Chloride inhibition of spinach nitrate reductase

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V_max and K_m for NO₃⁻ yielding V_max values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K_m values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K_m of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K_i of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO₃⁻ complex. Binding of Cl⁻ to the enzyme-NO₃⁻ complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.     

Electrochemically driven catalysis of the bacterial molybdenum enzyme YiiM

The Mo-dependent enzyme YiiM enzyme from Escherichia coli is a member of the sulfite oxidase family and shares many similarities with the well-studied human mitochondrial amidoxime reducing component (mARC). We have investigated YiiM catalysis using electrochemical and spectroscopic methods. EPR monitored redox potentiometry found the active site redox potentials to be Mo^VI/V –0.02 V and Mo^V/IV –0.12 V vs NHE at pH 7.2. In the presence of methyl viologen as an electrochemically reduced electron donor, YiiM catalysis was studied with a range of potential substrates. YiiM preferentially reduces N-hydroxylated compounds such as hydroxylamines, amidoximes, N-hydroxypurines and N-hydroxyureas but shows little or no activity against amine-oxides or sulfoxides. The pH optimum for catalysis was 7.1 and a bell-shaped pH profile was found with pK_a values of 6.2 and 8.1 either side of this optimum that are associated with protonation/deprotonations that modulate activity. Simulation of the experimental voltammetry elucidated kinetic parameters associated with YiiM catalysis with the substrates 6–hydroxyaminopurine and benzamidoxime.     

Benzyl viologen cation radical: First example of a perfectly selective anion ionophore of the carrier type

Measurements were made of the effect of dicationic (oxidized) and monocationic radical (reduced) forms of benzyl viologen (BV) and methyl viologen (MV) on the ion conductance across planar phospholipid bilayers under conditions of constant voltage. BV⁺ at 60 μM greatly increased ion conductance whereas BV²⁺, MV⁺ and MV²⁺ did not. Ion permeability ratios relative to nitrate were determined in the BV⁺ system. BV⁺ appears to be the first example of a perfectly anion-selective ionophore of the carrier type. BV⁺ probably functions both as an electron carrier and ionophore for nitrate while catalyzing the dithionite-nitrate reductase reaction in $\text{Paracoccus}\text{denitrificans}$.     

Kinetic parameters for hydrogen evolution by the NAD-linked hydrogenase of Alcaligenes eutrophus

The hydrogen-evolving reaction of the purified soluble NAD-linked hydrogenase of Alcaligenes eutrophus was used to determine kinetic parameters of the enzyme. The H₂-evolving activity with methyl viologen as electron mediator was 20-fold as compared to that with NADH. In the assay with dithionite-reduced methyl viologen (K _m 0.7 mM) the hydrogenase was most active at a redox potential of –560 mV and exhibited a pH optimum of 7.0. The K _m for protons, the second substrate for H₂ evolution, was 6.2 nM. With electrochemically reduced methyl viologen the pH optimum was shifted to pH 6.0. Double-reciprocal plots of reaction rates versus proton concentrations intercepted at the ordinate for different methyl viologen concentrations. At different pH values such an intercept was also observed with the dye as the varied substrate. The kinetic data are diagnostic for an ordered bisubstrate mechanism where both substrates are bound before the product H₂ is released. Hydrogenase coupled to thylakoid membranes resulted in a constant H₂ evolution rate over 6 h. The system appeared to be limited by the capacity of the thylakoid membranes.     

