Dose-dependent inhibition of emergency queen rearing by synthetic 9-ODA in the honey bee,Apis mellifera carnica

In the honey bee colony queen rearing is usually suppressed by releaser effects of the queen's pheromone. This is part of the dominance hierarchy maintaining the monogynous homeostasis. Under queenless conditions, the queen's control over the construction of emergency queen cells by the workers can be substitued by exposure to only one component of the mandibular pheromone secretion of a queen, the main compound (E)-9-oxo-2-decenoic acid. A novel and simple synthesis of (E)-9-oxo-2-decenoic acid is described, and a bioassay was developed by which a dose-dependent effect of synthetic (E)-9-oxo-2-decenoic acid presented on a dummy bee was evaluated.Abbreviation 9-ODA (E)-9-oxo-2-decenoic acid In memoriam Viktor Schwartz (1907–1992), Professor of Zoology and Developmental Biology, University of Tübingen, who introduced smoothened bee stings into microsurgery     

Factors determining the release of Nasonov pheromone by honeybees at the hive entrance

ABSTRACT. Worker bees recently denied access to their colony expose their Nasonov glands, thereby releasing pheromone, at the entrance to their hive. Odours of the following induced this response: empty comb, purified beeswax, honey, pollen, propolis, a live queen, the (E)-9-hydroxy-2-decenoic acid component of a queen's mandibular glands, live drones and workers, inert material on which workers had walked inside the hive, and synthetic Nasonov pheromone. The total odour of a foreign colony also induced worker bees to expose their Nasonov glands but was less effective than the odour of their own colony. Odours of the following were not effective: the (E)-9-oxo-2-decenoic acid component of a queen's mandibular glands, recently killed drones and workers, worker brood (eggs, larvae, pupae).     

Identification of novel decenoic acids in heated butter

Novel decenoic acids such as (E)-4-decenoic acid and (E)- and (Z)-5-,6-decenoic acid were detected as minor components in heated butter using GC and GC/MS. The formation mechanism of these novel decenoic acids is discussed on the basis of the result of the reaction of delta-decalactone with active clay in a model experiment.     

Thiafatty acids as tracers to investigate biosynthetic pathways of lepidopteran sex pheromones

In order to investigate the potential utility of thiafatty acids as tracers for biosynthetic studies of moth sex pheromones, a series of thiatetradecanoic acids, namely 8-, 9-, 10-, 11-, 12- and 13-thiatetradecanoic, were prepared and their metabolism was investigated in pheromone glands of Spodoptera littoralis. Analysis by gas chromatography coupled to mass spectrometry of extracts from pheromone glands treated with the above acids showed that only 8-thiatetradecanoic acid and 13-thiatetradecanoic acid were metabolized by desaturation and were incorporated into the sex pheromone biosynthetic pathway. 13-Thiatetradecanoic acid was converted into (E)- and (Z)-13-thiatetradec-11-enoic acids, (Z,E)-13-thiatetradeca-9,11-dienoic acid, 11-thiadodecanoic acid, (E)- and (Z)-11-thiadodec-9-enoic acids and 15-thiahexadecanoic acid. 8-Thiatetradecanoic acid gave rise to two monoenoic thiafatty acids and two dienoic thiafatty acids, which were assigned to (Z)- and (E)-8-thiatetradec-11-enoic acids, (Z,E)-8-thiatetradeca-9,11-dienoic acid and (E,E)-8-thiatetradeca-10,12-dienoic acid. The other thiafatty acids tested, 9-, 10-, 11- and 12-thiatetradecanoic acids, were not metabolized by desaturation, although the corresponding products of beta-oxidation and chain elongation were detected. The occurrence of sulfoxides was not detected in this case, in disagreement with results on the metabolism of some thiaacids previously reported by other authors in yeast, Saccharomyces cerevisiae.     

Effect of fatty acids on the membrane fluidity of cultured chick dorsal root ganglion measured by fluorescence photobleaching recovery

The effect of fatty acids on the membrane fluidity in tissue cultured chick embryo dorsal root ganglion was studied by fluorescence recovery method. Lateral motion of the lipid was measured by observing the fluorescent probe, 5-(octadecylthiocarbamoylamino) fluorescence, F18. The effective lateral diffusion coefficient of the membrane was around 0.30 X 10(-8) cm2/sec in control cells, 0.42 X 10(-8) cm2/sec in 2-decenoic acid treated cells, and 0.35 X 10(-8) cm2/sec in valeric acid treated cells. From these results it is concluded that effective mobilities of the membrane complex increased about 40% by the external application of 2-decenoic acid, while valeric acid increased it only 12%. From the physiological results that 2-decenoic acid inhibits the Na-channel, it is suggested that this increase in the membrane fluidity might affect the Na-channel.     

