Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: how to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24 °C, 38 °C and 55 °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38 °C and 55 °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55 °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2 μM gibberellic acid.     

Interactions of free copper (II) ions alone or in complex with iron (III) ions with erythrocytes of marine fish Dicentrarchus labrax

As a consequence of human activity, various toxicants - especially metal ions - enter aquatic ecosystems and many fish are exposed to considerable levels. As the free ion and in some complexes, there is no doubt that copper promotes damage to cellular molecules and structures through radical formation. Therefore, we have investigated the influence of copper uptake by the red blood of the sea bass (Dicentrarchus labrax), and its oxidative action and effects on cells in the presence of complexed and uncomplexed Fe³⁺ ions.Erythrocytes were exposed to various concentrations of CuSO₄, Fe(NO₃)₃, and K₃Fe(CN)₆ for up to 5 h, and the effects of copper ions alone and in the combination with iron determined. The results show that inside the cells cupric ion interacts with hemoglobin, causing methemoglobin formation by direct electron transfer from heme Fe²⁺ to Cu²⁺. Potassium ferricyanide as a source of complexed iron decreases Met-Hb formation induced by copper ions unlike Fe(NO₃)₃. We also found that incubation of fish erythrocytes with copper increased hemolysis of cells. But complexed and uncomplexed iron protected the effect of copper. CuSO₄ increased the level of lipid peroxidation and a protective effect on complexed iron was observed. Incubation of erythrocytes with copper ions resulted in the loss of a considerable part of thiol content at 10 and 20 μM. This effect was decreased by potassium ferricyanide and Fe(NO₃)₃ only after 1 and 3 h of incubation. The level of nuclear DNA damage assayed by comet assay showed that 20 μM CuSO₄ as well as 20 μM Fe(NO₃)₃ and 10 mM K₃Fe(CN)₆ induce single- and double-strand breaks. The lower changes were observed after the exposure of cells to K₃Fe(CN)₆. The data suggest that complexed iron can act protectively against copper ions in contrast to Fe(NO₃)₃.     

Performance of electron acceptors in catholyte of a two-chambered microbial fuel cell using anion exchange membrane

The performance of the cathodic electron acceptors (CEA) used in the two-chambered microbial fuel cell (MFC) was in the following order: potassium permanganate (1.11 V; 116.2 mW/m²) > potassium persulfate (1.10 V; 101.7 mW/m²) > potassium dichromate, K₂Cr₂O₇ (0.76 V; 45.9 mW/m²) > potassium ferricyanide (0.78 V; 40.6 mW/m²). Different operational parameters were considered to find out the performance of the MFC like initial pH in aqueous solutions, concentrations of the electron acceptors, phosphate buffer and aeration. Potassium persulfate was found to be more suitable out of the four electron acceptors which had a higher open circuit potential (OCP) but sustained the voltage for a much longer period than permanganate. Chemical oxygen demand (COD) reduction of 59% was achieved using 10 mM persulfate in a batch process. RALEX™ AEM-PES, an anion exchange membrane (AEM), performed better in terms of power density and OCP in comparison to Nafion®117 Cation Exchange Membrane (CEM).     

Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and ¹H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.     

Comparison of the SOD-like activity of hexacoordinate Mn(II), Fe(II) and Ni(II) complexes having isoindoline-based ligands

Recently, a series of Fe(II) complexes have been published by our group with 3 N-donor 1,3-bis(2′-Ar-imino)isoindoline ligands containing various Ar-groups (pyridyl, 4-methylpyridyl, thiazolyl, benzimidazolyl and N-methylbenzimidazolyl). The superoxide scavenging activity of the compounds showed correlation with the Fe(III)/Fe(II) redox potentials. Analogous, electroneutral chelate complexes with Mn(II) and Ni(II) in 2:1 ligand:metal composition are reported here. Each Mn(II) complex exhibits one reversible redox wave that is assigned as the Mn(III)/Mn(II) redox transition. The E_1/2 spans a 180 mV range from − 98 (Ar = 3-methylpyridyl) to 82 mV (Ar = thiazolyl) vs. the Fc⁺/Fc depending on the Ar-sidearm. The SOD-like (SOD=superoxide dismutase)activity of all complexes was determined according to the McCord-Fridovich method. The Mn(II) isoindolinates have IC₅₀ values - determined with 50 μM cytochrome c Fe(III) - that range from (3.22 ± 0.39) × 10^− 6 (Ar = benzimidazolyl) to (10.80 ± 0.54) × 10^− 6 M (Ar = N-methylbenzimidazolyl). In contrast with the Fe(II) complexes, the IC₅₀ concentrations show no significant dependence on the E_1/2 values in this narrow potential range emphasizing that the redox potential is not the governing factor in the Mn(II)-containing scavengers. The analogous Ni(II) compounds show no redox transitions in the thermodynamically relevant potential range (− 0.40 to 0.65 V vs. SCE) and accordingly, their superoxide scavenging activity (if any) is below the detection level.     

