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PubMed1987

Changes in physicochemical properties of mitochondrial membranes during the formation process of megamitochondria induced by hydrazine.

Changes in some biochemical and physico-chemical properties of rat liver mitochondrial membranes during the formation process of megamitochondria induced by hydrazine were analyzed. Hepatic mitochondria obtained from rats placed on a 1% hydrazine diet for 3 days became slightly enlarged and sometimes elongated, while they became gigantic after 7 days of hydrazine intoxication. Changes were observed in mitochondria from rats treated with hydrazine for 3 days. Total amounts of phospholipids extracted from mitochondria and submitochondrial fractions were increased. Among phospholipid species, relative amounts of acidic phospholipids were increased. Contents of Ca2+ in mitochondria were increased. Differential scanning calorimetric analysis of mitochondria, especially that of the outer membrane fraction, showed that the thermotropic lipid phase transition temperatures were elevated accompanying the broadening of thermograms and the increase in transition enthalpy. Contents of water in mitochondria were increased significantly with the ratio of freezable water to unfreezable water unchanged. Among the changes observed was that the total amount of phospholipids (except for that of the outer membrane fraction) and the contents of water and Ca2+ nearly returned to normal in megamitochondria after 7 days of hydrazine intoxication. Relative amounts of phospholipids and thermotropic lipid phase transition temperatures of megamitochondria did not return to normal levels and yet changes were smaller than those obtained from 3 days of hydrazine intoxication. The fluidity of mitochondrial membranes was not affected by hydrazine treatment. These data would suggest that hydrazine-induced megamitochondrial formation is not due simply to the swelling of mitochondria, but might be due to the fusion of adjacent mitochondria by Ca2+-acidic phospholipid interactions, and once megamitochondria are formed the mitochondrial membranes are stabilized.

Animals↗

PubMed2004

Highly sensitive determination of hydrazine ion by ion-exclusion chromatography with ion-exchange enhancement of conductivity detection.

An ion-exclusion chromatography method with ion-exchange enhancement of conductivity was developed for the selective separation and sensitive determination of hydrazine ion from alkali/alkaline earth metal cations and ammonium ion. Hydrazine ion was separated by ion-exclusion/penetration effect from other cations on a weakly basic anion-exchange column in the OH- form (TSKgel DEAE-5PW). Moreover, two different ion-exchange resin columns were inserted between the separating column and conductimetric detector in order to improve the sensitivity of hydrazine ion. The first enhancement column packed with a strongly basic anion-exchange resin in the SO4(2-) form (TSKgel SAX) for hydrazine ion can convert from N2H5OH to (N2H5)2SO4. Moreover, the second enhancement column packed with a strongly acidic cation-change resin in the H+ form (TSKgel SCX) can convert to H2SO4. As a result, the sensitivity of hydrazine ion using two conductivity enhancement columns could be 26.8-times greater than using the separating column alone. This method was effectiveness also for the enhancement of ammonium ion (6.1-times) and sodium ion (1.2-times). The calibration graph of hydrazine ion detected as H2SO4 was linear over the concentration range of 0.001-100 ppm (r2 = 0.9988). The detection limit of hydrazine ion in this system was 0.64 ppb. Therefore, hydrazine ion in real boiler water sample could be accurately determined, avoiding the interference of other cations.

Calibration↗

PubMed2000

Flow injection monitoring and analysis of mixtures of hydrazine compounds using filter-supported bilayer lipid membranes with incorporated DNA.

This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring and analysis of mixtures of hydrazine compounds using stabilized systems of filter-supported bilayer lipid membranes (BLMs) composed of egg phosphatidylcholine (egg PC) with incorporated DNA. Injections of hydrazines were made into flowing streams of a carrier electrolyte solution, and a transient current signal with a duration of seconds reproducibly appeared in less than one min after exposure of the DNA-modified lipid membranes to the hydrazines. The magnitude of this signal was linearly related to the concentration of hydrazines, which could be determined at sub-micromolar levels. Repetitive cycles of injection of hydrazines have shown no signal degradation during each cycle (30 sequential injections). The time of appearance of the transient response was different for each hydrazine and increased in the order of hydrazine, methylhydrazine or dimethylhydrazine, and phenylhydrazine. The difference in time of response has allowed selective detection and analysis of these hydrazines in mixtures.