Autocatalytic inactivation of plant cytochrome P-450 enzymes: selective inactivation of the lauric acid in-chain hydroxylase from Helianthus tuberosus L. by unsaturated substrate analogs

Lauric acid in-chain hydroxylation is inhibited in microsomes from Jerusalem artichoke tubers (Helianthus tuberosus L.) incubated with 9-decenoic, 11-dodecenoic, or 11-dodecynoic acids. 9-Decenoic acid is at best a weak competitive inhibitor of the in-chain hydroxylase, but inactivates the enzyme in a time-dependent, pseudo-first-order process with a rate constant of approximately 1.1 X 10(-3) s-1. In contrast, 11-dodecenoic acid causes a slower, time-dependent loss of the hydroxylase activity, but is a potent competitive inhibitor of the enzyme (Ki = 2 microM). Neither agent decreases the microsomal concentration of cytochrome b5, NADH-cytochrome b5 reductase, or NADPH cytochrome P-450 reductase. Cinnamic acid 4-hydroxylation, catalyzed by a cytochrome P-450 enzyme, is not affected by concentrations of 9-decenoic acid that suppress lauric acid hydroxylation. 11-Dodecenoic acid is much less specific and, at higher concentrations, markedly reduces the microsomal cytochrome P-450 content, and the hydroxylation of both lauric and cinnamic acids.     

Structure and amount of positional isomers of monounsaturated fatty acids in human depot fat

Positional isomers of decenoic, dodecenoic, and tetradecenoic acids of human adipose tissue have been separated by gas-liquid chromatography and their amounts determined by oxidative cleavage. The following acids have been shown to be present: 7-decenoic, 8-decenoic, 9-decenoic, 7-, 8-, and 9-dodecenoic, and 7-, 8-, 9-, and 11-tetradecenoic acids. Among all isomers of the even-numbered acids from C(10) to C(18) the cis-9-isomer predominates. With increasing chain length, however, the content of 9-isomer decreases and the number of isomers increases. No 3- or 5-enoic acids could be detected. The origin and biosynthesis of all isomers are discussed.     

Site-directed mutagenesis of the PsaC subunit of photosystem I. F(b) is the cluster interacting with soluble ferredoxin.

The two [4Fe-4S] clusters F(A) and F(B) are the terminal electron acceptors of photosystem I (PSI) that are bound by the stromal subunit PsaC. Soluble ferredoxin (Fd) binds to PSI via electrostatic interactions and is reduced by the outermost iron-sulfur cluster of PsaC. We have generated six site-directed mutants of the green alga Chlamydomonas reinhardtii in which residues located close to the iron-sulfur clusters of PsaC are changed. The acidic residues Asp(9) and Glu(46), which are located one residue upstream of the first cysteine liganding cluster F(B) and F(A), respectively, were changed to a neutral or a basic amino acid. Although Fd reduction is not affected by the E46Q and E46K mutations, a slight increase of Fd affinity (from 1.3- to 2-fold) was observed by flash absorption spectroscopy for the D9N and D9K mutant PSI complexes. In the FA(2) triple mutant (V49I/K52T/R53Q), modification of residues located next to the F(A) cluster leads to partial destabilization of the PSI complex. The electron paramagnetic resonance properties of cluster F(A) are affected, and a 3-fold decrease of Fd affinity is observed. The introduction of positively charged residues close to the F(B) cluster in the FB(1) triple mutant (I12V/T15K/Q16R) results in a 60-fold increase of Fd affinity as measured by flash absorption spectroscopy and a larger amount of PsaC-Fd cross-linking product. The first-order kinetics are similar to wild type kinetics (two phases with t((1)/(2)) of <1 and approximately 4.5 microseconds) for all mutants except FB(1), where Fd reduction is almost monophasic with t((1)/(2)) < 1 microseconds. These data indicate that F(B) is the cluster interacting with Fd and therefore the outermost iron-sulfur cluster of PSI.     

