Reduced Methyl Viologen Research Articles

Photoelectrochemical CO2 Reduction to Formate with the Sacrificial Reagent Free System of Semiconductor Photocatalysts and Formate Dehydrogenase

AbstractEfficient sacrificial reagent free (and no external bias) photoelectrochemical CO2 reduction to formate with a hybrid system consisting of semiconductor‐based photocatalytic TiO2 nanoparticles, methylviologen (MV) as an electron carrier, and biocatalytic formate dehydrogenase (FDH) was investigated. To develop the sacrificial reagent free photoelectrochemical cell for the CO2 reduction to formate, the photoanode based on TiO2 film and cathode based on the carbon fabric paper (CFP) were separated into two compartments using a proton‐permeable Nafion film to prevent gas diffusion. When the anode was irradiated with Xenon lamp (>300 nm), MV reduction and CO2 reduction to formate with FDH on the cathode side proceeded in this system. For MV reduction with photoelectrochemical cell, MV was reduced using TiO2 film at each pH (6, 7 and 8) without bias, the reduction of MV did not progressed at pH 6. In contrast, MV reduction was progressed at pH 7 or 8. For CO2 reduction to formate in the presence FDH on the cathode side with photoelectrochemical cell, the rate of formate production was estimated to be 31.9 nmol h−1 at pH 8 under continuous irradiation without bias. Moreover, the rates of formate production at pH 7 and 8 were estimated to be 56.5 and 168.8 nmol h−1, respectively under continuous irradiation with bias (0.4 V vs. cathode). By using this system, we have succeeded in constructing CO2 reduction to formate using water as an electron donor, which does not require a sacrificial reagent.

Effect of conductive substrate on the photoelectrochemical properties of Cu2O film electrodes for methyl viologen reduction

Cuprous oxide (Cu2O) is a candidate for low-cost p-type semiconductor oxides for photoelectrochemical solar energy conversion. The Cu2O crystalline films electrodeposited on conductive substrates have been extensively studied as photocathodes because of their moderate photoelectrochemical performance, but their incident photon-to-current conversion efficiency (IPCE) was still limited because of the incompatibility between minority carrier diffusion length and light absorption depth in planar structures. One of the strategies to solve this issue is to decrease the thickness of the Cu2O films with keeping the loading amount. Herein, we used a sintered titanium microfiber felt as a three-dimensional macroporous scaffold for Cu2O crystalline thin films because it exhibits a large surface area compared with the conventional flat substrate. The effect of the Cu2O film thickness on the photocathode properties was investigated using methyl viologen as an acceptor of the photoexcited electrons. The Cu2O thin films deposited on Ti microfibers were superior to that of the Cu2O films deposited on dense planar substrates such as fluorine-doped tin oxide (FTO)-coated glass and titanium sheets. It was revealed that the large Cu2O–electrolyte and Cu2O–substrate interfaces resulted in the enhancement of IPCE for the photoelectrochemical cathodic reaction.

Visible-light driven hydrogen production using chlorophyll derivatives conjugated with a viologen moiety in the presence of platinum nanoparticles.

The utilization of the light-harvesting and electron-transferring function of chlorophylls (Chls) has received attention for visible-light driven hydrogen production. In this work, a series of Chl derivatives based on pyropheophorbide-a (Pyro-a) conjugated with a viologen moiety, including a Pyro-a methyl ester directly bonded with the viologen at the 3-position 1, its 31-methylene analog 2 and Pyro-a connected with the viologen in the 17-substituent 3, were synthesized from chemical modification of naturally occurring Chl-a and characterized in terms of their photochemical and photophysical properties. As the photoexcited singlet state of the Pyro-a moiety was strongly quenched by the viologen moiety in a molecule, the effective photoinduced intramolecular electron transfer from Pyro-a to the bonded viologen moiety occurred. Moreover, these molecules were applied as a photosensitizer in the system for visible-light driven hydrogen production with platinum nanoparticles via intramolecular reduction of the bonded viologen moiety. Efficient photoreduction of external methyl viologen and successive hydrogen production on platinum nanoparticles were achieved using the synthetic conjugate of Pyro-a with the viologen moiety as a photosensitizer. In particular, effective visible-light driven hydrogen production was accomplished using 3 and platinum nanoparticles via the reduction of external methyl viologen.

