Effects of benzyl viologen on increasing NADH availability, acetate assimilation, and butyric acid production by Clostridium tyrobutyricum.

Clostridium tyrobutyricum produces butyric and acetic acids from glucose. The butyric acid yield and selectivity in the fermentation depend on NADH available for acetate reassimilation to butyric acid. In this study, benzyl viologen (BV), an artificial electron carrier that inhibits hydrogen production, was used to increase NADH availability and butyric acid production while eliminating acetic acid accumulation by facilitating its reassimilation. To better understand the mechanism of and find the optimum condition for BV effect on enhancing acetate assimilation and butyric acid production, BV at various concentrations and addition times during the fermentation were studied. Compared with the control without BV, the addition of 1 µM BV increased butyric acid production from glucose by ∼50% in yield and ∼29% in productivity while acetate production was completely inhibited. Furthermore, BV also increased the coutilization of glucose and exogenous acetate for butyric acid production. At a concentration ratio of acetate (g/L) to BV (mM) of 4, both acetate assimilation and butyrate biosynthesis increased with increasing the concentrations of BV (0-6.25 µM) and exogenous acetate (0-25 g/L). In a fed-batch fermentation with glucose and ∼15 g/L acetate and 3.75 µM BV, butyrate production reached 55.9 g/L with productivity 0.93 g/L/h, yield 0.48 g/g, and 97.4% purity, which would facilitate product purification and reduce production cost. Manipulating metabolic flux and redox balance via BV and acetate addition provided a simple to implement metabolic process engineering approach for butyric acid production from sugars and biomass hydrolysates.

Reciprocal regulation of sulfite oxidation and nitrite reduction by mitochondrial sulfite oxidase.

The oxygen-independent nitrate-nitrite-nitric oxide (NO) pathway is considered as a substantial source of NO in mammals. Dietary nitrate/nitrite are distributed throughout the body and reduced to NO by the action of various enzymes. The intermembrane spaced (IMS), molybdenum cofactor-dependent sulfite oxidase (SO) was shown to catalyze such a nitrite reduction. In this study we asked whether the primary function of SO - sulfite oxidation - and its novel function - nitrite reduction - impact each other. First, we utilized benzyl viologen as artificial electron donor to investigate steady state NO synthesis by SO and found fast (kcat = 14 s-1) nitrite reduction of SO full-length and its isolated molybdenum domain at pH 6.5. Next, we determined the impact of nitrite on pre-steady state kinetics in SO catalysis and identified nitrite as a pH-dependent inhibitor of SO reductive and oxidative half reaction. Finally, we report on the time-dependent formation of the paramagnetic Mo(V) species following nitrite reduction and demonstrate that sulfite inhibits nitrite reduction. In conclusion, we propose a pH-dependent reciprocal regulation of sulfite oxidation and nitrite reduction by each substrate, thus facilitating quick responses to hypoxia induced changes in the IMS, which may function in protecting the cell from reactive oxygen species production.

Carboxydotrophic growth of Geobacter sulfurreducens.

This study shows that Geobacter sulfurreducens grows on carbon monoxide (CO) as electron donor with fumarate as electron acceptor. Geobacter sulfurreducens was tolerant to high CO levels, with up to 150 kPa in the headspace tested. During growth, hydrogen was detected in very slight amounts (∼5 Pa). In assays with cell-free extract of cells grown with CO and fumarate, production of hydrogen from CO was not observed, and hydrogenase activity with benzyl viologen as electron acceptor was very low. Taken together, this suggested that CO is not utilized via hydrogen as intermediate. In the presence of CO, reduction of NADP(+) was observed at a rate comparable to CO oxidation coupled to fumarate reduction in vivo. The G. sulfurreducens genome contains a single putative carbon monoxide dehydrogenase-encoding gene. The gene is part of a predicted operon also comprising a putative Fe-S cluster-binding subunit (CooF) and a FAD-NAD(P) oxidoreductase and is preceded by a putative CO-sensing transcription factor. This cluster may be involved in a novel pathway for CO oxidation, but further studies are necessary to ascertain this. Similar gene clusters are present in several other species belonging to the Deltaproteobacteria and Firmicutes, for which CO utilization is currently not known.

Improvement of the butanol production selectivity and butanol to acetone ratio (B:A) by addition of electron carriers in the batch culture of a new local isolate of Clostridium acetobutylicum YM1.