Photophysics of ruthenium(II) complexes carrying amino acids in the ligand 2,2′-bipyridine and intramolecular electron transfer from methionine to photogenerated Ru(III)

New ruthenium(II) complexes carrying methionine and phenylalanine in the bipyridine ligand, [Ru(bpy)₂(4-Me-4′-(CONH-l-methionine methyl ester)-2,2′-bipyridine)](PF₆)₂ (IV) and [Ru(bpy)₂(4-Me-4′-(CONH-l-phenylalanine ethyl ester)-2,2′-bpy)](PF₆)₂(V) have been synthesized and characterized and their photophysical properties studied. Flash photolysis measurements of complex IV, in the presence of an electron acceptor, methyl viologen (MV²⁺) show that an intermolecular electron transfer from the excited state of Ru(II) in complex IV, to MV²⁺ takes place, forming Ru(III) and the methyl viologen cation radical, MV⁺. The formation of MV⁺ in this system is confirmed using time-resolved transient absorption spectroscopy. This intermolecular electron transfer is followed by intramolecular electron transfer from the thioether moiety (methionine) to the photogenerated Ru(III), regenerating Ru(II).     

Redox reactions of the pyranopterin system of the molybdenum cofactor

This work provides the first extensive study of the redox reactivity of the pyranopterin system that is a component of the catalytic site of all molybdenum and tungsten enzymes possessing molybdopterin. The pyranopterin system possesses certain characteristics typical of tetrahydropterins, such as a reduced pyrazine ring; however, it behaves as a dihydropterin in redox reactions with oxidants. Titrations using ferricyanide and dichloroindophenol (DCIP) prove a 2e^–/2H⁺ stoichiometry for pyranopterin oxidations. Oxidations of pyranopterin by Fe(CN)₆ ^3– or DCIP are slower than tetrahydropterin oxidation under a variety of conditions, but are considerably faster than observed for oxidations of dihydropterin. The rate of pyranopterin oxidation by DCIP was studied in a variety of media. In aqueous buffered solution the pyranopterin oxidation rate has minimal pH dependence, whereas the rate of tetrahydropterin oxidation decreases 100-fold over the pH range 7.4–8.5. Although pyranopterin reacts as a dihydropterin with oxidants, it resists further reduction to a tetrahydropterin. No reduction was achieved by catalytic hydrogenation, even after several days. The reducing ability of the commonly used biological reductants dithionite and methyl viologen radical cation was investigated, but experiments showed no evidence of pyranopterin reduction by any of these reducing agents. This study illustrates the dual personalities of pyranopterin and underscores the unique place that the pyranopterin system holds in the spectrum of pterin redox reactions. The work presented here has important implications for understanding the biosynthesis and reaction chemistry of the pyranopterin cofactor in molybdenum and tungsten enzymes.Abbreviations DCIP dichloroindophenol - H₄DMP 6,7-dimethyltetrahydropterin - MV⁺ methyl viologen radical cation     

High efficient cyclic electron flow and functional supercomplexes in Chlamydomonas cells

A very high rate for cyclic electron flow (CEF) around PSI (~180 s^?1 or 210 s^?1 in minimum medium or in the presence of a carbon source respectively) is measured in the presence of methyl viologen (MV) in intact cells of Chlamydomonas reinhardtii under anaerobic conditions. The observation of an efficient CEF in the presence of methyl viologen is in agreement with the previous results reports of Asada et al. in broken chloroplasts (Plant Cell Physiol. 31(4) (1990) 557–564). From the analysis of the P700 and PC absorbance changes, we propose that a confinement between 2 PC molecules, 1 PSI and 1 cytb₆f corresponding to a functional supercomplex is responsible for these high rates of CEF. Supercomplex formation is also observed in the absence of methyl viologen, but with lower maximal CEF rate (about 100 s^?1) suggesting that this compound facilitates the mediation of electron transfer from PSI acceptors to the stromal side of cytb₆f. Further analysis of CEF in mutants of Chlamydomonas defective in state transitions shows the requirement of a kinase-driven transition to state 2 to establish this functional supercomplex configuration. However, a movement of the LHCII antennae is not involved in this process. We discuss the possible involvement of auxiliary proteins, among which is a small cytb₆f-associated polypeptide, the PETO protein, which is one of the targets of the STT7 kinase.     

