One protein, two enzymes revisited: a structural entropy switch interconverts the two isoforms of acireductone dioxygenase

Acireductone dioxygenase (ARD) catalyzes different reactions between O2 and 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (acireductone) depending upon the metal bound in the active site. Ni2+ -ARD cleaves acireductone to formate, CO and methylthiopropionate. If Fe2+ is bound (ARD'), the same substrates yield methylthioketobutyrate and formate. The two forms differ in structure, and are chromatographically separable. Paramagnetism of Fe2+ renders the active site of ARD' inaccessible to standard NMR methods. The structure of ARD' has been determined using Fe2+ binding parameters determined by X-ray absorption spectroscopy and NMR restraints from H98S ARD, a metal-free diamagnetic protein that is isostructural with ARD'. ARD' retains the beta-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the beta-sandwich and decreases order at the other upon interconversion of ARD and ARD', causing loss of the C-terminal helix in ARD' and rearrangements of residues involved in substrate orientation in the active site.     

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.     

Theoretical study of the decomposition of ethyl and ethyl 3-phenyl glycidate

The mechanism of the decomposition of ethyl and ethyl 3-phenyl glycidate in gas phase was studied by density functional theory (DFT) and MP2 methods. A proposed mechanism for the reaction indicates that the ethyl side of the ester is eliminated as ethylene through a concerted six-membered cyclic transition state, and the unstable intermediate glycidic acid decarboxylates rapidly to give the corresponding aldehyde. Two possible pathways for glycidic acid decarboxylation were studied: one via a five-membered cyclic transition state, and the other via a four-membered cyclic transition state. The results of the calculations indicate that the decarboxylation reaction occurs via a mechanism with five-membered cyclic transition state.

The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates.

Electron spin resonance spectra are observed during the enzymatic reduction of many nitrophenyl derivatives by rat hepatic microsomes or mitochondria. The spectra indicate that nitroaromatic anion radicals are present and are freely rotating in aqueous solution at a steady-state concentration of 0.1-6 muM. The rate of formation of p-nitrobenzoate (NBZO) dianion radical in microsomal incubates is consistent with the radical being an obligate intermediate in the reduction of NBZO to p-aminobenzoic acid. A model system consisting of NBZO, NADPH, and FMN, but no heme-containing compounds, also reduced NBZO to the NBZO dianion free radical. The steady-state concentration of the anion radicals in microsomal systems is not altered by CO. This observation, together with the results from the model system, suggests that the formation of nitroaromatic anion radicals is mediated through a flavine and not cytochrome P-450. The oxidation of the anion radical intermediate by O2 to the parent nitro compound is proposed to account for the well-known O2 inhibition of microsomal nitroreductase.     

Mechanism of electron transfer to coordinated dioxygen of oxyhemoglobins to yield peroxide and methemoglobin. Protein control of electron donation by aquopentacyanoferrate(II)