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

The heme-based oxygen-sensor enzyme from Escherichia coli (Ec DOS) is a heme-regulated phosphodiesterase with activity on cyclic-di-GMP and is composed of an N-terminal heme-bound sensor domain with the PAS structure and a C-terminal functional domain. The activity of Ec DOS is substantially enhanced by the binding of O₂ to the Fe(II)-protoporphyrin IX complex [Fe(II) complex] in the sensor domain. The binding of O₂ to the Fe(II) complex changes the structure of the sensor domain, and this altered structure becomes a signal that is transduced to the functional domain to trigger catalysis. The first step in intra-molecular signal transduction is the binding of O₂ to the Fe(II) complex, and detailed elucidation of this molecular mechanism is thus worthy of exploration. The X-ray crystal structure reveals that Phe113 is located close to the O₂ molecule bound to the Fe(II) complex in the sensor domain. Here, we found that the O₂ association rate constants (>200 × 10⁻³ μM⁻¹ s⁻¹: F113L; 26 × 10⁻³ μM⁻¹ s⁻¹: F113Y) of the Fe(II) complexes of Phe113 mutants were markedly different from that (51 × 10⁻³ μM⁻¹ s⁻¹) of the wild-type enzyme, and auto-oxidation rates (0.00068 min⁻¹: F113L; 0.039 min⁻¹: F113Y) of the Phe113 mutants also differed greatly from that (0.0062 min⁻¹) of the wild-type enzyme. We thus suggest that Phe113, residing near the O₂ molecule, has a critical role in optimizing the Fe(II)-O₂ complex for effective regulation of catalysis by the oxygen-sensor enzyme. Interactions of CO and cyanide anion with the mutant proteins were also studied.     

The iron component of sodium nitroprusside blocks NMDA-induced glutamate accumulation and intracellular Ca2+ elevation

These studies were designed to compare the effects of nitric oxide (NO) generating compounds with those of several iron containing, compounds which do not generate NO on glutamate receptor function. Stimulation of primary cultures of cerebellar granule cells with N-methyl-D-aspartate (NMDA) or kainate results in the elevation of intracellular calcium ([Ca²⁺]_i) and cGMP and the release of glutamate. The iron containing compounds, sodium nitroprusside (SNP), potassium ferrocyanide (K₄Fe(CN)₆) and potassium ferricyanide (K₃Fe(CN)₆) decrease the NMDA-induced release of glutamate. SNP is the only compound of the above 3 agents which generates NO. A non-iron, NO generating compound, S-nitroso-N-acetylpenicillamin (SNAP), has no effect on the NMDA-induced glutamate release. Potassium ferrocyanide (Fe II), but not potassium ferricyanide (Fe III), blocks NMDA-induced cGMP elevations after 3 min exposure times. This contrasts with the NO generating compounds (both SNP and SNAP) which elevate cGMP levels. Furthermore, both potassium ferrocyanide (Fe II) and SNP (Fe II) suppress the elevation of [Ca²⁺]_i induced by NMDA but neither potassium ferricyanide (Fe III) nor SNAP are effective in this regard. These effects are also independent of cyanide as another Fe II compound, ferrous sulfate (FeSO₄) is also able to suppress NMDA-induced elevations of [Ca²⁺]_i SNP was unable to suppress kainate receptor functions. Collectively, these results indicate that Fe II, independently of NO, has effects on NMDA receptor function.     