DNA↗

PubMed1983

S-adenosylmethionine metabolism and DNA methylation in hydrazine-treated rats.

The treatment of rats with hepatotoxic doses of hydrazine (NH2-NH2) induces the rapid formation of 7-methylguanine and O6-methylguanine in liver DNA. The methyl moiety in these reactions might be derived from the cellular S-adenosylmethionine pool because radioactivity administered to these rats as methionine rapidly appears in the DNA as methylated guanine. An increased incorporation of radioactivity into 5-methylcytosine was previously reported followed by subsequent suppression. This increased radiolabeling of 5-methylcytosine coincided with time of maximal DNA guanine methylation. To determine the nature of S-adenosylmethionine metabolism during the period of DNA methylation induced by hydrazine treatment, and to determine if the increased radiolabeling of 5-methylcytosine at this time reflected an actual increase in 5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethionine levels and turnover were assayed. Liver S-adenosylmethionine concentrations varied slightly between control rats and hydrazinetreated rats during the first five hours after hydrazine administration, and no difference was detectable in the incorporation of administered [3H]methionine into S-adenosylmethionine. Because S-adenosylmethionine specific radioactivity in hydrazine-treated rats was not different from control rats, the previously observed increased radiolabeling of 5-methylcytosine appeared to represent an actual increase in synthesis. This conclusion was supported by finding that incorporation of radioactive thymidine into DNA was also accelerated immediately following hydrazine administration, again followed by a decrease. 5-Methylcytosine sythesis, therefore, appears to follow DNA synthesis during hydrazine toxicity, and formation of 7-methylguanine and O6-methylguanine in liver DNA of hydrazine-treated rats occurs during a short period of increased DNA sythesis and 5-methylcytosine formation very early in hydrazine toxicity.

Animals↗

PubMed1998

The anaerobic oxidation of hydrazine: a novel reaction in microbial nitrogen metabolism.

Hydrazine is rarely found as an intermediate in microbial nitrogen conversions. In this study the conversion of hydrazine by the anaerobic ammonium oxidation (Anammox) culture, in which hydrazine has been proposed as an intermediate, was investigated. This study demonstrated the biological nature of hydrazine conversion by the Anammox culture. In batch cultures with hydrazine it was observed that 3 mol N2H4 was disproportionated to 4 mol NH4+ and 1 mol N2. Hydrazine with nitrite as an electron acceptor showed a conversion of 3 mol N2H4 and 4 mol NO2- to 5 mol N2, with a specific activity of 5.5 nmol min-1 (mg volatile suspended solids)-1. Addition of hydrazine to a biofilm reactor for 80 days showed that it was not possible to grow Anammox with hydrazine.

Alcaligenes↗

PubMed1992

Studies on the disposition and metabolism of hydrazine in rats in vivo.

1. Rats were given various doses of hydrazine orally and their plasma and liver hydrazine levels were determined (at various times up to 270 min after dosing) by gas chromatography/mass spectrometry. 2. The increase in the peak plasma level and in the area under the plasma concentration-time curve (AUC) were not directly proportional to the dose. 3. The ratio of plasma to liver hydrazine varied with dose suggesting saturation of an uptake mechanism might be occurring. 4. In a separate experiment hydrazine was still detectable in the plasma and liver 24 h after dosing with hydrazine i.p. 5. Rats were given the same doses of hydrazine and urine was collected for 24 h after dosing and assayed for hydrazine and acetylhydrazine. The proportion of hydrazine and acetylhydrazine excreted declined with dose. 6. Liver samples were taken for histopathological examination 96 h after dosing. Only after the highest dose (81 mg kg-1) was there evidence of fatty liver, 96 h after a single dose, and a reduction in both liver and body weight.

Animals↗

PubMed2002

Mechanisms of carcinogenicity of aryl hydrazines, aryl hydrazides, and arenediazonium ions.