A new self-assembled inorganic-organic hybrid layered compound containing hexarhenium cluster: [MV][{Mn(CH3OH)2}{Re6Se8(CN)6}]

A new layered compound, [MV][{Mn(CH₃OH)₂}{Re₆Se₈(CN)₆}] (1) consists of a layer alternately knitted by hexarhenium cluster and Mn complex, and MV²⁺ cations (methyl viologen dication = 1,1′-dimethyl-4,4′-bipyridilium dication) reside between the layers. The title compound 1 is the first layered framework containing cyano-hexarhenium clusters with photoactive guest molecules, MV²⁺. The MV²⁺ can be partly exchanged by H₂TMB²⁺ (N,N,N′,N′-tetramethylbenzidine dication) to form a compound [H₂TMB²⁺]_x[MV²⁺]_1−x [{Mn(CH₃OH)₂}{Re₆Se₈(CN)₆}] (2) showing an electronic interaction between the layered framework and [H₂TMB]²⁺ cation.     

Esterolytic Properties of Leucine-Proteinase, the Leucine-Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves : A Steady-State and Pre-Steady-State Study

Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-α-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 ± 0.5°C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of k_cat and k_cat/K_m reflect the ionization of two groups with pK_a values ranging between 6.5 and 6.9, and 8.1 and 8.3 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); and (c) values of K_m are pH independent. Among the substrates examined, N-α-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 × 10⁻¹⁰ molar at the optimum pH value (approximately 7.5).     

Purification and characterization of ferredoxin–nitrite reductase from the eukaryotic microalga Monoraphidium braunii

Nitrite reductase (NiR; EC 1.7.7.1) from the eukaryotic microalga Monoraphidium braunii has been purified to electrophoretic homogeneity, resulting in a preparation with a specific activity of 3574 nkat mg^–1 and a purification factor of 2553-fold. The enzyme is a single polypeptide chain with a molecular mass of 63 kDa, and absorption maxima at 690, 573, 385 and 280 nm. Kinetic data indicate K_m values of 0.7 mM for nitrite, 10 μM for M. braunii ferredoxin (Fd) and 0.26 mM for methyl viologen. The enzyme showed an optimum pH of 7.5 in 100 mM Tris–HCl buffer and an optimum temperature of 40 °C. NiR activity was inhibited by the sulfhydryl reagent p-hydroxymercuribenzoate and the chelating reagent KCN. Immunological studies revealed the presence of common antigenic determinants, at the Fd-binding domain, in NiR and glutamate synthase (EC 1.4.7.1) from M. braunii.     

Feruloyl esterase from <Emphasis Type="Italic">Alternaria tenuissima</Emphasis> that hydrolyses lignocellulosic material to release hydroxycinnamic acids

An extracellular feruloyl esterase from the culture filtrates of the isolated fungus Alternaria tenuissima was successfully purified to apparent homogeneity by anion-exchange and size-exclusion chromatography. Peptide fragments of purified enzyme (designated as AltFAE; molecular weight of 30.3 kDa determined by SDS-PAGE) were identified by mass spectrometry using a NanoLC-ESI-MS/MS system. Michaelis-Menten constants (K_M) and catalytic efficiencies (k_cat/K_M) were determined for typical substrates of feruloyl esterase, and the lowest K_M of 50.6 μM (i.e., the highest affinity) and the highest k_cat/K_M (3.1 × 10⁵ s^—1 M^–1) were observed for methyl ^p-coumarate and methyl ferulate, respectively. Not least, AltFAE catalyzed conversion of lignocellulosic material (e.g. wood meal) to release hydroxycinnamic products, i.e. ferulic- and ^p-coumaric acids.     

