Artificial Electron Donors Research Articles

INFLUENCE HYDROGEN SULFIDE ON CA2 + -DEPENDENT POTASSIUM PERMEABILITY OF THE MEMBRANE OF HUMAN ERYTHROCYTES

The purpose of the research was to study the influence of hydrogen sulfide donor NaHS on red blood cells membrane potential changes caused by the activation of Ca2+ dependent K+ channels in the presence of calcium ionophore or artificial electron donor system ascorbate phenazine methosulfate. Materials and methods. We used the packed erythrocytes obtained from venous blood of 25 healthy volunteers at the age of 20-27 years. Registration of the membrane potential of erythrocytes in the presence of Ca2+ ionophore (A23187) or artificial electron donor system ascorbate – FMS was performed with potentiometric method based on the fact that in the presence of the protonophore distribution of H + depends on the membrane potential Em like as Em = RT/F (pHi – pH0 ). Here pHi and pH0 are pH of the incubation medium and cytoplasm, respectively. An amplitude of hyperpolarizing response (HR) was calculated as an integral characteristic of Ca2+-dependent permeability for K+. Results. The addition of NaHS at concentrations from 0.2 mM to 0.005 mM into the cells incubation medium caused a change in the amplitude of hyperpolarizing response of erythrocyte membranes induced by both methods. In the presence of 0.005 mM NaHS the amplitude of A23187-dependent HR increased significantly, whereas the amplitude of the redox-dependent HR decreased. Suppression of A23187-dependent HR in the presence of higher concentrations of NaHS was more pronounced than the suppression of the amplitude of redox-dependent response. The amplitude of A23187-dependent HR increased under the joint action of hydrogen sulfide and the blocker of Na+, K+, 2Cl- -cotransport (NKCC) bumetanide, and the amplitude of redox-dependent HR decreased in comparison with the parameters obtained in the absence of a blocker. Conclusion. It is found that hydrogen sulfide exerts a modulatory effect on K (Ca2+) - channels of erythrocyte membrane. The effect of H2 S depends on the method of activation of the studied channels. A23187-dependent HR was more sensitive to H2S in comparison to redox-induced HR of erythrocyte membrane. The effect of NaHS on the amplitude of the HR in the presence of NKCC blocker bumetanide also depended on the method of stimulation of channels.

Effect of ammonia on the photosynthetic activity of Arthrospira and Chlorella: A study on chlorophyll fluorescence and electron transport

Although ammoniacal nitrogen is the preferred nitrogen source for microalgae/cyanobacteria, at elevated concentrations and high pH values it may negatively impact photosynthesis and growth. Chlorophyll (Chl) fluorescence analysis is a useful tool to monitor the influence of various stress conditions on the photosynthetic activity of plants or microalgae/cyanobacteria. In this study, we investigated the effect of ammoniacal nitrogen on Chl fluorescence in microalgae/cyanobacteria. Chl fluorescence analysis revealed that the parameters related to flux ratios and specific energy fluxes of photochemistry were gradually inhibited as the free ammonia (FA) concentration increased. Photosynthetic electron transport activity was measured using artificial electron acceptors, donors or inhibitors. These analyses suggest that ammonia has multiple impacts on the photosynthetic apparatus; photosystems I (PSI) and II (PSII), the electron transport chain, the oxygen-evolution complex (OEC) as well the dark respiration were gradually inhibited by increasing FA concentration. At high FA concentration, the PSI/PSII activity increased, suggesting that PSI was more tolerant to FA than PSII. Non-photochemical quenching (NPQ) decreased to zero at elevated FA concentrations. The Chl fluorescence data obtained in the presence of DCMU (diuron) suggest that the decrease of NPQ under ammonia inhibition/toxicity is due to the increase of PSI/PSII activity. The rapid response of Chl fluorescence transients to increases in FA may allow one to use pulse amplitude modulation (PAM) fluorescence as a tool to monitor ammonia inhibition/toxicity in cultures of microalgae/cyanobacteria.

Energy Conservation Associated with Ethanol Formation from H2 and CO2 in Clostridium autoethanogenum Involving Electron Bifurcation.