Aryl hydrazines carcinogenesis has been studied for over 25 years and remains poorly understood, although most aryl hydrazines are toxic, tumorigenic, or carcinogenic. In this article, aryl hydrazine carcinogenesis is reviewed comprehensively. The relevant chemistry and biochemistry of aryl hydrazines are first addressed and provide the framework for understanding how aryl hydrazines are metabolized, the reactive intermediates that are produced, and the biological reactive intermediates and products that are formed. Issues of DNA damage, mutagenicity, and enzyme activation are next addressed followed by a brief review of aryl hydrazine tumorigenicity studies. Because several related substrates are metabolized to the same intermediates as are aryl hydrazines, they are briefly discussed. The review concludes with a short discussion of the possible mechanism of carcinogenesis by aryl hydrazines.

Carcinogens↗

PubMed1978

Some studies on ascorbic acid metabolism in hydrazine-treated rats.

The metabolism of ascorbic acid was studied in hydrazine-treated rats. Hydrazine was administered i.p. at a dose of 1.28 mg/day (20% LD50) for each 100 g body weight for 7 days. Hydrazine administration at the present dose did not appear to have an effect on the total ascorbic acid level of liver, kidney, spleen and testis. The adrenal and plasma total ascorbic acid levels were, however, elevated. The activity of liver D-glucuronoreductase and that of liver and kidney dehydroascorbatases were diminished after hydrazine administration. The changes in the activities of liver enzymes were accompanied by a fall in the reduced ascorbic acid level and an elevation in the dehydroascorbic acid level. The uronolactonase activity of liver, on the other hand, remained independent of hydrazine treatment. It has been suggested that hydrazine treatment at the present dose reduced the biosynthesis of L-ascorbic acid from D-glucuronolactone as substrate. In spite of diminished synthesis, the normal level of total ascorbic acid in the liver of hydrazine-treated rats was maintained by reducing the degradation of L-ascorbic acid. The rise in the plasma total ascorbic acid level after hydrazine treatment was ascribed to reduced catabolism and urinary excretion of ascorbic acid, while the elevation in adrenal total ascorbic acid level might result from increased uptake of ascorbic acid by the gland from blood or from nonfunctional accumulation.

Alcohol Oxidoreductases↗

PubMed1994

Contribution of tumor necrosis factor-alpha and glucocorticoid in hydrazine sulfate-mediated protection against endotoxin lethality.

Hydrazine sulfate pretreatment has previously been shown in our laboratory to protect normal mice against endotoxin and D-galactosamine-sensitized mice against both exogenous tumor necrosis factor (TNF) and endotoxin. An intact pituitary is required for manifestation of the protective effects. Further, we have demonstrated that hydrazine sulfate pretreatment specifically modulates the TNF response to lipopolysaccharide (LPS) in mouse macrophages in vitro. This in vivo study was performed to test whether a reduced TNF response and/or increased glucocorticoid response may contribute to hydrazine sulfate protection against LPS-induced lethality in vivo. The results presented here establish that hydrazine sulfate pretreatment selectively attenuates circulating TNF levels following LPS challenge. Moreover, adrenalectomy abrogates hydrazine sulfate protection but does not prevent hydrazine sulfate attenuation of circulating TNF levels in response to LPS. Hydrazine sulfate-mediated protection is, however, restored permissively by corticosterone. Thus, the mechanism by which hydrazine sulfate protects against LPS lethality in adrenalectomized mice includes TNF modulation in response to endotoxin, as well as a pivotal requirement for glucocorticoid.

Adrenalectomy↗

PubMed1998

Mono- and Bis(hydrazine) Complexes of Osmium(II): Synthesis, Reactions, and X-ray Crystal Structure of the [Os(NH(2)NH(2))(2){P(OEt)(3)}(4)](BPh(4))(2) Derivative.