Reactions of human oxyhemoglobin A with iron(II) compounds have been investigated. Human oxyhemoglobin (HbO2) reacts with aquopentacyanoferrate(II), Fe(II)(CN)5H2O3-, to yield hydrogen peroxide, aquomethemoglobin and Fe(III)(CN)5H2O2-. The reaction follows a second order rate law, first order in the pentacyanide and in HbO2. Since reaction rates are lower in the presence of catalase, the H2O2 produced must promote metHb formation in reactions independent of pentacyanide. Changes in concentrations of effectors (e.g. H+, inositol hexaphosphate, Cl-, and Zn2+), alkylation of beta-93 cysteine with N-ethylmaleimide, and substitution at distal histidine (as in Hb Zurich with beta-63 His----Arg) in each case can markedly affect pentacyanide reaction rates demonstrating a fine control of rates by protein structure. Hexacyanoferrate(II) (ferrocyanide) reacts with HbO2 to produce cyano-metHb as well as aquo-metHb but the reaction with the hexacyanide is much slower than with the aquopentacyanide. Iron(II) EDTA converts HbO2 to deoxy-Hb with no evidence for formation of metHb as an intermediate. These findings support a mechanism in which the pentacyanide anion reacts directly with coordinated dioxygen. One-electron transfers to O2 from both pentacyanide iron(II) and heme iron(II) result in the formation of a mu-peroxo intermediate, HbFe(III)-O-O-Fe(III) (CN)5(3-). Hydrolysis of this intermediate yields metHb . H2O, H2O2, and FeIII(CN)5H2O2-. The reaction of HbO2 with Fe(CN)6(4-) must follow an outer sphere electron transfer mechanism. However, the very slow rate that is seen with Fe(CN)6(4-) could arise entirely from the pentacyanide produced from loss of one cyanide ligand from the hexacyanide. Fe(II)EDTA reacts rapidly with free O2 in solution but can not interact directly with the heme-bound O2 of HbAO2. The dynamic character of the O2 binding sites apparently permits access of the Fe2+ of the pentacyanide to coordinated dioxygen but the protein structure is not sufficiently flexible to allow the larger Fe2+EDTA molecule to react with bound O2. It is necessary for maintenance of the oxygen transport function of the red cell for reductants such as the methemoglobin reductase system, glutathione, and ascorbate to be able to reduce metHb to deoxy-Hb. It is also important for these reductants to be unable to donate an electron to HbO2 to yield H2O2 and metHb. Thus, a mechanistic requirement for the delivery of one-electron directly to the dioxygen ligand, if peroxide is to be produced, enables the protein to protect the oxygenated species from those electron donors normally present in the cell by denying these reductants steric access to coordinated O2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carbodiimide-intermediated esterification of the inorganic phosphates and the effect of tertiary amine base.

Detailed analysis of appropriate 31P nuclear-magnetic-resonance spectra shows that under the usual laboratory conditions, carbodiimide-induced condensation of orthophosphoric acid in a number of solvents leads to condensation only slightly beyond the metaphosphate composition in the presence of strong tertiary amines; whereas in the absence of amine, the condensation proceeds into the ultraphosphate region about halfway between the metaphosphate and phosphoric anhydride compositions. With amine, the principal product consists of the cyclic trimetaphosphate anion, with one of the nonbridging oxygen atoms substituted by the urea resulting from hydration of the carboiimide, i.e., (O2-) P-O-P(O2-) -O-P(O) [N[CH(CH3)2] see article [C(O)NHCH(CH3)2]] for the condensation with diisopropylcarbodiimide. Without amine, the major product is the 1,5-mu-oxotetrametaphosphate anion see article. The well-known carbodiimide-mediated phosphorylation of alcohols with orthophosphoric acid is shown to be directly attributable to the high reactivity of the phosphate branch groups of the carbodiimide-generated ultraphosphates.     

Synthetic, spectral, thermal and antimicrobial studies on some mixed 1,3-dithia-2-stannacyclopentane derivatives with dialkyldithiocarbamates

1,3-dithia-2-stannacyclopentane derivatives with dialkyldithiocarbamates of the types SCH(2)CH(2)SSn[S(2)CNR(2)]Cl (I) and SCH(2)CH(2)SSn[S(2)CNR(2)](2) (II) (where R = CH(3), C(2)H(5) and -CH(2)-CH(2)-) have been synthesized by the reaction of 2,2-dichloro-1,3-dithia-2-stannacyclopentane and sodium/ammonium salts of dialkyldithiocarbamates in 1:1 and 1:2 molar ratios, respectively, in anhydrous benzene. These newly synthesized derivatives have been characterized by elemental analyses (C, H, N, S and Sn), thermal [thermogravimetry (TG) and differential thermal analyses (DTA)] as well as spectral [UV, IR and multinuclear NMR ((1)H, (13)C and (119)Sn)] studies. The monodentate behaviour of the dialkyldithiocarbamate ligands was confirmed by IR and (119)Sn NMR spectral data and distorted tetrahedral structures have been suggested for both type (I) and (II) compounds. The free ligands and their tin complexes have also been screened for their antibacterial and antifungal activities. These results made it desirable to delineate a comparison between free ligands and their tin complexes. These exhibit higher antibacterial effect than some of the previously investigated antibiotics.     

