 S-1, respectively. These rate constants tend to increase as the electron-withdrawing power of the side chains increases, indicating that reduced electron density of the iron atom of heme in myoglobin favors the combination reaction for both oxygen and carbon monoxide. Equilibrium constants (L) between carbon monoxide and various myoglobins were also determined by measuring the partition coefficients (M) between oxygen and carbon monoxide for the myoglobins, and were also found to be closely related to the electronic properties (pK3 of porphyrin) of the heme side chains. The equilibrium association constants for carbon monoxide thus obtained increased with a decrease in pK3 value of the porphyrin. This order was completely opposite to the case of the oxygen binding reaction. The dissociation rate constants for oxygen (k) and carbon monoxide (l) were calculated from the equilibrium and the combination rate constants. The dissociation rate constants showed a similar characteristic to the combination rate constants and increased with the increase in electron attractivity of heme side chains. The concomitant increase in both the combination and dissociation rate constants with increase in electronegativity of the iron atom suggests that these reactions have different rate determining steps, although such a reaction process is contradictory to the generally accepted concept that in a reversible reaction, both on and off reactions proceed through the same transition state. In the on reaction sigma bond formation appears to be dominant, while in the off reaction eta bond break-up is more important.     

Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide reduces extracellular glutamate levels in primary rat cerebral cortex cell cultures

The effects of prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 (0.5 mg/kg s.c.), alone or in combination with carbon monoxide, on extracellular glutamate levels in primary rat cerebral cortical neuronal cultures, were investigated. Dam weight gain, pregnancy length and litter size at birth were not affected by prenatal treatment with WIN 55,212-2 and carbon monoxide alone or in combination. Basal and K(+)-evoked extracellular glutamate levels were reduced in cortical cultures from pups born to mothers exposed to WIN 55,212-2 and carbon monoxide alone or in combination compared to cultures from rats born to vehicle-treated mothers. In cultures obtained from rats exposed to vehicle or carbon monoxide alone during gestation, WIN 55,212-2 (0.01-100 nM) increased extracellular glutamate levels, displaying a bell-shaped concentration-response curve. In cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide the WIN 55,212-2 ( 1 nM)-induced increase in extracellular glutamate levels was lower than that observed in cultures from rats born to vehicle-treated mothers and similar at those observed at 10 and 100 nM concentrations. The selective CB1 receptor antagonist SR141716A (10 nM) counteracted the WIN 55,212-2-induced increase in extracellular glutamate levels in cultures exposed to vehicle or carbon monoxide during gestation, but failed to antagonise it in cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide. These findings provide evidence that prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide, alone or in combination, is associated with an impairment in cortical glutamatergic transmission. It could be speculated that such detrimental effects might be involved in the reported deficit in learning and memory associated with prenatal marijuana exposure.     

Kinetics of carbon monoxide binding to singly reduced human methemoglobin

The kinetics of reaction of singly reduced methemoglobin (HbFe3(3+)Fe2+) with carbon monoxide have been investigated by the pulse radiolysis method. The rate constant for carbon monoxide binding to this form of hemoglobin is 4.1 X 10(6) M-1 S-1 at 24 degrees in our solutions. This value compares with existing values for various forms of hemoglobin ranging from 4 X 10(6) to 6.5 X 10(6) M-1 S-1. Addition of inositol hexaphosphate to the solutions results in a lower rate constant for carbon monoxide binding amounting to 1.1 X 10(5) M-1 S-1.     

Kinetics of oxygen binding to ferrous myeloperoxidase

Myeloperoxidase (MPO), which is involved in host defence and inflammation, is a unique peroxidase in having a globin-like standard reduction potential of the ferric/ferrous couple. Intravacuolar and exogenous MPO released from stimulated neutrophils has been shown to exist in the oxyferrous form, called compound III. To investigate the reactivity of ferrous MPO with molecular oxygen, a stopped-flow kinetic analysis was performed. In the absence of dioxygen, ferrous MPO decays to ferric MPO (0.04 s(-1) at pH 8 versus 1.4 s(-1) at pH 5). At pH 7.0 and 25 degrees C, compound III formation (i.e., binding of dioxygen to ferrous MPO) occurs with a rate constant of (1.1+/-0.1) x 10(4)M(-1)s(-1). The rate doubles at pH 5.0 and oxygen binding is reversible. At pH 7.0, the dissociation equilibrium constant of the oxyferrous form is (173+/-12)microM. The rate constant of dioxygen dissociation from compound III is much higher than conversion of compound III to ferric MPO (which is not affected by the oxygen concentration). This allows an efficient transition of compound III to redox intermediates which actually participate in the peroxidase or halogenation cycle of MPO.     

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.     

Circulatory effects and kinetics following acute administration of carbon monoxide in a porcine model

Carbon monoxide is produced in the endothelial cells and has possible vasodilator activity through three different pathways. The aim of this study was to demonstrate circulatory effects after administration of saturated carbon monoxide blood and to describe the pharmacokinetics of carbon monoxide. Six pigs were anesthetized and 150 ml blood was removed. This blood was bubbled with carbon monoxide until the carboxyhemoglobin (COHb) levels were 90-99%. A specific amount of this blood was then injected back to the animal. At predetermined times; arterial and mixed venous blood was drawn and analyzed for carbon monoxide. Systemic and pulmonary vascular resistance index (SVRi and PVRi) were measured and exhaled air was sampled and measured for carbon monoxide. Blood samples were gathered over 300 minutes along with measurements of invasive pressures, heart rate, cardiac output, oxygen saturation (SpO2), Hb, temperature and blood gases. We conclude that this type of exposure to carbon monoxide appears to have little or no effect on general vasomotor tone and, after correcting for basal levels of carbon monoxide, elimination occurs through the lungs as predicted by a single compartment model. The half-life of carbon monoxide was determined to be 60.5 minutes (SEM 4.7).