Improvement in the butanol production selectivity or enhanced butanol:acetone ratio (B:A) is desirable in acetone-butanol-ethanol (ABE) fermentation by Clostridium strains. In this study, artificial electron carriers were added to the fermentation medium of a new isolate of Clostridium acetobutylicum YM1 in order to improve the butanol yield and B:A ratio. The results revealed that medium supplementation with electron carriers changed the metabolism flux of electron and carbon in ABE fermentation by YM1. A decrease in acetone production, which subsequently improved the B:A ratio, was observed. Further improvement in the butanol production and B:A ratios were obtained when the fermentation medium was supplemented with butyric acid. The maximum butanol production (18.20 ± 1.38 g/L) was gained when a combination of methyl red and butyric acid was added. Although the addition of benzyl viologen (0.1 mM) and butyric acid resulted in high a B:A ratio of 16:1 (800% increment compared with the conventional 2:1 ratio), the addition of benzyl viologen to the culture after 4 h resulted in the production of 18.05 g/L butanol. Manipulating the metabolic flux to butanol through the addition of electron carriers could become an alternative strategy to achieve higher butanol productivity and improve the B:A ratio.

The autocatalytic step is an integral part of the hydrogenase cycle.

We earlier proved the involvement of an autocatalytic step in the oxidation of H(2) by HynSL hydrogenase from Thiocapsa roseopersicina, and demonstrated that two enzyme forms interact in this step. Using a modified thin-layer reaction chamber which permits quantitative analysis of the concentration of the reaction product (reduced benzyl viologen) in the reaction volume during the oxidation of H(2), we now show that the steady-state concentration of the product displays a strong enzyme concentration dependence. This experimental fact can be explained only if the previously detected autocatalytic step occurs inside the catalytic enzyme-cycle and not in the enzyme activation process. Consequently, both interacting enzyme forms should participate in the catalytic cycle of the enzyme. As far as we are aware, this is the first experimental observation of such a phenomenon resulting in an apparent inhibition of the enzyme. It is additionally concluded that the interaction of the two enzyme forms should result in a conformational change in the enzyme-substrate form. This scheme is very similar to that of prion reactions. Since merely a few molecules are involved at some point of the reaction, this process is entirely stochastic in nature. We have therefore developed a stochastic calculation method, calculations with which lent support to the conclusion drawn from the experiment.

Chlamydomonas reinhardtii chloroplasts contain a homodimeric pyruvate:ferredoxin oxidoreductase that functions with FDX1.

Eukaryotic algae have long been known to live in anoxic environments, but interest in their anaerobic energy metabolism has only recently gained momentum, largely due to their utility in biofuel production. Chlamydomonas reinhardtii figures remarkably in this respect, because it efficiently produces hydrogen and its genome harbors many genes for anaerobic metabolic routes. Central to anaerobic energy metabolism in many unicellular eukaryotes (protists) is pyruvate:ferredoxin oxidoreductase (PFO), which decarboxylates pyruvate and forms acetyl-coenzyme A with concomitant reduction of low-potential ferredoxins or flavodoxins. Here, we report the biochemical properties of the homodimeric PFO of C. reinhardtii expressed in Escherichia coli. Electron paramagnetic resonance spectroscopy of the recombinant enzyme (Cr-rPFO) showed three distinct [4Fe-4S] iron-sulfur clusters and a thiamine pyrophosphate radical upon reduction by pyruvate. Purified Cr-rPFO exhibits a specific decarboxylase activity of 12 µmol pyruvate min⁻¹ mg⁻¹ protein using benzyl viologen as electron acceptor. Despite the fact that the enzyme is very oxygen sensitive, it localizes to the chloroplast. Among the six known chloroplast ferredoxins (FDX1-FDX6) in C. reinhardtii, FDX1 and FDX2 were the most efficient electron acceptors from Cr-rPFO, with comparable apparent K(m) values of approximately 4 µm. As revealed by immunoblotting, anaerobic conditions that lead to the induction of CrPFO did not increase levels of either FDX1 or FDX2. FDX1, being by far the most abundant ferredoxin, is thus likely the partner of PFO in C. reinhardtii. This finding postulates a direct link between CrPFO and hydrogenase and provides new opportunities to better study and engineer hydrogen production in this protist.

Photochromic hybrid containing in situ-generated benzyl viologen and novel trinuclear [Bi3Cl14]5-: improved photoresponsive behavior by the π···π interactions and size effect of inorganic oligomer.