Facilitated permeation of methyl viologen into chloroplasts in situ during electric pulse generation in excitable plant cell membranes

Generation of action potential (AP) in plasma membranes of characean algae has a strong impact on photoreactions occurring in chloroplasts. Under physiological conditions, AP suppresses electron transport in alkaline and acidic regions, although to a different extent; these changes are transient and reversible. In the presence of the artificial electron acceptor, methyl viologen (MV²⁺), AP-induced changes in electron transport in photosystem II become irreversible. Incubation of Chara corallina internodal cells with MV²⁺ has no effect on the chlorophyll P700 photooxidation kinetics in photosystem I reaction centers, suggesting that MV²⁺ is inaccessible for interactions with photosystem I, because its permeation into chloroplasts of a resting cell is hindered by membrane barriers. At the same time, AP generation in the presence of MV²⁺ is accompanied by irreversible modification of P700 photooxidation kinetics, as can be evidenced from differences in absorption changes at 810 and 870 nm (ΔA ₈₁₀ signals). These findings suggest that MV²⁺ permeation into chloroplasts in situ is facilitated during or after the AP generation. Similar to the ΔA ₈₁₀ signals, light-induced changes in membrane potential do not depend on the presence of MV²⁺ in the external medium until the first excitatory stimulus is applied. Electric photoresponses of the cell are irreversibly modified by AP generated in the presence of MV²⁺ at the expense of non-cyclic photosynthetic electron transport redirected to the MV²⁺ reduction. It is concluded that AP effects on chloroplast photosynthesis in situ are complex and involve permeability changes for MV²⁺ in membrane barriers of the “plasmalemma-chloroplast envelope” system.     

Partial purification of the NADPH-dependent aldehyde reductase from bovine cardiac muscle.

An aldehyde reductase catalyzing the NADPH-dependent reduction of long-chain aldehydes has been purified 690-fold from bovine cardiac muscle. Based on the results obtained during gel filtration, this enzyme has an apparent molecular weight of 34,000. The pI of the aldehyde reductase was 6.1 and the enzymatic activity had a sharp pH optimum at 6.4. The enzyme catalyzed the reduction of aromatic aldehydes and aliphatic aldehydes having eight or more carbon atoms. Short-chain aldehydes, aldoses, or ketoses or long-chain methyl ketones were not utilized as substrates by this enzyme. However, the methyl ketone, pentadecan-2-one, was a competitive inhibitor of this enzyme with an apparent K_i = 10 μm when tetradecanal was the variable substrate. The reaction was not reversible when ethanol or hexadecanol was employed as substrate, utilizing either NAD⁺, or NADP⁺ as a cofactor. The addition of 10 mm pyrazole to the incubation medium had no effect on the enzymatic activity.     

Kinetics of methyl viologen reduction by hydrogen catalyzed by hydrogenase from Desulfovibrio vulgaris

The reduction of methyl viologen by hydrogen with hydrogenase was studied kinetically. The initial rate of the reduction was expressed as

where k′, K₂, and K₃ are constants and [S′] is the concentration of methyl viologen.According to this equation, a sequential mechanism was proposed. By combining the mechanism of hydrogen production from the reduced methyl viologen, a reaction mechanism for the reduction and oxidation of methyl viologen was proposed.     

Purification and properties of human erythrocyte membrane NADH-cytochrome b5 reductase