Reaction of OsH(2)P(4) [P = P(OEt)(3), PPh(OEt)(2), PPh(2)OEt] with methyl triflate followed by the treatment with hydrazines gave the [OsH(RNHNH(2))P(4)]BPh(4) (1-3) (R = H, CH(3), C(6)H(5), 4-NO(2)C(6)H(4)) derivatives. Instead, the reaction of OsH(2)P(4) first with methyl triflate, then with triflic acid, and finally with an excess of the appropriate hydrazine afforded the bis(hydrazine) [Os(RNHNH(2))(2)P(4)](BPh(4))(2) (4, 5) (R = H, CH(3), C(6)H(5)) complexes. Also the [Os(NH(2)NH(2)){P(OEt)(3)}(5)](BPh(4))(2) (7) derivative was prepared. All the hydrazine complexes were fully characterized by IR and (1)H and (31)P NMR spectra, and a single-crystal X-ray structure determination of the complex [Os(NH(2)NH(2))(2){P(OEt)(3)}(4)](BPh(4))(2).C(2)H(5)OH (4a) is reported. The compound crystallizes in the space group P2(1)/c with a = 20.550(4) Å, b = 19.663(4) Å, c = 20.843(4) Å, beta = 99.84(9) degrees, and Z = 4. The coordination around the osmium atom is octahedral and the orientation of the ligands in the [Os(NH(2)NH(2))(2){P(OEt)(3)}(4)](2+) cation is determined by several strong hydrogen bonds involving hydrazine nitrogen and phosphite oxygen atoms. Amidrazone complexes [Os{eta(2)-NH=C(R1)N(R)NH(2)}{P(OEt)(3)}(4)](BPh(4))(2) (8, 9) (R = H, CH(3); R1 = CH(3), 4-CH(3)C(6)H(4)) were prepared by allowing nitrile complexes [Os(R1CN)(2)P(4)](BPh(4))(2) to react with hydrazine NH(2)NH(2) or methylhydrazine CH(3)NHNH(2). Reaction of complexes containing substituted hydrazine ligands of the type [OsH(RNHNH(2))P(4)]BPh(4) and [Os(RNHNH(2))(2)P(4)](BPh(4))(2) [P = P(OEt)(3); R = CH(3), C(6)H(5)] with Pb(OAc)(4) at -30 degrees C results in the selective oxidation of the hydrazine affording the corresponding diazene [OsH(RN=NH)P(4)]BPh(4) (10) and [Os(RN=NH)(2)P(4)](BPh(4))(2) (11) derivatives. The first bis(methyldiazene) complex [Os(CH(3)N=NH)(2){P(OEt)(3)}(4)](BPh(4))(2) (11b) was thus prepared.

Journal Article↗

PubMed2005

Acidity of di- and triprotected hydrazine derivatives in dimethyl sulfoxide and aspects of their alkylation.

The pK(a) values in DMSO for 22 di- and triprotected hydrazine NH acids and two monosubstituted hydrazines have been determined using potentiometric titration. The results of density functional theory calculations at the B3LYP/6-311+G level of gas-phase acidities of a representative selection of mono-, di-, and trisubstituted hydrazines are compared with both the relevant published and novel experimental titration data. In the course of this work, a rough estimation of the pK(a) value of hydrazine in DMSO (ca. 38.0) has been deduced. For typical triprotected compounds of this kind containing moderately electron-withdrawing carbamate and imidodicarbonate or arenesulfonylcarbamate functions the pK(a) values fall in the range 15.1-17.3, whereas for N,N'-diprotected hydrazines with a carbamate and an aromatic sulfonyl group the corresponding values are 12.7-14.5. Several of these triprotected derivatives have recently been applied preparatively in stepwise synthesis of substituted hydrazines using alkyl halides as electrophiles in the presence of a phase transfer catalyst, and a few of them, with varying success, have been examined in model experiments with benzyl alcohol, triphenylphosphine, and diethyl azodicarboxylate in the Mitsunobu reaction. The dependence of the reactivity on the intrinsic acidity of the hydrazines in this reaction is highlighted. Furthermore, the regioselective alkylation of an N,N'-diprotected hydrazine can be rationalized on the basis of the presented data.

Journal Article↗

PubMed1983

Determination of hydrazine in pharmaceuticals III: hydralazine and isoniazid using GLC.

