Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide.

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Mechanism of oxidation of carbon monoxide by bacteria

Double label experiments were performed employing ¹³CO and either H₂¹⁸O or ¹⁸O₂ in the presence of a CO utilizing bacterium. CO₂ generated was trapped and $\text{m}\text{e}$ ratios $\text{47}\text{45}$ showed that the second oxygen atom in the oxidation of CO to CO₂ by this bacterium comes neither from O₂ nor H₂O.     

Nitrate-dependent anaerobic carbon monoxide oxidation by aerobic CO-oxidizing bacteria

Two dissimilatory nitrate-reducing (Burkholderia xenovorans LB400 and Xanthobacter sp. str. COX) and two denitrifying isolates (Stappia aggregata IAM 12614 and Bradyrhizobium sp. str. CPP), previously characterized as aerobic CO oxidizers, consumed CO at ecologically relevant levels (<100 ppm) under anaerobic conditions in the presence, but not absence, of nitrate. None of the isolates were able to grow anaerobically with CO as a carbon or energy source, however, and nitrate-dependent anaerobic CO oxidation was inhibited by headspace concentrations >100-1000 ppm. Surface soils collected from temperate, subtropical and tropical forests also oxidized CO under anaerobic conditions with no lag. The observed activity was 25-60% less than aerobic CO oxidation rates, and did not appear to depend on nitrate. Chloroform inhibited anaerobic but not aerobic activity, which suggested that acetogenic bacteria may have played a significant role in forest soil anaerobic CO uptake.     

The dissociation of carbon monoxide from hemoglobin intermediate

To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (alpha beta-CO)2 and (alpha-CO beta)2, and the di- and monoliganded asymmetrical species (alpha-CO beta-CO)(alpha beta), (alpha-CO beta)(alpha beta-CO), (alpha beta-CO) (alpha beta), and (alpha-CO beta)(alpha beta). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at -25 degrees C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the alpha chains dissociate CO faster than the beta chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species.     

Attributes of atmospheric carbon monoxide oxidation by Maine forest soils

CO, one of the most important trace gases, regulates tropospheric methane, hydroxyl radical, and ozone contents. Ten to 25% of the estimated global CO flux may be consumed by soils annually. Depth profiles for (14)CO oxidation and CO concentration indicated that CO oxidation occurred primarily in surface soils and that photooxidation of soil organic matter did not necessarily contribute significantly to CO fluxes. Kinetic analyses revealed that the apparent K(m) was about 18 nM (17 ppm) and the V(max) was 6.9 micromol g (fresh weight)(-1) h(-1); the apparent K(m) was similar to the apparent K(m) for atmospheric methane consumption, but the V(max) was more than 100 times higher. Atmospheric CO oxidation responded sensitively to soil water regimes; decreases in water content in initially saturated soils resulted in increased uptake, and optimum uptake occurred at water contents of 30 to 60%. However, extended drying led to decreased uptake and net CO production. Rewetting could restore CO uptake, albeit with a pronounced hysteresis. The responses to changing temperatures indicated that the optimum temperature for net uptake was between 20 and 25 degrees C and that there was a transition to net production at temperatures above 30 degrees C. The responses to methyl fluoride and acetylene indicated that populations other than ammonia oxidizers and methanotrophs must be involved in forest soils. The response to acetylene was notable, since the strong initial inhibition was reversed after 12 h of incubation; in contrast, methyl fluoride did not have an inhibitory effect. Ammonium did not inhibit CO uptake; the level of nitrite inhibition was initially substantial, but nitrite inhibition was reversible over time. Nitrite inhibition appeared to occur through indirect effects based on abiological formation of NO.     

Anaerobic transformation of carbon monoxide by microbial communities of Kamchatka hot springs

Carbon monoxide (CO) is one of the common gaseous compounds found in hot volcanic environments. It is known to serve as the growth substrate for a number of thermophilic prokaryotes, both aerobic and anaerobic. The goal of this work was to study the process of anaerobic transformation of CO by microbial communities inhabiting natural thermal environments: hot springs of Uzon Caldera, Kamchatka. The anaerobic microbial community of Treshchinny Spring (80°C, pH 6.5) was found to exhibit two peaks of affinity for CO (K _S1 = 54 nM and K _S2 = 1 μM). The actual rate of anaerobic CO transformation by the microbial community of this spring, calculated after obtaining the concentration dependence curve and extrapolated to the natural concentration of CO dissolved in the hot spring water (20 nM), was found to be 120 μmol l⁻¹ of sediment day⁻¹. In all the hot springs studied, more than 90% of the carbon of ¹⁴CO upon anaerobic incubation was recovered as ¹⁴CO₂. From 1 to 5% of ¹⁴CO was transformed to volatile fatty acids (VFA). The number of microorganisms capable of anaerobic CO oxidation determined by dilution-to-extinction method reached 10⁶ cells ml⁻¹ of sediment. CO-transforming anaerobic thermophilic microorganisms isolated from the springs under study exhibited hydrogenogenic type of CO oxidation and belonged to the bacterial genera Carboxydocella and Dictyoglomus. These data suggest a significant role of hydrogenogenic carboxydotrophic prokaryotes in anaerobic CO transformation in Uzon Caldera hot springs.     