An NADH:(acceptor) oxidoreductase (EC 1.6.99.3) of human erythrocyte membrane was purified by DEAE-cellulose anion exchange, hydroxyapatite adsorption, and 5′-ADP-hexane-agarose affinity chromatographies after solubilization with Triton X-100. The purified reductase preparation was homogeneous and estimated to have an apparent molecular weight of 36,000 on SDS-polyacrylamide slab gel electrophoresis and of 144,000 on Sephadex G-200 gel filtration in the presence of 0.2% Triton X-100, whereas a soluble NADH-cytochrome b₅ reductase of human erythrocyte had a molecular weight of 32,000 by both methods, indicating the existence of a distinct membrane reductase. Digestion of the membrane reductase with cathepsin D yielded a new polypeptide chain which gave the same relative mobility as the soluble reductase on SDS-polyacrylamide slab gel electrophoresis. The membrane enzyme, the cathepsin-digested enzyme, and the soluble enzyme all cross-reacted with the antibody to rat liver microsomal NADH-cytochrome b₅ reductase. The enzyme had one mole FAD per 36,000 as a prosthetic group and could reduce K₃Fe(CN)₆, 2,6-dichlorophenolindophenol, cytochrome c, methemoglobin-ferrocyanide complex, cytochrome b₅ and methemoglobin via cytochrome b₅ when NADH was used as an electron donor. NADPH was less effective as an electron donor than NADH. The specific activity of the purified enzyme was 790 μmol ferricyanide reduced min^?1 mg^?1 and the turnover number was 40,600 mol ferricyanide reduced min^?1 mol^?1 FAD at 25 °C. The apparent K_m values for NADH and cytochrome b₅ were 0.6 and 20 μm, respectively, and the apparent V value was 270 μmol cytochrome b₅ reduced min^?1 mg^?1. These kinetic properties were similar to those of the soluble NADH-cytochrome b₅ reductase. The results indicate that the NADH:(acceptor) oxidoreductase of human erythrocyte membrane could be characterized as a membrane NADH-cytochrome b₅ reductase.     

Properties of Bromphenol Blue as an Electron Donor for Higher Plant NADH: Nitrate Reductase

Bromphenol blue, which was reduced with dithionite, was found to support nitrate reduction catalyzed by squash NADH:nitrate reductase at a rate about 5 times greater than NADH with freshly prepared enzyme and 10 times or more with enzyme having been frozen and thawed. Kinetic analysis of bromphenol blue as a substrate for squash nitrate reductase yielded apparent K_m values of 60 micromolar for bromphenol blue at 10 millimolar nitrate and 500 micromolar for nitrate at 0.2 millimolar bromphenol blue. With the same preparation of enzyme the apparent K_m values were 9 micromolar for NADH at 10 millimolar nitrate and 50 micromolar nitrate at 0.1 millimolar NADH. Bromphenol blue was found to be a noncompetitive inhibitor versus NADH with a K_i of 0.3 millimolar. When squash NADH:nitrate reductase activity was inactivated with p-hydroxymercuribenzoate or denatured by heating at 40°C, the bromphenol blue nitrate reductase activity was not lost. These results were taken to indicate that bromphenol blue and NADH donated electrons to nitrate reductase at different sites. When monoclonal antibodies prepared against corn and squash nitrate reductases were used to inhibit the nitrate reductase activities supported by NADH, bromphenol blue, and methyl viologen, differential inhibition was found which tended to indicate that the three electron donors were interacting with the enzyme at different sites. One monoclonal antibody prepared against squash nitrate reductase inhibited all three activities of both corn and squash nitrate reductase. It appears this antibody may bind to a highly conserved antigenic site in the nitrate binding region of the enzyme.     

Leucine aminopeptidase (bovine lens): Synthesis and kinetic properties of ortho-, meta-, and para-substituted leucyl-anilides