A GLC procedure has been developed for the determination of hydrazine in hydralazine and isoniazid drug raw materials, single and multicomponent tablets, injectables, and syrups. The method is based on the derivatization of hydrazine with benzaldehyde to form benzalazine. The minimum detectable amount of hydrazine in hydralazine and isoniazid raw materials and formulations is approximately 0.0003%. No hydrazine was found in the hydralazine raw material specimens examined. Traces of hydrazine (approximately 0.0003%) were found in some tablet lots and approximately 0.02% was found in an injectable product. A trace of hydrazine was found in one lot of isoniazid raw material and low levels (0.0012 and 0.0029%) were found in isoniazid tablet products. An isoniazid syrup contained approximately 0.2% hydrazine.

Chemistry, Pharmaceutical↗

PubMed2002

Content and redistribution of vitamin E in tissues of Wistar rats under oxidative stress induced by hydrazine.

Hydrazine toxicity is associated with generation of several kinds of free radicals and oxidative stress in cell. Experiments in vivo have demonstrated that oxidative stress could either diminish or increase concentration of vitamin E in some tissues. Thus in the present study we performed experiments to determine whether hydrazine-induced oxidative stress would change the tissue levels of the vitamin. Seven days of hydrazine intoxication led to accumulation of different amounts of vitamin E: 215% in the liver, 118% in the heart, 135% in the spleen, and 100% in the muscle over control value. There were no changes in the level of the vitamin in kidney and pancreas, despite its significant depletion in the serum. In tissue that accumulated vitamin E after hydrazine treatment, an increased of oxidative stress measured by the concentration of lipid-soluble fluorophore was observed. Significant increases of 107%, 46%, 72%, and 58% over control values were observed in the liver, heart, spleen, and muscle, respectively. Rats treated with hydrazine and pharmacological doses of alpha-tocopherol accumulated higher concentrations of vitamin E in all studied tissues compared with the alpha-tocopherol-only treated rats. However, in tissues with elevated levels of fluorophore as liver, heart, spleen, and muscle, the accumulation of vitamin E was 5.03, 4.5, 4.03, and 4.6 times higher than in alpha-tocopherol-treated rats, respectively. Vitamin E concentration was much higher than in kidney and pancreas, where the accumulation was only 2.31 and 2.6 times higher. On the other hand, 3 days of hydrazine treatment did not change either the level of lipid-soluble fluorophore or the level of vitamin E in the liver mitochondria, microsomes, and homogenate. In skeletal muscle vitamin E caused decreased lipofuscin accumulation, and in pancreas vitamin E increased lipofuscin accumulation. Our data indicate that hydrazine is able to modify significantly vitamin E status in different rat tissues.

Animals↗

PubMed1998

Human health perspective on environmental exposure to hydrazines: a review.

Hydrazines are colorless liquid compounds that have been found at various Department of Defense hazardous waste sites. They are designated as environmental contaminants causing adverse effects to public health and have been identified at many National Priorities List (NPL) hazardous waste sites and federal facilities sites in the United States. Three chemically similar hydrazines-hydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine--occur in the environment and cause adverse health effects to persons living near hazardous waste sites. Humans are exposed to hydrazines by drinking contaminated, water, by inhaling contaminated air, or by swallowing or touching contaminated dust. Human occupational data and studies in laboratory animals suggest that people exposed to hydrazines may develop adverse systemic health effects or cancer. Hydrazines have caused cancer in animals following acute- or intermediate- duration exposure by the oral and inhalation routes. The U.S. Environmental Protection Agency, the U.S. Department of Health and Human Services, the International Agency for Research on Cancer, and the World Health Organization have classified hydrazines as possible cancer-causing environmental contaminants.

Animals↗

PubMed1988

The question of carcinogenic effects of hydrazine.