M?ssbauer studies of the formation and reactivity of a quasi-stable peroxo intermediate of stearoyl-acyl carrier protein Delta 9-desaturase.

Stearoyl-ACP Delta(9)-desaturase (Delta 9D) is a diiron enzyme that catalyzes 18:0-ACP desaturation. Each subunit of homodimeric resting Delta 9D contains a diferric cluster, while chemical reduction by 4e(-) produces a diferrous cluster in each subunit. Reaction of 4e(-)-reduced Delta 9D with 18:0-ACP and O(2) yields a blue chromophore (lambda(max) approximately 700 nm) that exhibits a vibrational spectrum indicative of a micro-1,2-peroxo complex; this species has been designated peroxo Delta9D. In contrast to other enzymic peroxodiiron intermediates, peroxo Delta 9D is long-lived (t(1/2) approximately 30 min at 25 degrees C) and decays via an oxidase reaction without formation of either H(2)O(2) or product (18:1-ACP). In this work, optical, transient kinetic, and M?ssbauer techniques have been used to further investigate the origin and nature of this unusual peroxodiiron complex. Rapid mixing of 4e(-) Delta 9D with O(2)-equilibrated 18:0-ACP produced peroxo Delta 9D as revealed by a temperature-dependent, pseudo-first-order absorption increase at 700 nm (k = 46 s(-)(1) at 6 degrees C). The M?ssbauer spectrum of peroxo Delta 9D, accounting for 96% of the total iron, consists of two quadrupole doublets present in equal proportions: delta(1) = 0.68(1) mm/s, and Delta E(Q)(1) = 1.90(2) mm/s; delta(2) = 0.64(1) mm/s, and Delta E(Q)(2) = 1.06(2) mm/s. Decay of the 700 nm optical band (k = 0.004 min(-)(1) at 6 degrees C) correlates with the complete conversion of peroxo Delta 9D into a complex called peroxo-cycled Delta 9D, which exhibits two new doublets present in equal proportions: delta(1) = 0.57(2) mm/s, and Delta E(Q)(1) = 1. 91(3) mm/s; delta(2) = 0.52(2) mm/s, and Delta E(Q)(2) = 1.41(3) mm/s. Thus, peroxo Delta 9D contains two asymmetric diferric clusters and reacts to yield peroxo-cycled Delta 9D, also containing two asymmetric diferric clusters that most probably represent a substrate complex state. The clusters of both peroxo Delta 9D and peroxo-cycled Delta 9D have a diamagnetic ground state. Because peroxo Delta 9D and peroxo-cycled Delta 9D are observed only in the presence of 18:0-ACP, substrate binding appears to have introduced asymmetry into the Delta 9D diiron clusters. In situ photolysis of peroxo Delta 9D at 4.2 K in the M?ssbauer cryostat caused the release of O(2) and the reappearance of a diferrous Delta 9D.18:0-ACP complex with slightly changed parameters, suggesting a constrained cluster configuration was produced by the photolysis event. Annealing the photolyzed sample for 30 min at 77 K quantitatively restored the M?ssbauer spectrum of peroxo Delta 9D, showing that the released O(2) was effectively sequestered within the active site.     

A linoleic acid (8R)-dioxygenase and hydroperoxide isomerase of the fungus Gaeumannomyces graminis. Biosynthesis of (8R)-hydroxylinoleic acid and (7S,8S)-dihydroxylinoleic acid from (8R)-hydroperoxylinoleic acid.