Upgraded Bioelectrocatalytic N2 Fixation: From N2 to Chiral Amine Intermediates.

Enantiomerically pure chiral amines are of increasing value in the preparation of bioactive compounds, pharmaceuticals, and agrochemicals. ω-Transaminase (ω-TA) is an ideal catalyst for asymmetric amination because of its excellent enantioselectivity and wide substrate scope. To shift the equilibrium of reactions catalyzed by ω-TA to the side of the amine product, an upgraded N2 fixation system based on bioelectrocatalysis was developed to realize the conversion from N2 to chiral amine intermediates. The produced NH3 was in situ reacted with l-alanine dehydrogenase to generate alanine with NADH as a coenzyme. ω-TA transferred the amino group from alanine to ketone substrates and finally produced the desired chiral amine intermediates. The cathode of the upgraded N2 fixation system supplied enough reducing power to synchronously realize the regeneration of reduced methyl viologen (MV•+) and NADH for the nitrogenase and l-alanine dehydrogenase. The coproduct, pyruvate, was consumed by l-alanine dehydrogenase to regenerate alanine and push the equilibrium to the side of amine. After 10 h of reaction, the concentration of 1-methyl-3-phenylpropylamine achieved 0.54 mM with the 27.6% highest faradaic efficiency and >99% enantiomeric excess (eep). Because of the wide substrate scope and excellent enantioselectivity of ω-TA, the upgraded N2 fixation system has great potential to produce a variety of chiral amine intermediates for pharmaceuticals and other applications.

Identification of a nitrite reductase in Pseudomonas aeruginosa as a potential antimicrobial target

The opportunistic pathogen Pseudomonas aeruginosa utilizes a wide range of virulence factors to adapt to the host environment. With the antimicrobial pipeline drying up, understanding and targeting virulence factors for therapeutic development is an exciting alternative for the discovery of novel disease inhibitors. An integrated genome-wide transposon mutagenesis screening approach was performed in P. aeruginosa using multiple in vivo disease models with the aim to identify new virulence factors required for infection. A mutant attenuated in the production of multiple virulence determinants using in vitro assays was identified. This mutant also showed severe attenuation using in vivo models with up to an 80 % increased survival in murine chronic and acute lung infection models. The predicted protein coded by the mutated gene showed homology to nitrite and sulphite reductases. Using a methyl viologen reduction assay, we have shown that this gene encondes a nitrite reductase, operating in a siroheme and 4Fe-4S dependant manner. The preference for nitrite and the requirement of siroheme revealed that product of this gene is an assimilatory nitrite reductase and hence we propose it to be named as NirA. Work is now on-going to understand how NirA contributes to virulence and determine the crystal structure of this protein with a view to screen for novel inhibitors of this enzyme using a drug discovery platform available in our laboratories.

Sensitizing effects of BiVO4 and visible light induced production of highly reductive electrons in the TiO2/BiVO4 heterojunction

Abstract BiVO4 is an efficient and stable visible active photoanode material. However, due to its unfavorable conduction band position which falls below the H2 reduction potential, it fails to carry out the complete water splitting reaction. On the other hand, larger band gap TiO2 is able to photocatalytically split water, thanks to its negative conduction band energy. Aiming at verifying the possibility of sensitizing TiO2 with BiVO4 and employing the so obtained composite material in photocatalytic water splitting under visible light, we prepared and photoelectrochemically characterized TiO2/BiVO4 heterojunction electrodes. The photocatalytic reduction of methyl viologen, an electron acceptor probe with a reduction potential close to that of protons, was used to evaluate the reducing ability of the photoactive materials under visible light. An apparently counterintuitive electron transfer from photoexcited BiVO4 to the TiO2 conduction band occurs in the TiO2/BiVO4 heterojunction, resulting in TiO2 sensitization and production of highly reductive electrons, which appears to be favored by the band alignment occurring at the heterojunction.