Interaction of lead(II) with beta-cyclodextrin in alkaline solutions

Polarographic and UV-spectrophotometric investigations of Pb(II) complex formation with beta-cyclodextrin have showed that the complexation of Pb(II) ions begins at pH >10. The formation of lead(II) 1:1 complex with the beta-cyclodextrin anion was observed at pH 10-11.5. The logarithm of the stability constant of this complex compound is 15.9+/-0.3 (20 degrees C, ionic strength 1.0), and the molar extinction coefficient value is ca. 5500 (lambda(max)=260 nm). With further increase in solution pH the Pb-beta-cyclodextrin complex decomposes and converts to Pb(OH)(2) or Pb(OH)(3)(-) hydroxy-complexes. This process occurs with a decrease in Pb(II) complexation degree. The latter result could be explained by a decrease in the beta-cyclodextrin anion activity. Neither Pb(OH)(2) nor Pb(OH)(3)(-) encapsulation into beta-CD cavity was observed.     

Effects of O2 and CH4 on presence and activity of the indigenous methanotrophic community in rice field soil

The activity and distribution of methanotrophs in soil depend on the availability of CH4 and O2. Therefore, we investigated the activity and structure of the methanotrophic community in rice field soil under four factorial combinations of high and low CH4 and O2 concentrations. The methanotrophic population structure was resolved by denaturant gradient gel electrophoresis (DGGE) with different PCR primer sets targeting the 16S rRNA gene, and two functional genes coding for key enzymes in methanotrophs, i.e. the particulate methane monooxygenase (pmoA) and the methanol dehydrogenase (mxaF). Changes in the biomass of type I and II methanotrophic bacteria in the rice soil were determined by analysis of phospholipid-ester-linked fatty acid (PLFA) biomarkers. The relative contribution of type I and II methanotrophs to the measured methane oxidation activity was determined by labelling of soil samples with 14CH4 followed by analysis of [14C]-PLFAs. CH4 oxidation was repressed by high O2 (20.5%), and enhanced by low O2 (1%). Depending on the CH4 and O2 mixing ratios, different methanotrophic communities developed with a higher diversity at low than at high CH4 concentration as revealed by PCR-DGGE. However, a prevalence of type I or II populations was not detected. The [14C]-PLFA fingerprints, on the other hand, revealed that CH4 oxidation activity was dominated by type I methanotrophs in incubations with low CH4 mixing ratios (1000 p.p.m.v.) and during initiation of CH4 consumption regardless of O2 or CH4 mixing ratio. At high methane mixing ratios (10 000 p.p.m.v.), type I and II methanotrophs contributed equally to the measured CH4 metabolism. Collectively, type I methanotrophs responded fast and with pronounced shifts in population structure and dominated the activity under all four gas mixtures. Type II methanotrophs, on the other hand, although apparently more abundant, always present and showing a largely stable population structure, became active later and contributed to CH4 oxidation activity mainly under high CH4 mixing ratios.     

Role of mitochondria in ultraviolet-induced oxidative stress

The biological effects of ultraviolet radiation (UV), such as DNA damage, mutagenesis, cellular aging, and carcinogenesis, are in part mediated by reactive oxygen species (ROS). The major intracellular ROS intermediate is hydrogen peroxide, which is synthesized from superoxide anion ((*)O(2)(-)) and further metabolized into the highly reactive hydroxyl radical. In this study, we examined the involvement of mitochondria in the UV-induced H(2)O(2) accumulation in a keratinocyte cell line HaCaT. Respiratory chain blockers (cyanide-p-trifluoromethoxy-phenylhydrazone and oligomycin) and the complex II inhibitor (theonyltrifluoroacetone) prevented H(2)O(2) accumulation after UV. Antimycin A that inhibits electron flow from mitochondrial complex III to complex IV increased the UV-induced H(2)O(2) synthesis. The same effect was seen after incubation with rotenone, which blocks electron flow from NADH-reductase (complex I) to ubiquinone. UV irradiation did not affect mitochondrial transmembrane potential (DeltaPsi(m)). These data indicate that UV-induced ROS are produced at complex III via complex II (succinate-Q-reductase).     