Most acetogens can reduce CO2 with H2 to acetic acid via the Wood-Ljungdahl pathway, in which the ATP required for formate activation is regenerated in the acetate kinase reaction. However, a few acetogens, such as Clostridium autoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei, also form large amounts of ethanol from CO2 and H2. How these anaerobes with a growth pH optimum near 5 conserve energy has remained elusive. We investigated this question by determining the specific activities and cofactor specificities of all relevant oxidoreductases in cell extracts of H2/CO2-grown C. autoethanogenum. The activity studies were backed up by transcriptional and mutational analyses. Most notably, despite the presence of six hydrogenase systems of various types encoded in the genome, the cells appear to contain only one active hydrogenase. The active [FeFe]-hydrogenase is electron bifurcating, with ferredoxin and NADP as the two electron acceptors. Consistently, most of the other active oxidoreductases rely on either reduced ferredoxin and/or NADPH as the electron donor. An exception is ethanol dehydrogenase, which was found to be NAD specific. Methylenetetrahydrofolate reductase activity could only be demonstrated with artificial electron donors. Key to the understanding of this energy metabolism is the presence of membrane-associated reduced ferredoxin:NAD(+) oxidoreductase (Rnf), of electron-bifurcating and ferredoxin-dependent transhydrogenase (Nfn), and of acetaldehyde:ferredoxin oxidoreductase, which is present with very high specific activities in H2/CO2-grown cells. Based on these findings and on thermodynamic considerations, we propose metabolic schemes that allow, depending on the H2 partial pressure, the chemiosmotic synthesis of 0.14 to 1.5 mol ATP per mol ethanol synthesized from CO2 and H2. Ethanol formation from syngas (H2, CO, and CO2) and from H2 and CO2 that is catalyzed by bacteria is presently a much-discussed process for sustainable production of biofuels. Although the process is already in use, its biochemistry is only incompletely understood. The most pertinent question is how the bacteria conserve energy for growth during ethanol formation from H2 and CO2, considering that acetyl coenzyme A (acetyl-CoA), is an intermediate. Can reduction of the activated acetic acid to ethanol with H2 be coupled with the phosphorylation of ADP? Evidence is presented that this is indeed possible, via both substrate-level phosphorylation and electron transport phosphorylation. In the case of substrate-level phosphorylation, acetyl-CoA reduction to ethanol proceeds via free acetic acid involving acetaldehyde:ferredoxin oxidoreductase (carboxylate reductase).

The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae

The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb3 from Vibrio cholerae (CcoNOQPX) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQPX and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the KC-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

Engineering Hydrogen Gas Production from Formate in a Hyperthermophile by Heterologous Production of an 18-Subunit Membrane-bound Complex

Biohydrogen gas has enormous potential as a source of reductant for the microbial production of biofuels, but its low solubility and poor gas mass transfer rates are limiting factors. These limitations could be circumvented by engineering biofuel production in microorganisms that are also capable of generating H2 from highly soluble chemicals such as formate, which can function as an electron donor. Herein, the model hyperthermophile, Pyrococcus furiosus, which grows optimally near 100 °C by fermenting sugars to produce H2, has been engineered to also efficiently convert formate to H2. Using a bacterial artificial chromosome vector, the 16.9-kb 18-gene cluster encoding the membrane-bound, respiratory formate hydrogen lyase complex of Thermococcus onnurineus was inserted into the P. furiosus chromosome and expressed as a functional unit. This enabled P. furiosus to utilize formate as well as sugars as an H2 source and to do so at both 80° and 95 °C, near the optimum growth temperature of the donor (T. onnurineus) and engineered host (P. furiosus), respectively. This accomplishment also demonstrates the versatility of P. furiosus for metabolic engineering applications.

Mechanisms of muscular electrophysiological and mitochondrial dysfunction following exposure to malathion, an organophosphorus pesticide