Peroxynitrite-mediated oxidation of ferrous carbonylated myoglobin is limited by carbon monoxide dissociation

Peroxynitrite-mediated oxidation of ferrous nitrosylated myoglobin (Mb(II)-NO) involves the transient ferric nitrosylated species (Mb(III)-NO), followed by NO dissociation and formation of ferric myoglobin (Mb(III)). In contrast, peroxynitrite-mediated oxidation of ferrous oxygenated myoglobin (Mb(II)-O₂) involves the transient ferrous deoxygenated and ferryl derivatives (Mb(II) and Mb(IV)O, respectively), followed by Mb(III) formation. Here, kinetics of peroxynitrite-mediated oxidation of ferrous carbonylated horse heart myoglobin (Mb(II)-CO) is reported. Values of the first-order rate constant for peroxynitrite-mediated oxidation of Mb(II)-CO (i.e., for Mb(III) formation) and of the first-order rate constant for CO dissociation from Mb(II)-CO (i.e., for Mb(II) formation) are h = (1.2 ± 0.2) × 10⁻² s⁻¹ and k_off(CO) = (1.4 ± 0.2) × 10⁻² s⁻¹, respectively, at pH 7.2 and 20.0 °C. The coincidence of values of h and k_off(CO) indicates that CO dissociation represents the rate limiting step of peroxynitrite-mediated oxidation of Mb(II)-CO.     

Inhibition of dimethylnitrosamine-induced mutagenesis in Arabidopsis thaliana by diethyldithiocarbamate and carbon monoxide

Diethyldithiocarbamate and carbon monoxide markedly inhibited the frequency of embryonic and chlorophyll mutations induced by the metabolism-requiring mutagen dimethylnitrosamine in the higher plant Arabidopsis thaliana. In contrast, the monoamine oxidase substrates, tryptamine, benzylamine and 2-phenylethylamine, had no such effect. The mutagenicity of a direct-acting mutagen, N-methyl-N-nitrosourea, was not altered by these inhibitors or substrates.     

Regulation of anaerobic carbon monoxide oxidation activity in Rhodocyclus gelatinosus

Rhodocyclus gelatinosus grows anaerobically at the expense of carbon monoxide (CO). The CO-oxidation system was substrate-induced and in CO/light, cells grew at an exponential rate with ever increasing amounts of CO:MV oxidoreductase activity (the measure of CO oxidation). Once strain 1 reached a high cell density, the concentration of CO became limiting and gas oxidation activity suddenly decreased. Cell growth continued unaffected. To help explain this, it appeared that strain 1 variably used both CO oxidation and photometabolism to support growth in CO/light. Light intensity determined the upper limit of amounts of CO:MV oxidoreductase in a culture, while intermediate amounts were regulated by CO concentration. Thus, in darkness, cells produced the maximum CO oxidation activity, whereas in growth-saturating light, the minimum limit occurred. The lower the levels of CO:MV oxidoreductase in cells, the greater the content of bacteriochlorophyll. In this manner, strain 1 grew with a generation time of 6.7 independent of light intensity.     

Membrane association of the carbon monoxide oxidation system in Rhodopseudomonas gelatinosa.

A comparison of the distribution of CO oxidation activity between soluble and particulate protein fractions obtained after disruption of CO-grown Rhodopseudomonas gelatinosa 1 by French pressure cell breakage and osmotic lysis of spheroplasts suggested that, in situ, the enzyme complex was associated with the cell membrane. An improved, strictly anaerobic method is given for spectrophotometric measurement of CO oxidation activity based on the carbon monoxide:methyl viologen oxidoreductase reaction.     