Expression, purification and characterization of an active C491G variant of ferredoxin sulfite reductase from Synechococcus elongatus PCC 7942

Recently developed molecular wire technology takes advantage of [4Fe-4S] clusters that are ligated by at least one surface exposed Cys residue. Mutagenesis of this Cys residue to a Gly opens an exchangeable coordination site to a corner iron atom that can be chemically rescued by an external thiolate ligand. This ligand can be subsequently displaced by mass action using a dithiol molecular wire to tether two redox active proteins. We intend to apply this technique to tethering Photosystem I to ferredoxin sulfite reductase (FdSiR), an enzyme that catalyzes the six-electron reduction of sulfite to hydrogen sulfite and nitrite to ammonia. The enzyme contains a [4Fe-4S]2+/1+ cluster and a siroheme active site. FdSiRWT and an FdSiRC491G variant were cloned from Synechococcus elongatus PCC 7942 and expressed along with the cysG gene from Salmonella typhimurium using the pCDFDuet plasmid. UV/Vis absorbance spectra of both FdSiRWT and the FdSiRC491G variant displayed characteristic peaks at 278, 392 (Soret), 585 (α) and 714 nm (charge transfer band), and 278, 394 (Soret), 587 (α) and 714 nm (charge transfer band) respectively. Both enzymes in their as-isolated forms displayed an EPR spectrum characteristic of an S = 5/2 high spin heme. When reduced, both enzymes exhibited the signal of a low spin S = 1/2 [4Fe-4S]1+ cluster. The FdSiRWT and FdSiRC491G variant both showed activity using reduced methyl viologen and Synechococcus elongatus PCC 7942 ferredoxin 1 (Fd1) as electron donors. Based on these results, the FdSIRC491G variant should be a suitable candidate for wiring to Photosystem I.

SELENOPROTEIN O is a chloroplast protein involved in ROS scavenging and its absence increases dehydration tolerance in Arabidopsis thaliana

The evolutionary conserved family of Selenoproteins performs redox-regulatory functions in bacteria, archaea and eukaryotes. Among them, members of the SELENOPROTEIN O (SELO) subfamily are located in mammalian and yeast mitochondria, but their functions are thus far enigmatic. Screening of T-DNA knockout mutants for resistance to the proline analogue thioproline (T4C), identified mutant alleles of the plant SELO homologue in Arabidopsis thaliana. Absence of SELO resulted in a stress-induced transcriptional activation instead of silencing of mitochondrial proline dehydrogenase, and also high elevation of Δ(1)-pyrroline-5-carboxylate dehydrogenase involved in degradation of proline, thereby alleviating T4C inhibition and lessening drought-induced proline accumulation. Unlike its animal homologues, SELO was localized to chloroplasts of plants ectopically expressing SELO-GFP. The protein was co-fractionated with thylakoid membrane complexes, and co-immunoprecipitated with FNR, PGRL1 and STN7, all involved in regulating PSI and downstream electron flow. The selo mutants displayed extended survival under dehydration, accompanied by longer photosynthetic activity, compared with wild-type plants. Enhanced expression of genes encoding ROS scavenging enzymes in the unstressed selo mutant correlated with higher oxidant scavenging capacity and reduced methyl viologen damage. The study elucidates SELO as a PSI-related component involved in regulating ROS levels and stress responses.

Two electron utilization of methyl viologen anolyte in nonaqueous organic redox flow battery

Abstract Methyl viologen (MV) as a bench-mark anolyte material has been frequently applied in aqueous organic redox flow batteries (AORFBs) towards large-scale renewable energy storage. However, only the first reduction of MV was utilized in aqueous electrolytes because of the insoluble MV0 generated from the second reduction of MV. Herein, we report that methyl viologen with bis(trifluoromethane)sulfonamide counter anion, MVTFSI, can achieve two reversible reductions in a nonaqueous supporting electrolyte. Paired with (Ferrocenylmethyl)trimethylammonium bis(trifluoromethanesulfonyl)imide, FcNTFSI, as catholyte, the MVTFS/FcNTFSI nonaqueous organic redox flow battery (NOARFB) can take advantage of either one electron or two electron storage of the methyl viologen moiety and provide theoretical energy density of 24.9 Wh/L and a cell voltage of up to 1.5 V. Using a highly conductive LiTFSI/CH3CN supporting electrolyte and a porous Daramic separator, the NOARFB displayed excellent cycling performance, including up to a 68.3% energy efficiency at 40 mA/cm2, and more than 88% total capacity retention after 100 cycles.