Examination of microbial fuel cell start-up times with domestic wastewater and additional amendments

Rapid startup of microbial fuel cells (MFCs) and other bioreactors is desirable when treating wastewaters. The startup time with unamended wastewater (118 h) was similar to that obtained by adding acetate or fumarate (110-115 h), and less than that with glucose (181 h) or Fe(III) (353 h). Initial current production took longer when phosphate buffer was added, with startup times increasing with concentration from 149 h (25 mM) to 251 h (50 mM) and 526 h (100 mM). Microbial communities that developed in the reactors contained Betaproteobacteria, Acetoanaerobium noterae, and Chlorobium sp. Anode biomass densities ranged from 200 to 600 μg/cm² for all amendments except Fe(Ш) (1650 μg/cm²). Wastewater produced 91 mW/m², with the other MFCs producing 50 mW/m² (fumarate) to 103 mW/m² (Fe(III)) when amendments were removed. These experiments show that wastewater alone is sufficient to acclimate the reactor without the need for additional chemical amendments.     

On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0 × 10⁻⁸–1.0 × 10⁻⁶ M with a detection limit of 1.0 × 10⁻⁸ M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0 × 10⁻⁸, 5.0 × 10⁻⁷ and 1.0 × 10⁻⁶ M MTX (n = 11), respectively. In laboratory-built CE–CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.     

Coordination changes and auto-hydroxylation of FIH-1: uncoupled O2-activation in a human hypoxia sensor

Hypoxia sensing is the generic term for pO₂-sensing in humans and other higher organisms. These cellular responses to pO₂ are largely controlled by enzymes that belong to the Fe(II) α-ketoglutarate (αKG) dependent dioxygenase superfamily, including the human enzyme called the factor inhibiting HIF (FIH-1), which couples O₂-activation to the hydroxylation of the hypoxia inducible factor α (HIFα). Uncoupled O₂-activation by human FIH-1 was studied by exposing the resting form of FIH-1 (αKG + Fe)FIH-1, to air in the absence of HIFα. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (λ_max = 583 nm) arising from enzyme auto-hydroxylation of Trp²⁹⁶, forming an Fe(III)-O-Trp²⁹⁶ chromophore [Y.-H. Chen, L.M. Comeaux, S.J. Eyles, M.J. Knapp, Chem. Commun. (2008), doi:10.1039/B809099H]; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue (oxo)Met²⁷⁵. The kinetics of purple FIH-1 formation were independent of Fe(II) and αKG concentrations, however, product yield was saturable with increasing [αKG] and required excess Fe(II). Yellow FIH-1 was formed from (succinate + Fe)FIH-1, or by glycerol addition to (αKG + Fe)FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1 (αKG + Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O₂-activation by loss of an H₂O ligand from the metal center.     

Nitrogen and potassium variation on contaminant removal for a vertical subsurface flow lab scale constructed wetland

Lab scale constructed wetlands were used to evaluate organic load removal efficiency. Bioreactors were fed with synthetic wastewater (SW) with varying concentrations of nitrogen and potassium. Reactors were planted with species Phragmites australis. Fed theoretic COD was adjusted to 240.0 mg-O₂ L⁻¹, nitrogen levels were 10 and 40 mg-N L⁻¹ (ammonium sulfate), potassium levels were 5 and 31 mg-K L⁻¹ (potassium monobasic phosphate). The higher biomass yield, for 0.5 and 0.775 N:K ratios, was related with higher organic load removal. The ratio N:K showed significant differences for organic load abatement, when 1:0.5 and 1:0.775 N:K ratios were applied, 96.8% efficiency was obtained, whereas N:K ratio of 1:0.125 had efficiency of 92.1% and N:K ratio of 1:3.1 showed an efficiency of 90.5%. For planted bioreactor E_H decreased in 162.7 mV from sample port to 5 cm down to 35 cm depth, while for the bioreactor without plant showed an E_H decrement of only 17.7 mV.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

A mechanistic study of ferrioxamine B reduction by the biological reducing agent ascorbate in the presence of an iron(II) chelator