Two new member of (V)((2n+2)/2)[Bi(2n)Cl(8n+2)] series hybrids, (BzV)2[Bi2Cl10] (1) and (BzV)5[Bi3Cl14]2·(C6H5CH2)2O (2) (where BzV(2+) = N,N'-dibenzyl-4,4'-bipyridinium and (C6H5CH2)2O = dibenzyl ether) have been obtained, and compound 2 contains an unprecedented discrete trimer [Bi3Cl14](5-) counterion. The novel in situ-synthesized symmetric viologen cation with aromatic groups on both sides of 4,4'-bipy would provide more opportunities to create π···π interactions to optimize the photochromic property of the hybrid, and different bismuthated-halide oligomers enable us to discuss the size effect in this series of compounds. Both 1 and 2 are photochromic, and their photoresponsive rate is faster than that of reported viologen-metal halide hybrids. Experimental and theoretical data illustrated that the size of the inorganic oligomer can significantly influence the photoresponsive rate of the viologen dication, and the π···π interaction behaves as not only a powerful factor to stabilize the viologen monocation radical but also the second electron-transfer pathway, from a π-conjugated substituent to a viologen cation, for the photochromic process.

A simple chip free-flow electrophoresis for monosaccharide sensing via supermolecule interaction of boronic acid functionalized quencher and fluorescent dye.

Here, a simple micro free-flow electrophoresis (µFFE) was developed for fluorescence sensing of monosaccharide via supermolecule interaction of synthesized boronic acid functionalized benzyl viologen (ο-BBV) and fluorescent dye. The µFFE contained two open electrode cavities and an ion-exchange membrane was sandwiched between two polymethylmethacrylate plates. The experiments demonstrated the following merits of developed µFFE: (i) up to 90.5% of voltage efficiency due to high conductivity of ion-exchange membrane; (ii) a strong ability against influence of bubble produced in two electrodes due to open design of electrode cavities; and (iii) reusable and washable separation chamber (45 mm × 17 mm × 100 µm, 77 µL) avoiding the discard of µFFE due to blockage of solute precipitation in chamber. Remarkably, the µFFE was first designed for the sensing of monosaccharide via the supermolecule interaction of synthesized ο-BBV, fluorescent dye, and monosaccharide. Under the optimized conditions, the minimum concentration of monosaccharide that could be detected was 1 × 10(-11) M. Finally, the developed device was used for the detection of 0.3 mM glucose spiked in human urine. All of the results demonstrated the feasibility of monosaccharide detection via the µFFE.

The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity.

BACKGROUND: Escherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H2: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity. RESULTS: Here we report the identification of a new H2: benzyl viologen oxidoreductase enzyme activity in E. coli that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of E. coli mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes. CONCLUSIONS: The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of Escherichia coli have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme.

Efficient electron transfer from hydrogen to benzyl viologen by the [NiFe]-hydrogenases of Escherichia coli is dependent on the coexpression of the iron-sulfur cluster-containing small subunit.

Escherichia coli can both oxidize hydrogen and reduce protons. These activities involve three distinct [NiFe]-hydrogenases, termed Hyd-1, Hyd-2, and Hyd-3, each minimally comprising heterodimers of a large subunit, containing the [NiFe] active site, and a small subunit, bearing iron-sulfur clusters. Dihydrogen-oxidizing activity can be determined using redox dyes like benzyl viologen (BV); however, it is unclear whether electron transfer to BV occurs directly at the active site, or via an iron-sulfur center in the small subunit. Plasmids encoding Strep-tagged derivatives of the large subunits of the three E. coli [NiFe]-hydrogenases restored activity of the respective hydrogenase to strain FTD147, which carries in-frame deletions in the hyaB, hybC, and hycE genes encoding the large subunits of Hyd-1, Hyd-2, and Hyd-3, respectively. Purified Strep-HyaB was associated with the Hyd-1 small subunit (HyaA), and purified Strep-HybC was associated with the Hyd-2 small subunit (HybO), and a second iron-sulfur protein, HybA. However, Strep-HybC isolated from a hybO mutant had no other associated subunits and lacked BV-dependent hydrogenase activity. Mutants deleted separately for hyaA, hybO, or hycG (Hyd-3 small subunit) lacked BV-linked hydrogenase activity, despite the Hyd-1 and Hyd-2 large subunits being processed. These findings demonstrate that hydrogenase-dependent reduction of BV requires the small subunit.

A highly sensitive water-soluble system to sense glucose in aqueous solution.

A glucose sensing switch is formed by water soluble conjugated polymer (PP-S-BINOL) and boronic acid-functionalized benzyl viologen (o-BBV). The two-component system shows a high sensitivity for glucose sensing with a 17-fold increase in the fluorescence intensity in the presence of 100 mM glucose.

Deconstructing the electron transfer chain in a complex molybdoenzyme: Assimilatory nitrate reductase from Neurospora crassa.

Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV2+) and anthraquinone sulfonate (AQS-) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.

Characterization of a thioredoxin-thioredoxin reductase system from the hyperthermophilic bacterium Thermotoga maritima.