Photochemistry of uranyl complexes with the salicylidene-α-hydroxy acid chelates, X-Sal-AHA

Two dimeric uranyl complexes, K2[UO2(3,5-diCl-Sal-AHA)]2 (1) and Na2[UO2(3,5-ditbutyl-Sal-AHA)]2 (2), were synthesized from salicylidene-α-hydroxy acid-containing chelates of the X-Sal-AHA series. Both are photochemically reactive upon irradiation with UV light, with quantum yields of 4.2% and 2.9% for 1 and 2, respectively. Irradiation results in similar spectroscopic changes for 1 and 2, which differ for each depending on the presence of air. The photolysis products include an air-sensitive U(IV) species, which can reduce methyl viologen, and an aldehyde resulting from decarboxylation of the chelate. The photochemistry of 1 and 2 is more similar to that of the Fe(III) complexes of the X-Sal-AHA chelates than to the typical photochemical reaction of uranyl nitrate in solution.

Light-harvesting chlorophyll protein (LHCII) drives electron transfer in semiconductor nanocrystals

Type-II quantum dots (QDs) are capable of light-driven charge separation between their core and the shell structures; however, their light absorption is limited in the longer-wavelength range. Biological light-harvesting complex II (LHCII) efficiently absorbs in the blue and red spectral domains. Therefore, hybrid complexes of these two structures may be promising candidates for photovoltaic applications. Previous measurements had shown that LHCII bound to QD can transfer its excitation energy to the latter, as indicated by the fluorescence emissions of LHCII and QD being quenched and sensitized, respectively. In the presence of methyl viologen (MV), both fluorescence emissions are quenched, indicating an additional electron transfer process from QDs to MV. Transient absorption spectroscopy confirmed this notion and showed that electron transfer from QDs to MV is much faster than fluorescence energy transfer between LHCII and QD. The action spectrum of MV reduction by LHCII-QD complexes reflected the LHCII absorption spectrum, showing that light absorbed by LHCII and transferred to QDs increased the efficiency of MV reduction by QDs. Under continuous illumination, at least 28 turnovers were observed for the MV reduction. Presumably, the holes in QD cores were filled by a reducing agent in the reaction solution or by the dihydrolipoic-acid coating of the QDs. The LHCII-QD construct can be viewed as a simple model of a photosystem with the QD component acting as reaction center.

New Rh2(II,II) Complexes for Solar Energy Applications: Panchromatic Absorption and Excited-State Reactivity.

The new heteroleptic paddlewheel complexes cis-[Rh2(μ-form)2(μ-np)2][BF4]2, where form = p-ditolylformamidinate (DTolF) or p-difluorobenzylformamidinate (F-form) and np = 1,8-napthyridyine, and cis-Rh2(μ-form)2(μ-npCOO)2 (npCOO- = 1,8-naphthyridine-2-carboxylate), were synthesized and characterized. The complexes absorb strongly throughout the ultraviolet (λmax = 300 nm, ε = 20 300 M-1 cm-1) and visible regions (λmax = 640 nm ε = 3500 M-1 cm-1), making them potentially useful new dyes with panchromatic light absorption for solar energy conversion applications. Ultrafast and nanosecond transient absorption and time-resolved infrared spectroscopies were used to characterize the identity and dynamics of the excited states, where singlet and triplet Rh2/form-to-naphthyridine, metal/ligand-to-ligand charge-transfer (ML-LCT) excited states were observed in all four complexes. The npCOO- complexes exhibit red-shifted absorption profiles extending into the near-IR and undergo photoinitiated electron transfer to generate reduced methyl viologen, a species that persists in the presence of a sacrificial donor. The energy of the triplet excited state of each complex was estimated from energy-transfer quenching experiments using a series of organic triplet donors (E(3ππ*) from 1.83 to 0.78 eV). The singlet reduction (+0.6 V vs Ag/AgCl) potentials, and singlet and triplet oxidation potentials (-1.1 and -0.5 V vs Ag/AgCl, respectively) were determined. Based on the excited-state lifetimes and redox properties, these complexes represent a new class of light absorbers with potential application as dyes for charge injection into semiconductor solar cells and in sensitizer-catalyst assemblies for photocatalysis that operate with irradiation from the ultraviolet to ∼800 nm.

Hydrogen-Bonded Polymer-Porphyrin Assemblies in Water: Supramolecular Structures for Light Energy Conversion.

In this study, a new type of functional, self-assembled nanostructure formed from porphyrins and polyamidoamine dendrimers based on hydrogen bonding in an aqueous solution is presented. As the aggregates formed are promising candidates for solar-energy conversion, their photocatalytic activity is tested using the model reaction of methyl viologen reduction. The self-assembled structures show significantly increased activity as compared to unassociated porphyrins. Details of interaction forces driving the supramolecular structure formation and regulating catalytic efficiency are fundamentally discussed.

