Oxygen activation by cytochrome P450 monooxygenase

Unlike photosystem II (PSII) that catalyzes formation of the O-O bond, the cytochromes P450 (P450), members of a superfamily of hemoproteins, catalyze the scission of the O-O bond of dioxygen molecules and insert a single oxygen atom into unactivated hydrocarbons through a hydrogen abstraction-oxygen rebound mechanism. Hydroxylation of the unactivated hydrocarbons at physiological temperatures is vital for many cellar processes such as the biosynthesis of many endogenous compounds and the detoxification of xenobiotics in humans and plants. Even though it carries out the opposite of the water splitting reaction, P450 may share similarities to PSII in proton delivery networks, oxygen and water access channels, and consecutive electron transfer processes. In this article, we review recent advances in understanding the molecular mechanisms by which P450 activates dioxygen.     

Quenching of tryptophan fluorescence in human antithrombin III by iodide ion.

Iodide is an efficient quencher of antithrombin III intrinsic tryptophan fluorescence. The quenching pattern indicates that about 60% of the tryptophyl fluorescence originates from exposed residues in the multitryptophan-containing protein. In denaturing media all of the tryptophyls are solvent-exposed. The binding of heparin to antithrombin III influences the number of solvent-exposed tryptophan residues. By studying the dependence of the quenching on pH, information regarding the presence of charged residues adjacent to tryptophyls was obtained.     

Measurement of iodide in urine using the iodide-selective ion electrode

A simple and rapid way to measure the concentration of iodide in urine with an iodide-selective ion electrode was described. Potentiometric equilibrium was attained in less than 5 min, and a linear calibration curve was obtained over the potassium iodide (KI) concentration range of 10(-2) to 10(-6) M. The coefficients of variation ranged from 6.2 to 10.0% within assay, and 5.4 to 14.4% between assays. The serial dilution of 3 urine samples with different concentration of iodide showed good linear correlations passing through zero. In practice, the chloride ions in urine did not cause serious errors in the measurement of iodide at molar ratios of chloride ion to iodide up to 2 X 10(4). A good linear correlation was obtained between iodide concentrations in urine determined by the electrode method and by the conventional chemical method (r = 0.92). A linear correlation was also observed between the iodide concentrations of 24 h collected urine and those of single morning urine (r = 0.91). The normal iodide content in single morning urine specimens from 127 Japanese people was 5.3 to 62.0 X 10(-6) moles/g creatinine.     

Endocannabinoid activation of the TRPV1 ion channel is distinct from activation by capsaicin

Transient receptor potential vanilloid 1 (TRPV1) ion channel serves as the detector for noxious temperature above 42 °C, pungent chemicals like capsaicin, and acidic extracellular pH. This channel has also been shown to function as an ionotropic cannabinoid receptor. Despite the solving of high-resolution three-dimensional structures of TRPV1, how endocannabinoids such as anandamide and N-arachidonoyl dopamine bind to and activate this channel remains largely unknown. Here we employed a combination of patch-clamp recording, site-directed mutagenesis, and molecular docking techniques to investigate how the endocannabinoids structurally bind to and open the TRPV1 ion channel. We found that these endocannabinoid ligands bind to the vanilloid-binding pocket of TRPV1 in the “tail-up, head-down” configuration, similar to capsaicin; however, there is a unique interaction with TRPV1 Y512 residue critical for endocannabinoid activation of TRPV1 channels. These data suggest that a differential structural mechanism is involved in TRPV1 activation by endocannabinoids compared with the classic agonist capsaicin.     

Characteristics of iodide activation and inhibition of oxygen evolution by photosystem II