Crystal packing and hydrogen bonding in platinum(II) nucleotide complexes: X-ray crystal structure of [Pt(MeSCH(2)CH(2)SMe)(5'-GMP-N7)(2)].6H(2)O

We have synthesised the complex [Pt(CH(3)SCH(2)CH(2)SCH(3))(5'-GMP-N7)(2)].6H(2)O (1), where 5'-GMP is 5'-guanosine monophosphate, and determined its X-ray crystal structure. Pt(II) adopts a square-planar geometry in which the bases are coordinated head-to-tail (HT) in the Delta configuration. The nucleotide conformation in this complex is almost identical to that in the previously reported complex [Pt(en)(5'-GMP-N7)(2)].9H(2)O (2), in which there is outer sphere macrochelation via intramolecular H-bonding between the monoanionic phosphate groups and the coordinated ethylenediamine (en) NH. It is therefore apparent that intermolecular interactions rather than intramolecular H-bonding determines the orientation of the sugar-phosphate side-chain in these Pt(II) bisnucleotide complexes in the solid state.     

Comparative sensitivity of 2 carboxylesterases from the reindeer liver to various inhibitors

The sensitivity to the inhibitor of two forms of reindeer liver carboxylesterases differing in electrophoretic mobility and conventionally termed as "slow" and "fast" forms were investigated. The rate constants for the interaction of organophosphorous irreversible inhibitors--diisopropylfluorophosphate (DPP) and two methylthiophosphonic acid thioesters--C5H11O(CH3)P(O)S(CH2)SCH2C(O)OCH3 (Sh-205) and, C8H17O(CH3)P(O)S(CH2)SCH2C(O)OCH2 (Sh-207)--with the "fast" form are hundreds of times as high as those with the "slow" one. The rate constants for irreversible carbamate inhibitor interaction byehone with both carboxylesterase forms were approximately equal to 1.2 X 10(3) M-1 X min-1 and 2.0 X 10(3) M-1 X min-1, respectively. The reversible inhibitors potassium benzylate and kathapin also inhibited the "fast" carboxylesterase form in the indophenylacetate (IPA) hydrolysis reaction (770 and 1700-fold, respectively). On the contrary, N-methylpiperidinyl ester of benzyl acid inhibited the "slow" form three times stronger. Carbophos reversibly inhibited IPA hydrolysis in the presence of both enzyme forms, but the carboxyester carbophos group was hydrolyzed at a measurable speed only by the "slow" form.     

Metal chelates of 2-hydroxy-4-methylthiobutanoic acid in animal feeding: preliminary investigations on stability and bioavailability

The alpha-hydroxyacid 2-hydroxy-4-methylthiobutanoic acid (the so-called methionine hydroxy-analogue, MHA), largely used in animal nutrition as a source of methionine, forms stable metal chelates with divalent metals of formula [[CH(3)SCH(2)CH(2)CH(OH)COO](2)M].ZnH(2)O. Protonation and zinc(II) complex formation constants have been determined by pH-metry at 25 degrees C; the ternary system Zn(2+)/MHA/glycine was also studied by pH-metry and the formation constant of the species [ZnLA] was determined [log beta=6.57(11)]. Experiments in vitro with human intestinal CACO-2 cells indicated that the MHA/Fe chelate was taken up by the cells without any apparent toxic effect.     

Transfer of chirality by dithiophosphate ligands and chiral discrimination in the stereoselective formation of square-planar Ni(II) complexes

