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The nitric oxide (N = O) free radical exhibits potent cytocidal, mutagenic and vasodilatory properties. We have examined the hypothesis that the hydroxynitrosamino functionality (see sequence in text), which occurs naturally in antineoplastic and antihypertensive agents, will directly generate N = O following peroxidatic 1-electron oxidation. Cupferron (see sequence in text) is indeed an excellent (k greater than 10(7) m-1 s-1) substrate for horseradish peroxidase. The products are N = O and nitrosobenzene (phi - N = O) which are generated and consumed as follows. First, cupferron is oxidized by the classical peroxidatic mechanism to form an unstable nitroxide free radical (see sequence in text) which then forms N = O and phi - N = O spontaneously (see sequence in text). The N = O then reacts with phi - N = O to reform cupferron (see sequence in text) or with the enzyme to generate the characteristic peroxidase–N = O chromophore. Simultaneously, in a competitive reaction with O2, the N = O is converted to NO-2 (4N = O + O2 + 2H2O————4NO-2 + 4H+). The reactivity of hydroxynitrosamino compounds with horseradish peroxidase is in the order cupferron greater than hydroxynitrosaminomethane greater than alanosine. These model reactions, involving direct oxidation of the hydroxynitrosamino moiety, comprise a novel pathway for the biological production of N = O.

Horseradish peroxidase catalyzes the anaerobic oxidation of 2-nitropropane (probably as the nitronate R-) by H2O2 via Compounds I and II to form R-R. The oxidation rate is stimulated 10-fold by O2 and the products become acetone and NO-2. The aerobic oxidation of R- is a free radical chain reaction which is initiated by peroxidase and propagated by R because (a) R-R, reasonably, must arise as 2R leads to R-R, (b) over 90% of the enzyme-initiated reaction occurs free in solution, and (c) the Km value for R- is independent of the type of initiator. We present a scheme for initiation, propagation, and termination which explains product structure, the effects of resorcinol, CN-, ascorbate, superoxide dismutase, and catalase, as well as the ping-pong reaction kinetics. We used methanenitronate to investigate the locus of electron transfer from R- into the heme moiety of the enzyme because this donor reacts with Compound II to form an isoporphyrin, which then rearranges to a modified enzyme in which the ferriheme contains the nitromethyl group in covalent linkage at a methine carbon. The modified enzyme is 30-50% as active catalytically as the native enzyme. We argue that reduction of Compound II by R- occurs at the methine carbon by two competing pathways, namely, direct one electron transfer and, at about one-half the frequency, homolytic cleavage of a covalent heme-substrate adduct (isoporphyrin).

The CO2 adducts resulting from N-, O-, and S-carboxylation of suitable precursors are close analogues of carboxylate substrates in which -NH-CO2-, -O-CO2-, or -S-CO2- replaces -CH2-CO2- in the physiological substrate, -NOH-CO-2 replaces -CHOH-CO-2 and O-CO2- replaces -O-PO3H- R-XH + CO2 in equilibrium with R-X-CO2- + H+ X(-XH = -NH2, -NHOH, -OH or -SH). We find that aconitase catalyzes the CO2-dependent dehydration of N-hydroxy-DL-aspartate and erythro-beta-hydroxyl-L-aspartate with respective kcat values 62 and 90% of kcat for citrate and Km values of 3.6 and 3.2 mM, respectively. The CO2 adducts (carbamates) of the precursors would be structural and stereo analogues of the physiological substrate isocitrate. Detailed kinetic analyses of the behavior of intermediates and products show that aconitase catalyzes the formation of the enzyme-bound CO2 adducts from enzyme-bound precursors and CO2 and directs them, as well as the preformed CO2 adducts, into alpha, beta water elimination reactions formally identical to the isocitrate/cis-aconitate reaction. Six other enzymes of carbohydrate metabolism (succinate thiokinase and isocitrate, glucose-6-phosphate, succinate semialdehyde, glutamate, and malate dehydrogenase) utilize CO2 adducts as reactive substrate analogues. At least one of these (glucose-6-phosphate dehydrogenase) catalyzes the formation of the enzyme-bound CO2 adduct (presumed to be D-glucose 6-carbonate in this case) from enzyme-bound precursor (D-glucose) and CO2 in the manner of aconitase. The case of malate dehydrogenase is unique because the reactive malate analogue, -O2C-O-CHOH-CO-2, arises from nucleophilic attack of HCO-3 on the carbonyl of glyoxylate, rather than electrophilic attack of CO2 on the hydrated carbonyl of glyoxylate.