A thioredoxin reductase and a thioredoxin were purified to homogeneity from a cell extract of Thermotoga maritima. The thioredoxin reductase was a homodimeric flavin adenine dinucleotide (FAD)-containing protein with a subunit of 37 kDa estimated using SDS-PAGE, which was identified to be TM0869. The amino acid sequence of the enzyme showed high identities and similarities to those of typical bacterial thioredoxin reductases. Although the purified T. maritima thioredoxin reductase could not use thioredoxin from Spirulina as an electron acceptor, it used thioredoxin that was purified from T. maritima by monitoring the dithiothreitol-dependent reduction of bovine insulin. This enzyme also catalyzed the reduction of benzyl viologen using NADH or NADPH as an electron donor with apparent V(max) values of 1,111 +/- 35 micromol NADH oxidized min(-1)mg(-1) and 115 +/- 2.4 micromol NADPH oxidized min(-1)mg(-1), respectively. The apparent K(m) values were determined to be 89 +/- 1.1 microM, 73 +/- 1.6 microM, and 780 +/- 20 microM for benzyl viologen, NADH, and NADPH, respectively. Optimal pH values were determined to be 9.5 and 6.5 for NADH and NADPH, respectively. The enzyme activity increased along with the rise of temperature up to 95 degrees C, and more than 60% of the activity remained after incubation for 28 h at 80 degrees C. The purified T. maritima thioredoxin was a monomer with a molecular mass of 31 kDa estimated using SDS-PAGE and identified as TM0868, which exhibited both thioredoxin and thioltransferase activities. T. maritima thioredoxin and thioredoxin reductase together were able to reduce insulin or 5,5'-dithio-bis(2-nitrobenzoic acid) using NAD(P)H as an electron donor. This is the first thioredoxin-thioredoxin reductase system characterized from hyperthermophilic bacteria.

Methods for determining the reduction potentials of flavin enzymes.

A key factor for flavoenzyme activity is the reduction potential of the bound flavin. The reduction potentials of protein-bound flavins span approximately a 500-mV range consistent with flavoenzymes having critical roles in metabolism and a variety of biological processes. Redox potentials of flavoenzymes have traditionally been determined using an electrode-based system with either direct or indirect electrochemical coupling between the protein and the working electrode. An electrode independent method, however, is also now commonly used and involves calculating the unknown flavin reduction potential of the protein from the known reduction potential of a reference or indicator dye. Here, the "classic" potentiometric method and the xanthine/xanthine oxidase methods are described. Both methods rely on equilibrium between protein-bound flavin and redox dyes. The potentiometric method measures the equilibrated redox potential of the protein-dye mixture whereas the xanthine/xanthine oxidase technique relies on slow continuous enzymatic reduction to maintain a constant equilibrium between the protein and the dyes. Because electrochemical equipment is not required, the xanthine/xanthine oxidase method is more accessible and convenient for researchers seeking to determine reduction potentials of flavoproteins or other biological redox centers such as hemes. The xanthine/xanthine oxidase method has been used to determine flavin reduction potentials from +132 to -417mV, demonstrating it is suitable for characterizing the redox properties of most flavoproteins.

EPR spectroscopy of putative enzyme intermediates in the NO reductase and the auto-nitrosylation reaction of Desulfovibrio vulgaris hybrid cluster protein.

The hybrid cluster protein (Hcp) contains a unique 4Fe cluster that is a hybrid of µ-S and µ-O bridges. Escherichia coli Hcp has recently been found to carry NO reductase activity as well as S-nitrosylation activity in NO-based signaling. In other species, the physiological activity has not been established. No reaction mechanism of any Hcp has been proposed. Here, we show that Desulfovibrio vulgaris (Hildenborough) Hcp has nitric oxide reductase activity with benzyl viologen as electron donor. With EPR spectroscopy, we identify three unexpected putative reaction intermediates: both in reduced and oxidized Hcp, dinitrosyl iron complexes are formed. Also, the hybrid cluster in reduced Hcp, but not in oxidized Hcp, binds the product N2 O. Possible implications for a reaction mechanism are discussed.

Electro-fluorescence switching of bis-imidazolium ionic liquids.

Room-temperature ionic liquids (RTILs) containing bis-imidazolium salts were explored to control their optical properties of them in an organic device. The neat bis-imidazolium salts showed ionic conductivity of 3.5 x 10(-4) S/cm at room-temperature and the electrochemical window was exhibited within +/-2.5 V in a two electrode cell. The bis-imidazolium salts were transparent yellow and showed fluorescence upon excitation with light in the range of 360 to 500 nm. A two electrode organic ionic liquid cell was fabricated using a mixture of PEO200Blm-TFSI and electroactive molecules to control the emission property of the ionic liquid by electrochemical methods. The first example of the ionic liquid containing electro-fluorescence switch was explored with an electroactive benzyl viologen (BzV) blend of bis-imidazolium ionic liquid.