Muscle dysfunction in acute organophosphorus (OP) poisoning is a cause of death in human. The present study was conducted to identify the mechanism of action of OP in terms of muscle mitochondrial dysfunction. Electromyography (EMG) was conducted on rats exposed to the acute oral dose of malathion (400 mg/kg) that could inhibit acetylcholinesterase activity up to 70%. The function of mitochondrial respiratory chain and the rate of production of reactive oxygen species (ROS) from intact mitochondria were measured. The bioenergetic pathways were studied by measurement of adenosine triphosphate (ATP), lactate, and glycogen. To identify mitochondrial-dependent apoptotic pathways, the messenger RNA (mRNA) expression of bax and bcl-2, protein expression of caspase-9, mitochondrial cytochrome c release, and DNA damage were measured. The EMG confirmed muscle weakness. The reduction in activity of mitochondrial complexes and muscular glycogen with an elevation of lactate was in association with impairment of cellular respiration. The reduction in mitochondrial proapoptotic stimuli is indicative of autophagic process inducing cytoprotective effects in the early stage of stress. Downregulation of apoptotic signaling may be due to reduction in ATP and ROS, and genotoxic potential of malathion. The maintenance of mitochondrial integrity by means of artificial electron donors and increasing exogenous ATP might prevent toxicity of OPs.

Photocurrent Generation from Surface Assembled Photosystem I on Alkanethiol Modified Electrodes

Photosystem I (PSI) is a key component of oxygenic photosynthetic electron transport because of its light-induced electron transfer to the soluble electron acceptor ferredoxin. This work demonstrates the incorporation of surface assembled cyanobacterial trimeric PSI complexes into a biohybrid system for light-driven current generation. Specifically, this work demonstrates the improved assembly of PSI via electrophoretic deposition, with controllable surface assembled PSI density, on different self-assembled alkanethiol monolayers. Using artificial electron donors and acceptors (Os(bpy)(2)Cl(2) and methyl viologen) we demonstrate photocurrent generation from a single PSI layer, which remains photoactive for at least three hours of intermittent illumination. Photoelectrochemical comparison of the biohybrid systems assembled from different alkanethiols (hexanethiol, aminohexanethiol, mercaptohexanol, and mercaptohexanoic acid) reveals that the PSI generated photocurrent is enhanced by almost 5 times on negatively charged SAM surfaces as compared to positively charged surfaces. These results are discussed in light of how PSI is oriented upon electrodeposition on a SAM.

Oxygen, pH, and mitochondrial oxidative phosphorylation

The oxygen dependence of mitochondrial oxidative phosphorylation was measured in suspensions of isolated rat liver mitochondria using recently developed methods for measuring oxygen and cytochrome c reduction. Cytochrome-c oxidase (energy conservation site 3) activity of the mitochondrial respiratory chain was measured using an artificial electron donor (N,N,N',N'-tetramethyl-p-phenylenediamine) and ascorbate to directly reduce the cytochrome c, bypassing sites 1 and 2. For mitochondrial suspensions with added ATP, metabolic conditions approximating those in intact cells and decreasing oxygen pressure both increased reduction of cytochrome c and decreased respiratory rate. The kinetic parameters [K(M) and maximal rate (V(M))] for oxygen were determined from the respiratory rates calculated for 100% reduction of cytochrome c. At 22°C, the K(M) for oxygen is near 3 Torr (5 μM), 12 Torr (22 μM), and 18 Torr (32 μM) at pH 6.9, 7.4, and 7.9, respectively, and V(M) corresponds to a turnover number for cytochrome c at 100% reduction of near 80/s and is independent of pH. Uncoupling oxidative phosphorylation increased the respiratory rate at saturating oxygen pressures by twofold and decreased the K(M) for oxygen to <2 Torr at all tested pH values. Mitochondrial oxidative phosphorylation is an important oxygen sensor for regulation of metabolism, nutrient delivery to tissues, and cardiopulmonary function. The decrease in K(M) for oxygen with acidification of the cellular environment impacts many tissue functions and may give transformed cells a significant survival advantage over normal cells at low-pH, oxygen-limited environment in growing tumors.

Transmembrane electric potential difference in the protein-pigment complex of photosystem 2

The protein-pigment complex of photosystem 2 (PS2) localized in the thylakoid membranes of higher plants, algae, and cyanobacteria is the main source of oxygen on Earth. The light-induced functioning of PS2 is directly linked to electron and proton transfer across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). The major contribution to ΔΨ of the PS2 reaction center is due to charge separation between the primary chlorophyll donor P(680) and the quinone acceptor Q(A), accompanied by re-reduction of P(680)(+) by the redox-active tyrosine residue Y(Z). The processes associated with the uptake and release of protons on the acceptor and donor sides of the enzyme, respectively, are also coupled with ΔΨ generation. The objective of this work was to describe the mechanisms of ΔΨ generation associated with the S-state transitions of the water-oxidizing complex in intact PS2 complex and in PS2 preparation depleted of Mn(4)Ca cluster in the presence of artificial electron donors. The findings elucidate the mechanisms of electrogenic reactions on the PS2 donor side and may be a basis for development of an effective solar energy conversion system.