The fungus Gaeumannomyces graminis metabolized linoleic acid extensively to (8R)-hydroperoxylinoleic acid, (8R)-hydroxylinoleic acid, and threo-(7S,8S)-dihydroxylinoleic acid. When G. graminis was incubated with linoleic acid under an atmosphere of oxygen-18, the isotope was incorporated into (8R)-hydroxylinoleic acid and 7,8-dihydroxylinoleic acid. The two hydroxyls of the latter contained either two oxygen-18 or two oxygen-16 atoms, whereas a molecular species that contained both oxygen isotopes was formed in negligible amounts. Glutathione peroxidase inhibited the biosynthesis of 7,8-dihydroxylinoleic acid. These findings demonstrated that the diol was formed from (8R)-hydroperoxylinoleic acid by intramolecular hydroxylation at carbon 7, catalyzed by a hydroperoxide isomerase. The (8R)-dioxygenase appeared to metabolize substrates with a saturated carboxylic side chain and a 9Z-double bond. G. graminis also formed omega 2- and omega 3-hydroxy metabolites of the fatty acids. In addition, linoleic acid was converted to small amounts of nearly (65% R) racemic 10-hydroxy-8,12-octadecadienoic acid by incorporation of atmospheric oxygen. An unstable metabolite, 11-hydroxylinoleic acid, could also be isolated as well as (13R,13S)-hydroxy-(9E,9Z), (11E)-octadecadienoic acids and (9R,9S)-hydroxy-(10E), (12E,12Z)-octadecadienoic acids. In summary, G. graminis contains a prominent linoleic acid (8R)-dioxygenase, which differs from the lipoxygenase family of dioxygenases by catalyzing the formation of a hydroperoxide without affecting the double bonds of the substrate.     

Taxonomy and pathogenicity of Erwinia cacticida sp. nov.

A total of 108 pectolytic, soft-rotting Erwinia strains were collected from 11 types of cacti growing in Arizona, Texas, northern Mexico, and Australia between 1958 and 1989. Four strains were collected from soils beneath or close to naturally rotting saguaro cacti. Collectively, these strains caused soft rots of saguaro, organ pipe, and senita cacti, Opuntia (cactus) fruits and pads, tomato fruits, and potato slices, but only occasionally caused soft rots of slices of carrot roots. A numerical cluster analysis showed that 98 of the 112 strains formed a uniform group (cluster 1A) that was distinguished from other pectolytic erwinias by an API 20E code of 1205131, by negative reactions in API 50CHE tests for L-arabinose, myo-inositol, D-cellobiose, melibiose, and D-raffinose, and, in supplemental tests, by positive reactions for malonate and growth at 43 degrees C. The average levels of DNA relatedness of 22 cluster 1A strains to the proposed type strain (strain 1-12) as determined by the hydroxyapatite method were 88% in 60 degrees C reactions (with 1% divergence within related sequences) and 87% in 75 degrees C reactions. The levels of relatedness to the type strains of other Erwinia spp. were less than or equal to 38% in 75 degrees C reactions. Cluster 1A strains also had a characteristic cellular fatty acid profile containing cyclo-(11,12)-nonadecanoic acid (C19:0 Cyclo C11-12) and missing tridecanoic acid (C13:0), heptadecanoic acid (C17:0), and cis-9-heptadecenoic acid (C17:1 CIS 9), which separated them from other pectolytic erwinias. Collectively, these data indicate that the members of cluster 1A are members of a new species, which we name Erwinia cacticida. Three cactus strains in cluster 1B appear to represent a second new species that is closely related to E. cacticida; these strains are designated E. cacticida-like pending the availability of additional strains for testing. The remaining cactus strains (in cluster 4) have the physiological, DNA, and fatty acid profiles of Erwinia carotovora.     

Mandibular Gland Volatiles and Their Ontogenetic Patterns in Queen Honey Bees,Apis mellifera carnica

Extracts of mandibular glands taken from adult queens of the honey bee, Apis mellifera carnica, were analysed by gas chromatography-mass spectroscopy. More than 100 compounds could be identified among which oxygenated fatty acids with six, eight, 10 and 12 carbon atoms are particularly interesting since they show structural relationships to the queen substance, (E)-9-oxo-2-decenoic acid. Changes in the patterns of volatiles were followed up from emergence until the full dominant status of an egg-laying queen in a strong colony. Generally, the amount of volatiles per gland was found to increase with age. The final level of queen substance (9-ODA) content is reached at the postmating stage about 10 days after emergence. Ontogenetic patterns of concentrations were determined for those components regarded to predominantly contribute to the royal pheromone. Characteristic compositions of signals, possibly involved in the premating, mating and postmating dominance status of a honey bee queen are discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved     

The novel pathway for ketodiene oxylipin biosynthesis in Jerusalem artichoke (Helianthus tuberosus) tubers