In a number of older studies by various authors, hydrazine showed carcinogenic effects after toxic or subtoxic doses. Our own preliminary studies with maximum tolerable doses (rats, s.c. or i.t.), however, yielded negative results, as did the administration of maximum tolerable hydrazine doses simultaneously with benzo(a)pyrene treatment (rat i.t. and mouse i.p.). These preliminary experiments were therefore followed by a carcinogenicity study in rats, in which hydrazine was administered with the drinking water in concentrations of 0 mg/l (= control), 2, 10, and 50 mg/l. Each group comprised 50 male and 50 female Wistar rats. The investigation was continued until the spontaneous death of all the rats. The concentration of 2 mg/l was tolerated with hardly any damage; 10 mg/l proved to be the maximum tolerable dose, and 50 mg/l was clearly toxic although the survival times were not affected appreciably. Only under the toxicity of the highest concentration could a weak carcinogenic effect of hydrazine be detected, namely in the very late occurrence of small, usually benign liver cell tumours (overall 11.5%). A similar study carried out in mice revealed no macroscopic evidence of a carcinogenic effect of hydrazine (histology currently in preparation). According to these results, hydrazine is only weakly carcinogenic even after lifelong ingestion of unequivocally toxic doses. The classification of hydrazine as a "carcinogen" should therefore be re-evaluated.

Adenoma, Bile Duct↗

PubMed1997

Role of free radicals in the mechanism of the hydrazine-induced formation of megamitochondria.

The effect of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl(4-OH-TEMPO), a scavenger for free radicals, and 4-hydroxypyrazolo [3,4-d(pyrimidine)allopurinol], a xanthine oxidase inhibitor, on the hydrazine-induced changes of mitochondrial ultrastructure and those in the antioxidant system of the liver were investigated using rats as experimental animals. Animals were placed on a powdered diet containing 0.5% hydrazine for 7 d in the presence and absence of a combined treatment with 4-OH-TEMPO or allopurinol. Results obtained were as follows. 4-OH-TEMPO completely prevented the hydrazine-induced formation of megamitochondria in the liver, while it was partly prevented by allopurinol. The following changes observed in hydrazine-treated animals were improved almost completely by 4-OH-TEMPO:decreases in the body weight and liver weight; lowered rates of ADP-stimulated respiration and coupling efficiency of hepatic mitochondria; remarkable elevation of the level of lipid peroxidation. Improving effects of allopurinol were incomplete. The present results suggest that free radicals may play a key role in the mechanism of the hydrazine-induced formation of megamitochondria and that a part of free radicals generated during the hydrazine intoxication is ascribed to the degradation of purine nucleotides via xanthine oxidase. A general mechanism of the megamitochondria formation induced in various pathological conditions besides the case of hydrazine are discussed.

Allopurinol↗

PubMed2003

Effects of hydrazine derivatives on vascular smooth muscle contractility, blood pressure and cGMP production in rats: comparison with hydralazine.

Hydralazine is a hydrazine derivative used clinically as a vasodilator and antihypertensive agent. Despite numerous studies with the drug, its mechanism of action has remained unknown; guanylate cyclase activation and release of endothelial relaxing factors are thought to be involved in its vasodilator effect. Other hydrazine derivatives are known to stimulate guanylate cyclase and could therefore share the vasodilator activity of hydralazine, although such possibility has not been assessed systematically. In the present study, hydralazine, hydrazine, phenylhydrazine, and isoniazid were evaluated for vascular smooth muscle relaxation in rat aortic rings with and without endothelium, as well as after incubation with the guanylate cyclase inhibitor methylene blue. They were also tested for enhancement of cyclic guanosine monophosphate (cGMP) production by cultured rat aortic smooth muscle cells and for hypotension in the anesthetized rat. All hydrazines relaxed aortic rings, an action unaffected by endothelium removal and, in all cases except hydralazine, antagonized by methylene blue. Only phenylhydrazine increased cGMP production and only hydralazine markedly lowered blood pressure. It was concluded that hydralazine vascular relaxation is independent of endothelium and is not related to guanylate cyclase activation. The other hydrazines studied also elicit endothelium-independent relaxation, but the effect is related to guanylate cyclase. The marked hypotensive effect of hydralazine contrasts with its modest relaxant activity and is not shared by the other hydrazines. The fact that hydrazine and isoniazid produce methylene blue-sensitive relaxation, yet do not enhance cGMP production suggests the need for activating factors present in aortic rings but not in isolated cells.

Animals↗

PubMed2005

Trapping a hydrazine reduction intermediate on the nitrogenase active site.

A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.

Azotobacter vinelandii↗