Differential effects of UV-B radiation fluence rates on growth, photosynthesis, and phosphate metabolism in two cyanobacteria under copper toxicity

This work was undertaken to ascertain the impact of different fluence rates of ultraviolet-B (UV-B) radiation on two cyanobacterial biofertilizers, Phormidium foveolarum and Nostoc muscorum, growing under copper toxicity. Copper (2 and 5 µmol L−1) and high UV-B fluence rate (UV-BH; 1.0 µmol m−2 s−1) decreased the growth, pigment content, photosynthetic oxygen yield, phosphate uptake, and acid phosphatase activity in both the strains analyzed after 24 and 72 h of experiments, and combined exposure further enhanced the toxic effects. Respiration and alkaline phosphatase activities were stimulated appreciably. The damaging effect was shown on the order on pigments: phycocyanin > chlorophyll a > carotenoids, and on photosystems: whole chain photosynthetic reaction > photosystem II > photosystem I. Partial recovery in the photosystem II activity in the presence of artificial electron donors; diphenyl carbazide (DPC), hydroxylamine (NH2OH), and manganese chloride (MnCl2) pointed out the interruption of electron flow on the oxidation side of photosystem II. Unlike UV-BH, low UV-B fluence rate (UV-BL; 0.1 µmol m−2 s−1), rather than causing damaging effect partially, alleviated the toxic effects of Cu. This study suggests that the cyanobacterium P. foveolarum is less sensitive against UV-BH and excess Cu (2 and 5 µmol L−1), thus P. foveolarum may be used as a biofertilizer for sustainable agriculture.

Stimulatory effect of ascorbate, the alternative electron donor of photosystem II, on the hydrogen production of sulphur-deprived Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is capable of photoproducing molecular hydrogen following sulphur deprivation, which results in anaerobiosis and a suppression of oxygen evolution and thus an alleviation of the inhibitory effect of oxygen on the hydrogenase. At the same time it transiently maintains a limited supply of electrons arising from photosystem II (PSII) to the hydrogenase (Melis and Happe Plant Physiol 2001; 127:740–748). In this work, using fast chl a fluorescence and P700 measurements, we show that ascorbate (Asc), a naturally occurring PSII alternative electron donor, is capable of donating electrons to PSII in heat-treated and sulphur-deprived cells and this can be significantly accelerated by supplementing the culture with 10 mM Asc. It also enhances, about three-fold, the photoproduction of hydrogen in cells subjected to sulphur deprivation as shown by gas chromatography. Similar stimulation was obtained in the presence of diphenylcarbazide (DPC), an artificial PSII electron donor. Asc and DPC also facilitated the anaerobiosis of cells, probably via super reducing the oxygen evolving complex while feeding electrons to PSII reaction centres and the linear electron transport chain, and ultimately to the hydrogenase – as shown by the significant DCMU-sensitivity of the light-induced Asc- and DPC-dependent re-reduction of P700+ and hydrogen evolution.

Photosynthesis-inhibiting effects of 2-benzylsulphanylbenzimidazoles in spinach chloroplasts

AbstractInhibition of photosynthetic electron transport (PET) in spinach chloroplasts by nineteen 2-benzylsulphanylbenzimidazoles (BZA) was studied. BZA were found to inhibit photosynthetic electron transport (PET) and for their inhibitory efficiency, electronic properties of the R substituent on the benzyl moiety are decisive. The PET inhibiting activity of the studied BZA expressed as IC50 varied in the range from 28.5 μM (R = 3,5-(CF3)2) to 394.5 μM (R = 2,4-(NO2)2). For compounds with R = H, 4-CH3, 3-CH3, 3-OCH3, 4-F, 3-F, 4-Cl, 3-Cl, 2-Cl, 4-Br, 3-Br, 3,4-F2, 3,4-Cl2, 3-CF3, 3,5-(CF3)2 linear increase of the inhibitory activity with the increasing value of the substituent’s σ constant up to 0.86 was observed. Further increase of the σ constant resulted in a sharp activity decrease of the corresponding compounds (R = 2-F-6-Cl, 2-NO2, 3,5-(NO2)2, 2,4-(NO2)2). Using EPR spectroscopy and an artificial electron donor diphenyl carbazide it was found that the site of BZA action in the photosynthetic apparatus is situated on the donor side of PS 2, prior to the Z·/D· intermediate.