The new route of the plant lipoxygenase pathway, directed specifically towards the ketodiene formation, was detected during in vitro experiments with Jerusalem artichoke (Helianthus tuberosus) tubers. Through this pathway (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD) is reduced to corresponding 13-hydroxy acid (13-HOD), which is in turn dehydrogenated into ketodiene (9Z,11E,13S)-13-oxo-9,11-octadecadienoic acid (13-KOD). Dehydrogenation of 13-HOD into 13-KOD was not dependent on the presence of either NAD or NADP, but was strongly dependent on the presence of oxygen. Under anoxic conditions, 13-HOD dehydrogenation was blocked, but addition of 2,6-dichlorophenolindophenol restored it. Sulfite addition fully suppressed the aerobic dehydrogenation of 13-HOD. Hydrogen peroxide is a by-product formed by the enzyme along with 13-KOD. These data suggest that the ketodiene biosynthesis in H. tuberosus tubers is catalyzed by flavin dehydrogenase. (9S,10E,12Z)-9-Hydroxy-10,12-octadecadienoic acid (9-HOD) is dehydrogenated by this enzyme as effectively as 13-HOD, while alpha-ketol, (9Z)-12-oxo-13-hydroxy-9-octadecenoic acid, and ricinoleic acid did not act as substrates for dehydrogenase. The enzyme was soluble and possessed a pH optimum at pH 7.0-9.0. The only 13-HOD dehydrogenase known so far was detected in rat colon. However, unlike the H. tuberosus enzyme, the rat dehydrogenase is NAD-dependent.     

Products of metal exchange reactions of metallothionein

Hepatic metallothionein (MT) isolated from Cd-exposed animals always contains Zn (2-3 mol/mol of protein) in addition to Cd (4-5 mol/mol of protein), and the two metals are distributed in a nonuniform, but reproducible, manner among the seven binding sites of the protein's two metal-thiolate clusters. Different methodologies of preparing rabbit liver Cd, Zn-MT in vitro were investigated to provide insight into why such a distinct mixture of mixed-metal clusters is produced in vivo and by what mechanism they form. 113Cd NMR spectra of the products of stepwise displacement of Zn2+ from Zn7-MT by 113Cd2+ show that Cd binding to the clusters is not cooperative (i.e., clusters containing exclusively Cd are not formed in preference to mixed-metal Cd, Zn clusters), there is no selective occupancy of one cluster before the other, and many clusters are produced with a nonnative metal distribution indicating that this pathway is probably not followed in vivo. In contrast, the surprising discovery was made that the native cluster compositions and their relative concentrations could be reproduced exactly by simply mixing together the appropriate amounts of Cd7-MT and Zn7-MT and allowing intermolecular metal exchange to occur. This heretofore unknown metal interchange reaction occurs readily, and the driving force appears to be the relative thermodynamic instability of three-metal clusters containing Cd. With this new insight into how Cd,Zn-MT is likely to be formed in vivo we are able for the first time to postulate rational explanations for previous observations regarding the response of hepatic Zn and metallothionein levels to Cd administration.     

Age dependency of worker bee response to queen pheromone in a four-armed olfactometer

Summary Behavioral responses of differently aged worker beesApis mellifera to a queen pheromonal extract were analysed. The bees were tested individually in a four-armed olfactometer, one arm being scented with the pheromonal extract. This extract was prepared from heads of 14–17-day-old unmated queens. Among the components of the blend, 470 g 9-keto-2-(E)-decenoic acid, 200 g 9-hydroxy-2-(E)-decenoic acid and 5 gp-hydroxybenzoic acid methyl ester per queen equivalent were dosed. An age dependency in the worker bees' olfactory response to the components of the queen extract was shown, the strongest response occurring below the age of 5 days.     

Finger printing of acid lime varieties and clones having varied resistance to bacterial canker,using RAPD marker