A bacterial chloroform reductive dehalogenase: purification and biochemical characterization.

SummaryWe report herein the purification of a chloroform (CF)‐reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23‐fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X‐100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS‐PAGE and MALDI‐TOF/TOF. The purified dehalogenase reductively dechlorinated CF to dichloromethane in vitro with reduced methyl viologen as the electron donor at a specific activity of (1.27 ± 0.04) × 103 units mg protein−1. The optimum temperature and pH for the activity were 45°C and 7.2, respectively. The UV‐visible spectrometric analysis indicated the presence of a corrinoid and two [4Fe‐4S] clusters, predicted from the amino acid sequence. This is the first report of the production, purification and biochemical characterization of a CF reductive dehalogenase.

Inorganic/whole-cell biohybrid photocatalyst for highly efficient hydrogen production from water

To obtain a clean hydrogen production system, we have developed an inorganic-bio hybrid photocatalyst system based on the combination of anatase TiO2, methylviologen (MV) as an electron mediator, and a whole-cell biocatalyst consisting of [FeFe]-hydrogenase and maturase gene-harboring recombinant Escherichia coli; however, the apparent quantum yield at 300nm (AQY300) for hydrogen production was low (0.3%). The system consists of a two-step reaction: (1) photocatalytic MV reduction by TiO2, and (2) hydrogen production with reduced MV using a biocatalyst. The enhancement of step 1 under biocatalyst-friendly conditions was investigated in an attempt to further improve the reaction efficiency. Among the condition tested, the use of 100mM Tris-HCl (pH 7), 150mM NaCl, and 5% (v/v) glycerol with P-25 TiO2 especially enhanced the step 1 reaction by a 300-fold increase in the MV reduction rate compared with previously tested reaction condition (100mM Tris-HCl (pH 7), 150mM NaCl, 5% (v/v) glycerol, and 100mM ascorbate with anatase TiO2). Under the enhanced step 1 reaction, AQY300 and AQY350 for photocatalytic MV reduction reached 60.8% and 52.2%, respectively. The enhanced step 1 reaction thus significantly improved the overall photocatalytic hydrogen productivity of the hybrid system and AQY300 and AQY350 reached 26.4% and 31.2%, respectively. The inorganic-whole-cell biohybrid system can therefore provide noble metal-free, efficient, and clean hydrogen production.

Improving Photocatalytic Activity: Versatile Polyelectrolyte – Photosensitizer Assemblies for Methyl Viologen Reduction

AbstractIn this study, we introduce a versatile polyelectrolyte‐photosensitizer self‐assembled system based on non‐covalent interaction showing highly efficient photocatalytic behavior in terms of methyl viologen reduction. The photocatalytic performance of the dendrimer‐dye system under visible light irradiation is modified to be more than 6‐times higher compared to the performance of the pure photosensitizer Rose Bengal. By adjusting the pH the self‐assembled structure can be size‐controlled. At the same time the underlying interaction forces change from mainly electrostatic nature to hydrogen and halogen bonding, a combination of forces reported here for the first time for dendrimer based assemblies. While strong electrostatic interaction of oppositely charged components at low pH hinders catalytic activity, the structure exhibits highly increased efficiency upon structure formation at high pH. Thus, a pH and loading ratio dependent photocatalytic system with potential for efficient solar energy conversion via photocatalysis has been established.

Effect of Liposomes on the Kinetics and Mechanism of the Photocatalytic Reduction of Methyl Viologen.