The synthesis of a number of leucyl derivatives of substituted anilides and their properties as substrates and inhibitors of Zn²⁺-Mg²⁺ leucine aminopeptidase (EC 3.4.11.1) at pH 8.5 and 30 °C are described. The compounds include leucyl-X where X is o-, m-, or p-aminobenzenesulfonic acid, o-, m-, or p-anisidine, and m- or p-aminobenzenesulfonyl fluoride. The latter two sulfonyl fluorides, designed to be active site-directed irreversible inhibitors, turned out to be good substrates for leucine aminopeptidase. The K_m and V values of the above compounds as substrates for leucine aminopeptidase are reported. N-Leucyl-m-aminobenzenesulfonate exhibits desirable properties (solubility much greater than K_m, Δ? at 295 nm of 2000 m^?1 cm^?1, and V of 300 μmol min^?1 mg^?1) as a substrate for a spectrophotometric assay of leucine aminopeptidase. With the exception of N-leucyl-p-aminobenzenesulfonate, all of the above compounds are inhibitors of the hydrolysis of leucyl-p-nitroanilide by leucine aminopeptidase with K_i values approximately their K_m values when they are used as substrates. Despite wide variability in steric bulk, chemical composition, and electrical charge of the substituted anilides, the K_m values of the above compounds vary over a narrow range (0.5 to 4.8 mm), which indicates that the leucyl moiety plays the predominant role in the determination of K_m values. Although the K_m values of m- substituents are similar to those of o- substituents, the V values for m-substituents are much greater than those for o- substituents, which suggests that o-substituents interfere with the catalytic process. N-Leucyl-p-aminobenzenesulfonate and N-alanyl-p-aminobenzenesulfonate as well as the nonsubstrate p-aminobenzenesulfonate stimulate rather than inhibit the proteolysis of leucyl-p-nitroanilide. The stimulation has no effect on V but lowers the K_m for the hydrolysis of leucyl-p-nitroanilide, which is compatible with these compounds' serving as nonessential activators.     

Anionic modulation of the catalytic activity of hydrogenase from Chlamydomonas reinhardtii

Hydrogen production by cell-free extracts of Chlamydomonas reinhardtii is stimulated by anions when methyl viologen, reduced by dithionite, is used as the electron donor to hydrogenase. The increasing effectiveness of various anions closely follows their position in the Hofmeister chaotropic sequence. The most stimulatory anion tested, I^?, gives a six-fold increase in activity at a concentration of 0.5 n. The K_m of the enzyme for methyl viologen is not affected by anions, while the V is greatly increased. H₂ oxidation coupled to methyl viologen reduction is also greatly stimulated by anions. However, when reduced ferredoxin is used as the electron donor to hydrogenase, there is a very strong inhibition of H₂ production by salts. In this case, the V of the enzyme is unaffected, but there is a large increase in the K_m of the enzyme for ferredoxin. The most inhibitory salt tested, KI, decreases hydrogenase activity by 93% at a concentration of 0.2 n.     

Characterization of a robust glucose 1-dehydrogenase,SyGDH, and its application in NADPH regeneration for the asymmetric reduction of haloketone by a carbonyl reductase in organic solvent/buffer system

To realize coenzyme regeneration in the reduction of haloketones, a codon-optimized gene Sygdh encoding glucose 1-dehydrogenase (SyGDH) was synthesized based on the putative GDH gene sequence (Ta0897) in Thermoplasma acidophilum genomic DNA, and expressed in E. coli BL21(DE3). Recombinant SyGDH was purified to homogeneity by affinity chromatography with the specific activity of 86.3 U/mg protein towards D-glucose at the optimum pH and temperature of 7.5 and 40 °C. It was highly stable in a pH range of 4.5–8.0 and at 60 °C or below, and resistant to various organic solvents. The K_m and catalytic efficiency (k_cat/K_m) of SyGDH towards NADP+ were 0.67 mM and 104.0 mM⁻¹ s⁻¹, respectively, while those towards NAD+ were 157.9 mM and 0.64 mM⁻¹ s⁻¹, suggesting that it preferred NADP+ as coenzyme to NAD+. Additionally, using whole cells of E. coli/Sygdh-Sys1, coexpressing SyGDH and carbonyl reductase (SyS1), as the biocatalyst, the asymmetric reduction of 60 mM m-chlorophenacyl chloride coupled with the regeneration of NADPH in situ was conducted in DMSO/phosphate buffer (2:8, v/v) system, producing (R)-2-chloro-1-(3-chlorophenyl)ethanol with over 99.9% ee_p and 99.2% yield. Similarly, the reduction of 40 mM α-bromoacetophenone in n-hexane/buffer (6:4, v/v) biphasic system produced (S)-2-bromo-1-phenylethanol with over 99.9% ee_p and 98.3% yield.