Photoproduction of Catalase-Insensitive Peroxides on the Donor Side of Manganese-Depleted Photosystem II: Evidence with a Specific Fluorescent Probe

The photoproduction of organic peroxides (ROOH) in photosystem II (PSII) membranes was studied using the fluorescent probe Spy-HP. Two types of peroxide, highly lipophilic ones and relatively hydrophilic ones, were distinguished by the rate of reaction with Spy-HP; the former oxidized Spy-HP to the higher fluorescent form Spy-HPOx within 5 min, while the latter did so very slowly (the reaction was still not completed after 180 min). The level of photoproduction of these peroxides was significantly larger in the alkaline-treated, Mn-depleted PSII membranes than that in the untreated membranes, and it was suppressed by an artificial electron donor (diphenylcarbazide or ferrocyanide) and by the electron transport inhibitor diuron. Postillumination addition of Fe(2+) ions, which degrade peroxides by the Fenton mechanism, abolished the accumulation of Spy-HPOx, but catalase did not change the peroxide level, indicating that the detected species were organic peroxides, excluding H(2)O(2). These results agreed with our previous observation of an electron transport-dependent O(2) consumption on the PSII donor side and indicated that ROOH accumulated via a radical chain reaction that started with the formation of organic radicals on the donor side. Illumination (λ > 600 nm; 1500 μmol of photons m(-2) s(-1)) of the Mn-depleted PSII membranes for 3 min resulted in the formation of nearly 200 molecules of hydrophilic ROOH per reaction center, but only four molecules of highly lipophilic ROOH. The limited formation of the latter was due to the limited supply of its precursor to the reaction, suggesting that it represented structurally fixed peroxides, i.e., either protein peroxides or peroxides of the lipids tightly bound to the core complex. These ROOH forms, likely including several species derived from lipid peroxides, may mediate the donor side-induced photoinhibition of PSII via protein modification.

Homologous expression of a subcomplex of Pyrococcus furiosus hydrogenase that interacts with pyruvate ferredoxin oxidoreductase.

Hydrogen gas is an attractive alternative fuel as it is carbon neutral and has higher energy content per unit mass than fossil fuels. The biological enzyme responsible for utilizing molecular hydrogen is hydrogenase, a heteromeric metalloenzyme requiring a complex maturation process to assemble its O2-sensitive dinuclear-catalytic site containing nickel and iron atoms. To facilitate their utility in applied processes, it is essential that tools are available to engineer hydrogenases to tailor catalytic activity and electron carrier specificity, and decrease oxygen sensitivity using standard molecular biology techniques. As a model system we are using hydrogen-producing Pyrococcus furiosus, which grows optimally at 100°C. We have taken advantage of a recently developed genetic system that allows markerless chromosomal integrations via homologous recombination. We have combined a new gene marker system with a highly-expressed constitutive promoter to enable high-level homologous expression of an engineered form of the cytoplasmic NADP-dependent hydrogenase (SHI) of P. furiosus. In a step towards obtaining ‘minimal’ hydrogenases, we have successfully produced the heterodimeric form of SHI that contains only two of the four subunits found in the native heterotetrameric enzyme. The heterodimeric form is highly active (150 units mg−1 in H2 production using the artificial electron donor methyl viologen) and thermostable (t1/2 ∼0.5 hour at 90°C). Moreover, the heterodimer does not use NADPH and instead can directly utilize reductant supplied by pyruvate ferredoxin oxidoreductase from P. furiosus. The SHI heterodimer and POR therefore represent a two-enzyme system that oxidizes pyruvate and produces H2 in vitro without the need for an intermediate electron carrier.