Liposomes are interesting scaffolds for photocatalysis. In particular, charged liposomes were shown to increase the quantum efficiency of photocatalytic reactions involving charged porphyrin photosensitizers and charged electron acceptors. In this work, the effects of adding positively charged liposomes (DMPC/eDMPCCl 1:1) on the mechanism of the photocatalytic reduction of methyl viologen (MV(2+)) by cysteine in the presence of sodium meso-tetra-(4-sulfonato)porphyrinatozinc (Na41) were probed by modeling UV-vis spectroscopy data using a steady-state approximation. By varying the concentration of methyl viologen, we found that the liposomes not only prevent the formation of a 1:1 complex between ground-state photosensitizer 1(4-) and MV(2+) but also that they increase the cage-escape yield in the excited state. Furthermore, the electrostatic repulsion between the liposomes and MV(2+) diminishes by 1 order of magnitude the rate of oxidative quenching of the photosensitizer triplet excited state ((T)1(4-)) by MV(2+). By varying the amount of sacrificial electron donor (cysteine), the effect of liposome addition on the charge recombination reactions could also be studied. Because of the positive charge borne by the photoproduct MV(•+), it was also repelled from the membrane, which significantly slows charge recombination at the surface of the liposome. Overall, compared to a liposome-free solution, the rates of most elementary steps of the photocatalytic reduction of MV(2+) by cysteine are strongly modified when the negative photosensitizer is adsorbed on a positively charged liposome surface. These results not only explain the much higher efficiency of the liposome-containing system but also illustrate the power of supramolecular chemistry for the tuning of photocatalysis.

Modulation of Cu(2-x)S Nanocrystal Plasmon Resonance through Reversible Photoinduced Electron Transfer.

Copper sulfide (Cu(2-x)S) nanocrystals with nonstoichiometric composition exhibit plasmon resonance in the near-infrared region. Compositional changes and varying electron density markedly affect the position and intensity of the plasmon resonance. We report a photochemically induced phenomenon of modulating the plasmon resonance in a controlled fashion. As photogenerated reduced methyl viologen radicals transfer electrons to Cu(2-x)S in inert solutions, we observe a decrease in localized surface plasmon resonance (LSPR) absorbance at 1160 nm. Upon exposure to air, the plasmon resonance band recovers as stored electrons are scavenged away by oxygen. This cycle of electron charge and discharge of Cu(2-x)S nanocrystals is reversible and can be repeated through photoirradiation in N2 saturated solution followed by exposure of the suspension to air. The spectroscopic studies that provide mechanistic insights into the reversible charging and discharging of plasmonic Cu(2-x)S are discussed.

Cost-Effective Electrophoretic Deposition of Cu2ZnSnS4 Nanocrystals for Photovoltaic Films

For the first time, kesterite Cu2ZnSnS4 (CZTS) thin films were efficiently obtained by electrophoretic deposition (EPD) of CZTS nanocrystal (NC) dispersion in an environmentally-friendly solvent, isopropanol. By regulating the applied potential, deposition time, and NC suspension concentration, the EPD process was successfully optimized. Photoelectrochemical measurements (PECMs) were carried out to test the EPD film performance, where the photocurrent density generated from the reduction of methyl viologen (MV2+) was evaluated. EPD films showed a photocurrent three times higher than those obtained by dropcasting at the same concentration. X-ray diffraction and Raman spectroscopy confirmed the formation of a single phase Cu2ZnSnS4 kesterite structure, without impurities. Intensity modulated photocurrent spectroscopy was used to determine the hole-electron recombination and product separation kinetics in the photoprocesses. Lastly, UV-visible spectroscopy determined the bandgap as 1.45 eV, which is an ideal value for single junction solar cell devices. The EPD approach can be combined with our optimized one-pot synthesis of CZTS NCs, which avoids annealing steps after the deposition. Based on these findings, EPD is anticipated to become one of the most promising routes toward fabrication of solar cell devices.

Identification of Amino Acids at the Catalytic Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase.

An in silico model of the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942, and information about active sites in related enzymes, had identified Cys148, Met149, Met306, Asp163, and Arg351 as amino acids likely to be involved in either nitrate binding, prosthetic group binding, or catalysis. Site-directed mutagenesis was used to alter each of these residues, and differences in enzyme activity and substrate binding of the purified variants were analyzed. In addition, the effects of these replacements on the assembly and properties of the Mo cofactor and [4Fe-4S] centers were investigated using Mo and Fe determinations, coupled with electron paramagnetic resonance spectroscopy. The C148A, M149A, M306A, D163N, and R351Q variants were all inactive with either the physiological electron donor, reduced ferredoxin, or the nonphysiological electron donor, reduced methyl viologen, as the source of electrons, and all exhibited changes in the properties of the Mo cofactor. Charge-conserving D163E and R351K variants were also inactive, suggesting that specific amino acids are required at these two positions. The implications for the role of these five conserved active-site residues in light of these new results and previous structural, spectroscopic, and mutagenesis studies for related periplasmic nitrate reductases are discussed.