On the fifth day following inoculation into an unstirred liquid surface culture, Penicillium atrovenetum abruptly, and reproducibly, secretes large quantities (2 g/liter) of the toxic antibiotic 3-nitropropionate into the medium. Concomitantly and with the same time course, crude extracts of the fungus acquire the ability to catalyze the oxidation of 3-nitropropionate by O2. We purified this activity some 300-fold to homogeneity and find it to be a soluble, dimeric (Mr = 73,000) flavoprotein oxidase having FMN as prosthetic group with lambda max = 363 and 433 nm. The preferred substrates are propionate-3-nitronate (3-NP-2) and O2 while the reaction products are malonate semialdehyde, NO2-, NO3-, O2-., and H2O2. Of 13 nitronates tested only butyrate-4-nitronate is more than 2% as reactive as 3-NP-2. 3-NP-2 (0.1 mM) rapidly reduces E-FMN anaerobically to E-FMNH., the flavin semiquinone (t1/2 less than 5 s), but reduces E-FMNH. to the fully reduced enzyme (E-FMNH2) very slowly (t1/2 approximately 900 s). The steady state turnover number with 0.1 mM 3-NP-2 and infinite O2 is 350 s-1. Therefore, the enzyme must oscillate almost exclusively between E-FMN and E-FMNH. during aerobic turnover. (Formula: see text). The complicated and non-integral reaction stoichiometry provides further support for this free radical mechanism. Each mole of 3-NP-. generated enzymatically initiates the nonenzymatic autoxidation of at least 2.2 mol of 3-NP-2 through a free radical chain reaction. An appropriate name for the newly characterized enzyme is propionate-3-nitronate oxidase.

We identify the cyanogenic substrate for horseradish peroxidase (HRP) as a conjugated enamine and explore this unusual reaction using alpha-aminocinnamate (RH) as follows. 1) HRP catalyzes the oxidation of RH by O2 (and its peroxidation by H2O2 to form R-R) to produce, simultaneously, CN- and benzaldehyde cyanohydrin. 2) RH is transient and must be generated in situ. The properties of the cyanogenic reaction of HRP are independent of the method of preparation of RH (whether this be condensation of NH3 with phenylpyruvate, enzymatic hydrolysis of glycyldehydrophenylalanine, or oxidation of L-phenylalanine by L-amino acid oxidase). 3) The oxidation of RH is a free radical chain reaction initiated by HRP Compounds I and II (I (or II) + RH→R. + II (or HRP], propagated by RO2. (R. + O2→RO2., RO2. + RH→R. + RO2H), and terminated by recombination reactions such as 2R.→R2 and RO2.→R' + HO2. followed by R. + HO2.→RH + O2. KMnO4 and K3Fe(CN)6 can substitute for HRP. 4) The proximal precursor of CN- and cyanohydrin is postulated to be RO2H (phi-CH(-O2H)-CCO2-(= NH]. These results explain why cyanide is generated from the synergistic action of HRP and L-amino acid oxidase on aromatic L-amino acids and O2 and suggest that the requirement for a beta-aryl substituent on the enamine originates in the reaction of RH with HRP, or of R with O2, rather than the imine/enamine tautomerization of the L-amino acid oxidase product.

We find ethanenitronate (formula; see text) to be a H2O2- (and peracetic acid-) dependent suicide substrate for bovine liver catalase (E) which converts E to Em, a modified form of the enzyme. The catalytic and suicide pathways are related to E, Em, Compound I, and Compound II according to the following scheme. (formula; see text) The catalytic cycle generates free radical products (EN.) which then participate in an O2-dependent chain reaction. Within experimental error the exclusive target for inactivation by EN- is Compound II. This partitions in the ratio (k4 = 1.2 M-1 s-1)/(k3 = 1.6 M-1 s-1) to generate Em and E, respectively. The species Em acquires 1 eq of 14C/ferriheme from [1-14C]ethanenitronate which is firmly (presumably covalently) affixed to the protein moiety. According to the standard H2O2 assay, Em is 7% as active catalytically as E. We regard inactivation as resulting from that fraction of EN. in the E...EN. complex which fails to diffuse from the complex because it is trapped by reaction with a neighboring amino acid residue to generate Em irreversibly. (formula; see text) This mechanism is identical to that deduced previously for suicide inactivation of horseradish peroxidase by alkane nitronates (Porter, D. J. T., and Bright, H. J. (1983) J. Biol. Chem. 258, 9913-9924) with the exception that EN. is trapped in that case by a methine carbon at the edge of the ferriheme rather than by the apoenzyme. The labeled residue in the catalase apoenzyme probably resides at or near the site of reduction of Compound II.

Given that the oxidation of nitroethane by D-amino acid oxidase proceeds through a transient carbinolamine adduct at the N5 position of the active-site FAD cofactor (Porter, D. J. T., Voet, J. G., and Bright, H. J. (1973) J. Biol. Chem. 248, 4400-4416), it follows that 1-chloro-1-nitroethane should generate a stable amide at N5 and thereby function as a suicide inactivator of this enzyme. This hypothesis was validated as follows. 1-Chloro-1-nitroethane, as the nitronate ion (pKa = 7.0), inactivated D-amino acid oxidase completely with a Km value of 2 mM and a maximum rate of 0.02 s-1. The chloro and nitro groups were quantitatively recovered as free Cl- and NO2(-) after the enzyme was inactivated by 1.5 flavin equivalents of 1-chloro-1-nitroethane. Inactivation did not require O2 and was accompanied by bleaching of the flavin under both anaerobic and aerobic conditions. The modified coenzyme of the inactivated enzyme was identified as N5-acetyl-1,5-dihydro FAD. The enzyme catalyzes the oxidation of 1-chloro-1-nitroethane to acetate approximately 0.5 times as rapidly as the enzyme catalyzes suicide inactivation. The transient intermediate which is common to both the inactivation and oxidation pathways must be N5-(1-X-1-hydroxyethyl)-1,5-dihydro FAD, where X = nitro or chloro.