Inhibition of the Fenton reaction by nitrogen monoxide

The toxicity of iron is believed to originate from the Fenton reaction which produces the hydroxyl radical and/or oxoiron(2+). The effect of nitrogen monoxide on the kinetics of the reaction of iron(II) bound to citrate, ethylenediamine-N,N′-diacetate (edda), ethylenediamine-N,N,N′,N′-tetraacetate (edta), (N-hydroxyethyl)amine-N,N′,N′-triacetate (hedta), and nitrilotriacetate (nta) with hydrogen peroxide was studied by stopped-flow spectrophotometry. Nitrogen monoxide inhibits the Fenton reaction to a large extent. For instance, hydrogen peroxide oxidizes iron(II) citrate with a rate constant of 5.8×10³ M⁻¹ s⁻¹, but in the presence of nitrogen monoxide, the rate constant is 2.9×10² M⁻¹ s⁻¹ . Similar to hydrogen peroxide, the reaction of tert-butyl hydroperoxide with iron(II) complexes is also efficiently inhibited by nitrogen monoxide. Generally, nitrogen monoxide binds rapidly to a coordination site of iron(II) occupied by water. The rate of oxidation is influenced by the rate of dissociation of the nitrogen monoxide from iron(II). Electronic Supplementary Material Supplementary material is available for this article at     

Membrane topography of anaerobic carbon monoxide oxidation in Rhodocyclus gelatinosus.

Rhodocyclus gelatinosus 1 grows anaerobically in the dark at the expense of carbon monoxide. Topographical studies with methyl viologen as the membrane probe indicated that CO oxidation and H2 production sites were on the cytoplasmic side of the cell membrane. Membrane-associated hydrogen gas production appeared to be a unidirectional reaction. In the dark, strain 1 whole cells oxidized CO and incorporated about 306 pmol of 32Pi into ATP per min per mg of protein. With CO as the sole energy-yielding substrate, cells grew with a low growth yield coefficient of 3.7 g (dry weight) of cells per mg of CO oxidized.     

Metabolism of carbon monoxide by Rhodopseudomonas gelatinosa: cell growth and properties of the oxidation system.

Rhodopseudomonas gelatinosa 1 grew as an anaerobic facultative methylotroph with carbon monoxide as the sole carbon and energy source. Carbon from CO was assimilated into cell material via the ribulose 1,5-bisphosphate carboxylase cycle. The CO oxidation system in R. gelatinosa was induced during growth with the gas substrate. Light-grown cells did not oxidize CO. Surprisingly, when strain 1 cells grown in the dark with CO were transferred to growth with both CO and light, they continued to use CO and then photometabolized after the CO gas flow was stopped. This change in the energy-yielding substrate resulted in a diauxic growth response. The use of CO in preference to light energy forms the basis of a system in the cells that controls photosynthetic differentiation. CO oxidation was assayed as CO-methyl viologen oxidoreductase. Methyl viologen reduction only occurred with CO; the dye was not reduced with other C1 compounds. In vitro methyl viologen was reduced best at 24 degrees C and at pH values above 8.5. Whole cells exhibited a Km of 12.5 microM for CO and a Vmax of 3,800 nmol of CO oxidized per mg of protein per min. This was a low-potential oxidation reaction that readily reduced the viologen dye triquat (1,1'-trimethylene-2,2'-dipyridilium dibromide) (E degrees' = -548 mV).     

Observation of a stable intermediate form in the reaction of human hemoglobin with carbon monoxide

The reaction of human hemoglobin with carbon monoxide has been investigated near the equilibrium isosbestic wavelength (i.e. 426 nm). As previously reported by others [Gray, R.D. & Gibson, Q. H. (1971) J. Biol. Chem. 246, 5176-5178], in the presence of 0.1 M phosphate pH 7.0 a rise-and-fall kinetic pattern can be observed at this wavelength, which indicates the presence of at least one spectroscopically detectable intermediate species. In this paper we demonstrate that (a) the intermediate species is thermodynamically stable; (b) both phases refer to bimolecular processes; (c) only the initial fast phase is observed when deoxyhemoglobin is reacted with substoichiometric amounts of CO (i.e. final [CO]/[heme] less than or equal to 0.5); (d) only the second slow phase is observed when hemoglobin that is partially saturated with CO (Y less than or equal to 0.5) is reacted with saturating CO concentrations; (e) the CO dissociation rate constant measured on the intermediate formed after a partial CO saturation at a final Y approximately 0.4 has a value similar to that observed starting from the fully liganded form. These results can be accounted for by a two-state allosteric model [Monod, J., Wyman, J. & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118] under the assumption that either (a) 426 nm is an isosbestic wavelength for the T0-R spectral changes but not for the T0-T liganded reaction; or (b) a functional heterogeneity of the two types of subunits is present in the T state and at this wavelength this feature is spectroscopically detectable.