The iron overload drug desferal (desferrioxamine B) forms the stable iron complex ferrioxamine B. The reduction potential of ferrioxamine B (E^o = −482 mV versus NHE pH 7) prohibits its reduction by biological reducing agents such as ascorbate, but it was found that the iron(II) chelator 2,2′-bipyridine (bipy) facilitates this reduction. Evidence is given to support the formation of a ternary complex between iron, bipy, and desferrioxamine B as the key step in facilitating the reduction. The equilibrium constant for the formation of the ternary complex was found to be 8.9 × 10⁷ and ternary complex formation is explained in terms of a three step mechanism. The mechanism for the reduction of ferrioxamine B is discussed in terms of rapidly established pre-equilibria which include ternary complex formation, ascorbic acid deprotonation, and encounter complex formation between ascorbate and the ternary complex. These equilibria are followed by rate limiting reduction of the ternary complex. Bipy was found to be a similar facilitator to sulfonated bathophenanthroline for the reduction of ferrioxamine B by ascorbate.     

Measuring the antioxidant capacity of blood plasma using potentiometry

The use of potentiometry to measure plasma antioxidant capacity to contribute to oxidative stress evaluation is presented. In this assay, plasma (n = 60) diluted (0.3 to 1 ml) in phosphate buffer, pH 7.4, NaCl 9%, was submitted to potentiometry. A platinum wire was the working electrode and saturated calomel the reference. The results are presented as the difference between sample and buffer potential (ΔE). ΔE presented a good inverse correlation with added increasing concentrations of ascorbate (2.5−75 μmol/L; R = −0.99), urate (9.0−150 μmol/L; R = −0.99), and bilirubin (0.78−13 μmol/L; R = −0.99). Increase in the antioxidant capacity decreased ΔE. Depletion of the antioxidant capacity by tert-butylhydroperoxide (6.5−50 μmol/L) presented a direct correlation (0.97) with ΔE. Furthermore, ΔE presented an inverse correlation (R = −0.99) with increased antioxidant capacity of plasma (FRAP) induced by the addition of ascorbate (2.5−75 μmol/L). The response of the potentiometric method proved be adequate for measuring the plasma antioxidant depletion induced by acute exhaustive exercise in rats (control, n = 15; exercised, n = 15). This exercise decreased the concentration of urate (p < 0.05), decreased FRAP (p < 0.5), increased TBARS (p < 0.5), and decreased the potentiometer sensor response (p = 6.5 × 10⁻³). These results demonstrate the adequacy of potentiometry for evaluating the antioxidant capacity of blood plasma samples.     

Migration and retention of dissolved iron in three mesocosm wetlands

Three mesocosm wetlands (250 cm × 100 cm × 100 cm) with different wetland plants (Calamgrostis angustifolia, CA, Carex lasiocarpa, CL, and C. angustifolia/C. lasiocarpa mixture, AL, respectively) and hydrologic regimes were set to test migration and retention of exogenous dissolved iron ((NH₄)₂Fe(SO₄)₂of 40 mg Fe(II) L⁻¹) in the Sanjiang Plain Wetland in northeast China. The experiment was designed as two stages: open migration period (OMP) for 1.5 d and close retention period (CRP) for 28.5 d. Based on the outflow Fe(II) concentration during the OMP, retention efficiencies (RE) and iron retention fluxes adjusted by area (RF_ad) in the three mesocosm wetlands were calculated, and the migration of iron were modeled using the first-order kinetic model. Outflow pH decreased gradually from a weak alkaline condition to a weak acid condition during the OMP, and then increased during the CRP, while outflow Eh and DO decreased during the experiment. The three mesocosm wetlands had considerable RE ranging from 75% to 98%, with the averaged RF_ad of 4.31 ± 0.17, 4.20 ± 0.16, and 4.37 ± 0.13 g m⁻² h⁻¹ for CA, CL, and AL, respectively. The reduction conditions in the mesocosm wetlands developed after 4 d or 12 d and the former retained iron during the OMP became mobile and discharged primarily in the form of Fe(III). The first-order kinetic model could simulate the outflow concentration of dissolved iron during the OMP (R² = 0.91, 0.69, and 0.68 for CA, CL, and AL, respectively), while the outflow dissolved iron during the CMP was difficult to model because the changed pH and Eh conditions in the mesocosm wetlands cause the former precipitated iron to be mobile after several days.     

pH- and DNA-induced dual molecular light switches based on a novel ruthenium(II) complex