In the formation reaction of Ni(2+) with the chiral racemic ligand, (R)(R)bdtp(-)/(S)(S)bdtp(-), bdtp(-) = [SSPOCH)CH(3))CH(CH(3))O](-), cyclo- O,O'-[1,2-dimethylethylene] dithiophosphato ion, the meso-complex Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)-bdtp] is stereoselectively produced. The meso-complex was compared with the enantiopure crystals of (+)(589)Ni[(R)(R)(lambda)bdtp](2) or (-)(589)Ni[(S)(S)(delta)bdtp](2), as well as racemic crystals, rac-(+/-)Ni[bdtp](2), which were prepared from the solution containing the two enantiomers in a 1:1 ratio. Dissociation constants in solutions indicate different stability of the meso and enantiopure complexes depending on the solvent, whereas a more efficient crystal packing, weak H-bonding, and nonbonding interactions contribute to stabilization of the meso-species over the racemic one. Molecular structures show that the outer five-membered ligand ring adopts the half-chair conformation C(2) with either the lambda or the delta chirality and the methyl groups are in equatorial (e) positions. Enantiopure ligands of (+)(589)Ni[(R)(R)(lambda)bdtp](2) and (-)(589)Ni[(S)(S)(delta)bdtp](2) induce chirality into the symmetric SSNiSS chromophore with slightly helical distortion. Thus, their CD spectra exhibit weak negative or positive Cotton effects at 662 nm. CD spectra in L(+)- and D(-)diethyltartrate of the meso-complex and racemic crystal, rac-(+/-)Ni[bdtp](2), exhibit different weak Cotton effects of opposite sign. Complexes dissociate in methanol; rac-(+/-)Ni[bdtp](2) in methanol undergoes a crystallization-induced second-order asymmetric transformation which finally yields crystals of the meso-Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)bdtp] complex.     

Reaction of Co(II)bleomycin with dioxygen.

The reaction of Co(II)bleomycin with dioxygen has been investigated. Dioxygen binds to the Co(II) complex within the time of mixing according to electron spin resonance and uv-visible spectroscopy and dioxygen analysis. Then, two dioxygenated cobalt centers react, releasing 1 mol of O2 and forming an intermediate characterized by a few highly shifted 1H NMR resonances and loss of the ESR spectrum. This is thought to be a dioxygen-bridged dimer of cobalt bleomycin molecules. Time-dependent absorbance and dioxygen measurements yield the same second order rate constant for this step of the reaction. According to uv-visible and NMR spectral analysis, the intermediate decays into diamagnetic products in a first order rate process. High performance liquid chromatography and 1H NMR studies demonstrate that the product contains two bleomycin species of equal concentration. One component is Co(III)bleomycin, designated Form II. The other is the peroxide adduct of Co(III)bleomycin, Form I, as determined by direct determination of hydrogen peroxide, which is slowly released from the product at low pH. In contrast, hydrogen peroxide is readily detected during the reaction of Co(II)Blm with O2. In isolation, Form I is unstable at pH 7 and is converted within 24 h into a mixture of Form I and Form II.     

Nonlinear "hydrophobicity-antiesterase activity" model for various types of organophosphorous compounds

The dependence of antiesteratic activity on the structure of insecticides (RO)2P(O)SCH(COOEt)SP(O)(OR)2 (I) and (RO)2P(O)SCH(COOEt)OP(S)(OR)2 (II) was examined. Nonlinear regression equations (parabolic and bilinear) "hydrophobicity-antiesteratic activity" were derived. Basing on the studies of the relationships between hydrophobicity and individual constants, the detailed mechanisms were proposed for the interaction of type (I) and (II) compounds with the esterase active centers. The mechanisms implicate different kinds of sorbtion for compounds of type I and II. Applicability of bilinear models, similar to that of Kubinyi type, for analyzing the structure-antienzyme activity dependences was demonstrated. Thus, several equations were obtained starting from the literature data on inhibition of esterases with diverse organophosphorus compounds.     

Novel method of evaluating biological 19-hydroxylation and aromatization of androgens

[19C3H]Androstenedione of high specific activity has been prepared. In liver incubation the isotope was shown to be stable to biological processes other than 19-hydroxylation. Incubation of the new substrate with human placental microsomes yielded 3H2O, 3HCOOH and estrogens devoid of radioactivity. The formation of 3H2O and 3HCOOH was close to the expected 2:1 ratio indicating that the material can be used to discriminate between 19-hydroxylation which yields 3H2O and aromatization which results in 3HCOOH. Comparison of the formation of 3H2O from [1 beta, 2 beta 3H]androstenedione and of 3HCOOH from [19C3H3]androstenedione in placental microsomal incubation showed that the aromatization of the former was 3.2 times faster indicating an isotope effect of that magnitude for the aromatization of [19C3H] vs [19CH3]androstenediones. The new substrate will be an effective probe and discriminant of both 19-hydroxylation and aromatization of androgens in vivo and in vitro, reactions which have been reported to be dissociated in specific tissues.     