[Effect of oxidative stress inductors on the photosynthetic apparatus in cyanobacterium Synechocystis sp. PCC 6803 Prq20 mutant resistant to methyl viologen].

The damaging effect of oxidative stress inductors: methyl viologen, benzyl viologen, cumene hydroperoxide, H2O2, menadion, and high irradiance on the photosynthetic apparatus of cyanobacterium Synechocystis sp. PCC 6803 in cells of the wild type strain and the methyl viologen-resistant Prq20 mutant with the disrupted function of the regulatory gene prqR has been investigated by measuring the delayed fluorescence of chlorophyll a and the rate of CO2dependent -O2 gas exchange. It has been shown that the damage to the photosynthetic apparatus in the Prq20 mutant as compared with the wild type was less in the presence of methyl viologen and benzyl viologen. Reasons for the enhanced resistance of the photosynthetic apparatus in the mutant Prq20 to methyl viologen and benzyl viologen are discussed.

[Deep venous thrombosis of the upper limb in a violin player: The "bow syndrome"]. / Thrombose veineuse profonde du membre supérieur chez une violoniste : « le syndrome de l'archet ¼.

BACKGROUND: Exercise-induced thrombosis is a rare cause of deep venous thrombosis (DVT) of the upper limb and usually affects young subjects without comorbid conditions. The diagnosis may be challenging. CASE REPORT: A 23-year-old female right-handed French teacher and amateur violin player presented with edema of the root of the right arm associated with erythrocyanosis of the extremity and collateral circulation of the shoulder. History taking revealed oral contraception and recent change in violin playing habits. D-dimers were negative. A second duplex-Doppler was required before visualization of a DVT in the right subclavian vein. The patient was given low-molecular-weight heparin alone, followed by rivaroxaban. The outcome was very favorable at 48h. The patient was seen at 4 months and had not had a recurrent episode. DISCUSSION: The diagnosis of DVT of the upper limb is basically clinical. There is a clinical probability score for the introduction of anticoagulation even if the duplex-Doppler fails to visualize DVT, a situation that can occur due to the clavicular superposition in this region. Exercise-induced DVT should be suspected in patients with minimally intense but repeated exercise (hyper-abduction), e.g. as here playing the violin. Anticoagulation is the treatment of choice. The role for surgery and pharmacomechanical strategies remains to be defined. CONCLUSION: Exercise-induced thrombosis (Paget-Schroetter syndrome) should be suspected in young patients free of any comorbidity who develop a thrombosis of the upper limb. Studies comparing different therapeutic options would be useful to achieve more homogeneous management practices despite the heterogeneous clinical presentations.

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors.

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced. 2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the "resting" oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis. 3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a3 (k1obs = 7.5 s-1 for the oxygen-pulsed oxidase). 4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the "oxygenated" compound, but there were small differences between the two spectra.

In vitro complementation of assimilatory NAD(P)H-nitrate reductase from mutants of Chlamydomonas reinhardii.

In vitro complementation of the soluble assimilatory NAD(P)H-nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) was attained by mixing cell-free preparations of Chlamydomonas reinhardii mutant 104, uniquely possessing nitrate-inducible NAD(P)H-cytochrome c reductase, and mutant 305 which possesses solely the nitrate-inducible FMNH2- and reduced benzyl viologen-nitrate reductase activities. Full activity and integrity of NAD(P)H-cytochrome c reductase from mutant 104 and reduced benzyl viologen-nitrate reductase from mutant 305 are needed for the complementation to take place. A constitutive and heat-labile molybdenum-containing cofactor, that reconstitutes the NAD(P)H-nitrate reductase activity of nit-1 Neurospora crassa but is incapable of complementing with 104 from C. reinhardii, is present in the wild type and 305 algal strains. The complemented NAD(P)H-nitrate reductase has been purified 100-fold and was found to be similar to the wild enzyme in sucrose density sedimentation, molecular size, pH optimum, kinetic parameters, substrate affinity and sensitivity to inhibitors and temperature. From previous data and data presented in this article on 104 and 305 mutant activities, it is concluded that C. reinhardii NAD(P)H-nitrate reductase is a heteromultimeric complex consisting of, at least, two types of subunits separately responsible for the NAD(P)H-cytochrome c reductase and the reduced benzyl viologen-nitrate reductase activities.