A novel Ru(II) complex, [Ru(bpy)₂(btppz)]Cl₂, where bpy = 2,2′-bipyridine and btppz = benzo[h]tripyrido[3,2-a:2′,3′-c:2″,3″-j]phenazine, has been synthesized and characterized. The pH effects on UV-visible (UV-vis) absorption and emission spectra of the complex have been studied and ground- and excited-state ionization constants of the complex have been derived. The calf thymus DNA (ct-DNA) binding properties of the complex were investigated with UV-vis absorption and luminescence spectrophotometric titrations, steady-state emission quenching by [Fe(CN)₆]⁴⁻, DNA competitive binding with ethidium bromide, DNA melting experiments, reverse salt titrations and viscosity measurements. The complex was demonstrated to act as dual molecular switches: pH-induced “on-off” emission switch with an on-off intensity ratio of ∼54 which is favorably compared with those reported for structurally analogous Ru(II) complexes, and a DNA molecular light switch with a luminescence enhancement factor of 22 as it intercalatively bound to the DNA.     

Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism

Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b₅₆₁ family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at g_max = 3.7 corresponding to a highly anisotropic species, and another at g_max = 3.18; these species are similar to those observed in other cytochromes of the b₅₆₁ family, and were reducible by ascorbate. In addition another signal was observed in some preparations at g_max = 2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of + 80 mV ± 30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b₅₆₁ of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.     

Cyclic Photophosphorylation in the Mykotrophic Orhid Neottia nidus-avis

The mykotrophic orchid Neottia nidus-avis (L.) Rich. is not able to evolve oxygen in the light. Plastid preparations from the lip (labellum) of the orchid perform a photosystem I-dependent photoreduction of methylviologen with the artificial electron donor couple 2,6-dichlorophenol indophenol ascorbate. Photosystem II reactions such as the ferricyanide Hill reaction or the photoreduction of 2,6-dichlorophenol indophenol with diphenylcarbazide as the electron donor are not functioning. The plastids exhibit phenazine methosulfate-mediated cyclic photophosphorylation. After infiltration with ³²P-labeled phosphate the labellum forms ³²P-ATP in the light. This rate of ATP formation is enhanced by additional infiltration of phenazine methosulfate prior to illumination. The brown color of the plant is caused by an absorption shift of carotenoids to longer wavelength. By comparison of absorption spectra with the fluorescence excitation spectra of plastid preparations and of the extracted pigments we show that no appreciable energy transfer from carotenoids to chlorophyll occurs.     

Kinetic studies on the reversible ring opening-closure reaction of the triazine ligand and structural properties of palladium(II) complexes

A Pd(II) complex containing didentate triazine ligand L¹ (2-(2-methylphenyl)-3-(2-pyridyl)-2,3-dihydronaphtho[2,1-e][1,2,4]triazine) [PdCl₂(L¹)] (1) was prepared, and the structure was determined by X-ray crystallography. The absorption spectrum of complex 1 in dichloromethane changed gradually with isosbestic points when methanol was added to the solution, and [PdCl(L²)] (2) (L² = N-[methoxy(2-pyridyl)methyl]-1-(2-methylphenylazo)-2-naphthylamide) was obtained from the resulting solution after the reaction was completed. Addition of hydrogen chloride to the solution of complex 2 led to the recovery of complex 1. Thus, a reversible ring opening and closure reaction of the triazine ligand was observed. The progress of the ring opening reaction was monitored by observing the absorbance changes at several wavelengths of the visible spectra as a function of the concentration of methanol. The absorbance plots were fitted successfully with a mechanism that includes the consumption of two methanol molecules and the release of HCl, whose concentration is equivalent to that of the produced Pd complex . In dichloromethane with a low dielectric constant, the polar HCl molecule will be stabilized by forming an adduct with methanol. The equilibrium constant was determined as at T = 25.0 °C. The kinetics of the reaction of [PdCl₂(L¹)] with methanol was investigated by monitoring the absorbance change of the reacting solution with time. We obtained rate constant values of k₁ = (2.40 ± 0.07) × 10⁻³ s⁻¹ and k₂ = (5.8 ± 0.1) × 10⁻³ M⁻¹ s⁻¹ at T = 25.0 °C on the observed pseudo-first-order rate constant of the forward reaction, k_f = k₁ + k₂ [CH₃OH].