Group-specific quantification of methanotrophs in landfill gas-purged laboratory biofilters by tyramide signal amplification-fluorescence in situ hybridization

The aim of this study was to quantitatively analyse methanotrophs in two laboratory landfill biofilters at different biofilter depths and at temperatures which mimicked the boreal climatic conditions. Both biofilters were dominated by type I methanotrophs. The biofilter depth profiles showed that type I methanotrophs occurred in the upper layer, where relatively high O(2) and low CH(4) concentrations were present, whereas type II methanotrophs were mostly distributed in the zone with high CH(4) and low O(2) concentrations. The number of type I methanotrophic cells declined when the temperature was raised from 15 degrees C to 23 degrees C, but increased when lowered to 5 degrees C. A slight decrease in type II methanotrophs was also observed when the temperature was raised from 15 degrees C to 23 degrees C, whereas cell numbers remained constant when lowered to 5 degrees C. The results indicated that low temperature conditions favored both type I and type II methanotrophs in the biofilters.     

The role of esterases in the toxicity of organothiophosphorus insectoacaricides containing a fragment of mercaptoacetic acid

Interaction of insectoacaricide Me (EtO)P(S)SCH2SCH2COOMe (I), its activation metabolites (P = O (II), S = O, and P = O, S = O (III) analogues), and a detoxication product (-COOH analoque (IV) with rat liver carboxylesterase, acetylcholinesterase and butyrylcholinesterase of warm-blooded animals, as well as with cholinesterase and carboxylesterase of American cockroach has been studied. Low toxicity of (I) towards warm-blooded animals and American cockroach is shown to result from its rapid hydrolysis with corresponding carboxylesterases to form (IV). Monothiophosphonates (II) and (III) are not hydrolyzed by carboxylesterases but inhibit them irreversibly. High toxicity of (I) towards aphids can be ascribed to low activity of the carboxylesterase of that insect.     

DNA adduct formation by alachlor metabolites

The extent of DNA adduct formation by alachlor [ArN(CH2OCH3)C(O)CH2Cl wherein Ar is 2,6-diethylphenyl] and its metabolites is used as a guide to deduce the causal agent(s) in the carcinogenicity of this major herbicide. [14C-phenyl]Alachlor is compared to its two metabolic cleavage products, [14C-phenyl]2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) [ArNHC(O)CH2Cl] and [14C-phenyl]2,6-diethylaniline (DEA) (ArNH2), and to [14C-methoxy]alachlor in various in vitro and in vivo systems. Horseradish peroxidase and hydrogen peroxide activate DEA, but not CDEPA or alachlor, for formation of adducts with calf thymus DNA, which probably involves 2,6-diethylnitrosobenzene (ArNO) as an intermediate. Mouse liver microsomes and NADPH are both required to enhance the binding from each labeled preparation to calf thymus DNA; 4-fold higher labeling is observed from [14C-methoxy]- than from [14C-phenyl]alachlor. This 4-fold preferential DNA labeling from the 14C-methoxy compound is likewise found in the liver of mice treated intraperitoneally. Mouse liver protein and hemoglobin are also labeled, in vivo, with [14C-phenyl]alachlor, -CDEPA and -DEA, and, as with the DNA, the labeling of these proteins is 1.5- to 2-fold higher with [14C-methoxy]alachlor. Metabolic studies indicate that ArN(CH2OCH2OH)C(O)CH2Cl is an intermediate in forming CDEPA and presumably formaldehyde in the mouse liver microsomal mixed-function oxidase system and in yielding the O-glucuronide of ArN(CH2OH)C(O)CH2Cl in the urine of alachlor-treated mice. These findings point to the N-CH2OCH2OH metabolite or formaldehyde as a reactive intermediate in forming a DNA-adduct and as a candidate proximate carcinogen.