Citrus is an important fruit crop having divergent genetic variation within the species. The germplasm identification and characterisation is an important link between the conservation and utilisation of genetic resources. Conventionally, variety/clone identification has relied on morphological characters such as growth habit, leaf, floral and fruit characters etc. Investigation through RAPD (random amplified polymorphic DNA) markers was carried out for determination of genetic variation among 12 acid lime clones having varied resistance to bacterial canker disease. DNA was extracted from the leaf of 12 acid lime clones and was subjected to PCR using 20 random primers (nine from OPM and 11 from OPA series) which yielded a total of 127 distinct DNA fragments, out of which 103 were polymorphic. Genetic similarity was evaluated based on the presence or absence of bands. The bands obtained were polymorphic, with sizes ranging from 750 bp to 2.5 kb. Cluster analysis using the similarity coefficient showed that the Balaji, RHRL-124 and PKM-1 formed one cluster and the remaining clones formed a second cluster, which in turn were divided into TAL 94-9, TAL 94-10, TAL 94-11 and TAL 94-12 which formed the first subcluster; the Nalgonda selection and local acid lime formed a second subcluster; TAL 94-8, RHRL-49 and RHRL-122 did not resemble any other clones. Among the 12 acid lime clones, Balaji, RHRL-124, RHRL-122 and PKM-1 were found to be moderately resistant to bacterial canker. Correlation of RAPD data with canker disease incidence in the moderately resistant acid lime clones viz., Balaji, RHRL-124 and PKM-1 were formed as one cluster, and all susceptible clones formed as a second cluster viz., except TAL-94-9, RHRL-122, which were found to be moderately resistant and did not form a cluster with any other acid lime clone.     

Analysis of genetic diversity and population structure of Chinese yak breeds （Bos grunniens） using microsatellite markers

Nine Chinese yak breeds (Maiwa,Tianzhu White,Qinghai Plateau,Sibu,Zhongdian,Pall,Tibetan High Mountain,Jiulong,and Xin-jiang) and Gayal were analyzed by means of 16 microsatellite markers to determine the level of genetic variation within populations,genetic relationship between populations,and population structure for each breed.A total of 206 microsatellite alleles were observed.Mean F-statistics (0.056) for 9 yak breeds indicated that 94.4% of the genetic variation was observed within yak breeds and 5.6% of the genetic variation existed amongst breeds.The Neighbor-Joining phylogenetic free was constructed based on Nei's standard genetic dis-tances and two clusters were obtained.The Gayal separated from the yaks far away and formed one cluster and 9 yak breeds were grouped together.The analysis of population structure for 9 yak breeds and the Gayal showed that they resulted in four clusters; one clus-ter includes yaks from Tibet Autonomous Region and Qinghai Province,one cluster combines Zhongdian,Maiwa,and Tianzhu White,and Jiulong and Xinjiang come into the third cluster.Pali was mainly in the first cluster (90%),Jiulong was mainly in the second cluster (87.1%),Zhongdian was primarily in the third cluster (83%),and the other yak breeds were distributed in two to three clusters.The Gayal was positively left in the fourth cluster (99.3%).     

A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1'Z)-12-(1'-hexenyloxy)-9,11-dodecadienoic [(omega5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of alpha-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1'Z,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dodecadienoic [(omega5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, (1)H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.     

EPR characterization of the iron-sulfur-containing NADH-ubiquinone oxidoreductase of the Escherichia coli aerobic respiratory chain

The energy coupled NADH-ubiquinone (Q) oxidoreductase segment of the respiratory chain of Escherichia coli GR19N has been studied by EPR spectroscopy. Previously Matsushita et al. [(1987) Biochemistry 26, 7732-7737] have demonstrated the presence of two distinct NADH-Q oxidoreductases in E. coli membrane particles and designated them NADH dh I and NADH dh II. Although both enzymes oxidize NADH, only NADH dh I is coupled to the formation of the H+ electrochemical gradient. In addition to NADH, NADH dh I oxidizes nicotinamide hypoxanthine dinucleotide (deamino-NADH), while NADH dh II does not. In membrane particles we have detected EPR signals arising from four low-potential iron-sulfur clusters, one binuclear, one tetranuclear, and two fast spin relaxing g perpendicular = 1.94 type clusters (whose cluster structure has not yet been assigned). The binuclear cluster, temporarily designated [N-1]E, shows an EPR spectrum with gx,y,z = 1.92, 1.935, 2.03 and the Em7.4 value of -220 mV (n = 1). The tetranuclear cluster, [N-2]E, elicits a spectrum with gx,y,z = 1.90, 1.91, 2.05 and an Em7.4 of -240 mV (n = 1). These two clusters have been shown to be part of the NADH dh I complex by stability and inhibitor studies. When stored at 4 degrees C, both clusters are extremely labile as is the deamino-NADH-Q oxidoreductase activity. Addition of deamino-NADH in the presence of piericidin A results in nearly full reduction of [N-2]E within 17 s. In membrane particles pretreated with piericidin A, the cluster [N-1]E is only partly reducible by deamino-NADH and shows an altered line shape.(ABSTRACT TRUNCATED AT 250 WORDS)