Oxygen evolution by photosystem II (PSII) is activated by chloride and other monovalent anions. In this study, the effects of iodide on oxygen evolution activity were investigated using PSII-enriched membrane fragments from spinach. In the absence of Cl(-), the dependence of oxygen evolution activity on I(-) concentration showed activation followed by inhibition in both intact PSII and NaCl-washed PSII, which lacked the PsbP and PsbQ subunits. Using a substrate inhibition model, the range of values of the Michaelis constant K(M) in intact PSII (0.5-1.5 mM) was smaller than that in NaCl-washed PSII (1.5-5 mM), whereas values of the inhibition constant K(I) in intact PSII (9-17 mM) were larger than those in NaCl-washed PSII (1-4 mM). Studies of I(-) inhibition of Cl(-)-activated oxygen evolution in intact PSII revealed that I(-) was primarily an uncompetitive inhibitor, with uncompetitive constant K(i)' = 37 mM and Cl(-)-competitive constant K(i) > 200 mM. This result indicated that the activating Cl(-) must be bound for inhibition to take place, which is consistent with the substrate inhibition model for I(-) activation. The S(2) state multiline and g = 4.1 EPR signals in NaCl-washed PSII were examined in the presence of 3 and 25 mM NaI, corresponding to I(-)-activated and I(-)-inhibited conditions, respectively. The two S(2) state signals were observed at both I(-) concentrations, indicating that I(-) substitutes for Cl(-) in formation of the signals and that advancement to the S(2) state was not prevented by high I(-) concentrations. A model is presented that incorporates the results of this study, including the action of both chloride and iodide.     

Oxidation of NADH by chloramines and chloramides and its activation by iodide and by tertiary amines

Irreversible oxidation of reduced nicotinamide nucleotides by neutrophil-derived halogen oxidants (HOCl, chloramines, HOBr, etc.) is likely to be a highly lethal process, because of the essential role of NAD(P)H in important cell functions such as mitochondrial electron transport, and control of the cellular thiol redox state by NADPH-dependent glutathione reductase. Chloramines (chloramine-T, NH(2)Cl, etc.) and N-chloramides (N-chlorinated cyclopeptides) react with NADH to generate the same products as HOCl, i.e., pyridine chlorohydrins, as judged from characteristic changes in the NADH absorption spectrum. Compared with the fast oxidation of NADH by HOCl, k approximately 3 x 10(5) M(-1) s(-1) at pH 7.2, the oxidation by chloramines is about five orders of magnitude slower; that by chloramides is about four orders of magnitude slower. Apparent rate constants for oxidation of NADH by chloramines increase with increasing proton or buffer concentration, consistent with general acid catalysis, but oxidation by chloramides proceeds with pH-independent kinetics. In presence of iodide the oxidation of NADH by chloramines or chloramides is faster by at least two orders of magnitude; this is due to reaction of iodide with the N-halogen to give HOI/I(2), the most reactive and selective oxidant for NADH among HOX species. Quinuclidine derivatives (QN) like 3-chloroquinuclidine and quinine are capable of catalyzing the irreversible degradation of NADH by HOCl and by chloramines; QN(+)Cl, the chain carrier of the catalytic cycle, is even more reactive toward NADH than HOCl/ClO(-) at physiological pH. Oxidation of NADH by NH(2)Br proceeds by fast, but complex, biphasic kinetics. A compilation of rate constants for interactions of reactive halogen species with various substrates is presented and the concept of selective reactivity of N-halogens is discussed.     

Oxygen sensing by ion channels and chemotransduction in single glomus cells

We have monitored cytosolic [Ca2+] and dopamine release in intact fura- 2-loaded glomus cells with microfluoroimetry and a polarized carbon fiber electrode. Exposure to low PO2 produced a rise of cytosolic [Ca2+] with two distinguishable phases: an initial period (with PO2 values between 150 and approximately 70 mm Hg) during which the increase of [Ca2+] is very small and never exceeds 150-200 nM, and a second phase (with PO2 below approximately 70 mm Hg) characterized by a sharp rise of cytosolic [Ca2+]. Secretion occurs once cytosolic [Ca2+] reaches a threshold value of 180 +/- 43 nM. The results demonstrate a characteristic relationship between PO2 and transmitter secretion at the cellular level that is comparable with the relation described for the input (O2 tension)output (afferent neural discharges) variables in the carotid body. Thus, the properties of single glomus cells can explain the sensory functions of the entire organ. In whole-cell, patch- clamped cells, we have found that in addition to O2-sensitive K+ channels, there are Ca2+ channels whose activity is also regulated by PO2. Ca2+ channel activity is inhibited by hpoxia, although in a strongly voltage-dependent manner. The average hypoxic inhibition of the calcium current in 30% +/- 10% at -20 mV but only 2% +/- 2% at +30 mV. The differential inhibition of K+ and Ca2+ channels by hypoxia helps to explain why the secretory response of the cells is displaced toward PO2 values (below approximately 70 mm Hg) within the range of those normally existing in arterial blood. These data provide a conceptual framework for understanding the cellular mechanisms of O2 chemotransduction in the carotid body.     

Oxygen requirement for cupric ion induced hemolysis

The Cu(II) induced lysis of rabbit erythrocytes occurs in two phases. First there is a lag period of 60 min where few cells lyse, followed by a period of rapid lysis where most of the remaining cells undergo hemolysis. Lysis is effectively inhibited if the incubation is conducted in nitrogen or if the erythrocytes are pre-saturated with carbon monoxide. These results suggest that oxygenated hemoglobin is necessary for lysis. It has been reported that copper binds to oxyhemoglobin and releases superoxide ion. This additional oxidative stress can initiate lipid peroxidation which leads to cell hemolysis.     

Oxygen activation during oxidation of methoxyhydroquinones by laccase from Pleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH(2)) and 2, 6-dimethoxy-1,4-benzohydroquinone (DBQH(2)) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH(2) more efficiently than it oxidized MBQH(2); both the affinity and maximal velocity of oxidation were higher for DBQH(2) than for MBQH(2). Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q(*-) + O(2) <--> Q + O(2)(*-)). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O(2)(*-) on quinone formation rates. Then, the production of H(2)O(2) in laccase reactions, as a consequence of O(2)(*-) dismutation, confirmed that semiquinones autoxidized. The highest H(2)O(2) levels were obtained with DBQH(2), indicating that DBQ(*-) autoxidized to a greater extent than did MBQ(*-). Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O(2)(*-) consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O(2)(*-), besides undergoing dismutation, reacts with Mn(2+), Fe(3+), and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 microM concentrations of MBQ(*-) and DBQ(*-) from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ(*-) autoxidation rose to 13.8% and that of DBQ(*-) increased to 39.9%.     

Dynamic iodide trapping by tumor cells expressing the thyroidal sodium iodide symporter

The thyroidal sodium iodide symporter (NIS) in combination with various radioactive isotopes has shown promise as a therapeutic gene in various tumor models. Therapy depends on adequate retention of the isotope in the tumor. We hypothesized that in the absence of iodide organification, isotope trapping is a dynamic process either due to slow efflux or re-uptake of the isotope by cells expressing NIS. Iodide efflux is slower in ARH-77 and K-562 cells expressing NIS compared to a thyroid cell line. Isotope retention half times varied linearly with the number of cells expressing NIS. With sufficient NIS expression, iodide efflux is a zero-order process. Efflux kinetics in the presence or absence of perchlorate also supports the hypothesis that iodide re-uptake occurs and contributes to the retention of the isotope in tumor cells. Iodide organification was insignificant. In vivo studies in tumors composed of mixed cell populations confirmed these observations.     

Oxygen activation by cytochrome oxidase: a new spectral intermediate observed by flow-flash

The reaction of cytochrome-c-oxidase with O2 has been reinvestigated at 2 degrees C by a flow laser pulsed method. The experiments indicate that a new spectral intermediate, populated with a rate constant of approximately 5000/s, is observed in a wavelength range in which heme a is the only chromophore (445 and 430 nm). The results are interpreted with reference to previous data obtained in the near infrared region, and it is suggested that breakage of the (enzyme bound) dioxygen bond is associated to a spectral perturbation of the cytochrome a3-CuB binuclear center.     

6-aminohexanoate and chloride ion in the activation by urokinase of porcine plasminogens

The rate of activation by urokinase of porcine plasminogen is accelerated by 6-aminohexanoate, although the maximally enhanced rate is 10-fold less than that of human plasminogen without the amino acid. 6-Aminohexanoate facilitates only activation of native porcine plasminogen (asp-plasminogen), but has no effect on activation of des-kringle1-4-plasminogen. Sodium chloride, on the other hand, inhibits activation by urokinase of both porcine asp-plasminogen and des-kringle1-4-plasminogen. It is concluded that 6-aminohexanoate exerts its effect via kringle1-4 domains of plasminogen, whereas Cl- acts, at least in part, through effects on the kringle5 or proteinase domains.