Role of HoxE subunit in Synechocystis PCC6803 hydrogenase

Cyanobacterial NAD(P)(+)-reducing reversible hydrogenases comprise five subunits. Four of them (HoxF, HoxU, HoxY, and HoxH) are also found in the well-described related enzyme from Ralstonia eutropha. The fifth one (HoxE) is not encoded in the R. eutropha genome, but shares homology with the N-terminal part of R. eutropha HoxF. However, in cyanobacteria, HoxE contains a 2Fe-2S cluster-binding motif that is not found in the related R. eutropha sequence. In order to obtain some insights into the role of HoxE in cyanobacteria, we deleted this subunit in Synechocystis PCC6803. Three types of interaction of the cyanobacterial hydrogenase with pyridine nucleotides were tested: (a) reductive activation of the NiFe site, for which NADPH was found to be more efficient than NADH; (b) H(2) production, for which NADH appeared to be a more efficient electron donor than NADPH; and (c) H(2) oxidation, for which NAD(+) was a much better electron acceptor than NADP(+). Upon hoxE deletion, the Synechocystis hydrogenase active site remained functional with artificial electron donors or acceptors, but the enzyme became unable to catalyze H(2) production or uptake with NADH/NAD(+). However, activation of the electron transfer-independent H/D exchange reaction by NADPH was still observed in the absence of HoxE, whereas activation of this reaction by NADH was lost. These data suggest different mechanisms for diaphorase-mediated electron donation and catalytic site activation in cyanobacterial hydrogenase.

A carbonic anhydrase inhibitor induces bicarbonate-reversible suppression of electron transfer in pea photosystem 2 membrane fragments

The effects of suppression of the carbonic anhydrase (CA) activity by a CA-inhibitor, acetazolamide (AA), on the photosynthetic activities of photosystem II (PS II) particles from higher plants were investigated. AA along with CA-activity inhibits the PS II photosynthetic electron transfer and the AA-induced suppression is totally reversed by the addition of bicarbonate (3–5 mM). Similar effect of recovery in the PS II photosynthetic activity was also revealed upon the addition of known artificial electron donors (potassium ferrocyanide and TMPD). Significance and possible functions of CA for the PS II donor side are discussed.

The Hydrogenase Subcomplex of the NAD+‐Reducing [NiFe] Hydrogenase from Ralstonia eutropha – Insights into Catalysis and Redox Interconversions

AbstractThe O2‐tolerant, NAD+‐reducing soluble [NiFe] hydrogenase (SH) from Ralstonia eutropha H16, HoxHYFUI2, is a complex enzyme, harboring multiple redox cofactors: a [NiFe] active site, an electron relay of iron‐sulfur clusters, and two noncovalently bound flavin mononucleotides (FMN). The interplay and functional role of these cofactors is so far not understood in detail. In the present study, the isolated HoxHY module was investigated, which represents the smallest active subcomplex of a [NiFe] hydrogenase. Direct electrochemical studies and solution assays showed that the as‐isolated HoxHY is initially catalytically inactive, but after reductive activation at low potentials, exhibits both H2 oxidation and H+ reduction, consistent with the role of the SH in bidirectional catalysis. The overpotential relative to E(2H+/H2) is minimal, facilitating coupling of the closely spaced 2H+/H2 and NAD+/NADH half reactions in the SH. Methyl viologen reduction assays revealed that H2 oxidation by HoxHY is enhanced on addition of excess FMN, in line with results from optical spectroscopy which indicate that FMN is present at substoichiometric levels in as‐isolated HoxHY. X‐ray absorption spectroscopy suggested one 4Fe4S cluster in addition to the active site in HoxHY. FTIR investigations confirmed that the active site iron atom has a “standard” ligation, i.e., one CO and two cyanide ligands. At least two novel oxidized states were detected by FTIR, both of which could be reductively activated by artificial electron donors, such as dithionite, and by the native electron donor H2 in the presence of additional FMN. The flavin cofactor also appears to stabilize the active site, providing further evidence for its importance in HoxHY. All reduced states of the [NiFe] site previously identified for standard [NiFe] hydrogenases and for the native SH within living cells were detected in FTIR spectra of HoxHY with the exception of the intermediate Nia‐C species. Electrochemical experiments show that incubation of active HoxHY with O2 at high potentials causes slow inactivation, but activity is recovered within seconds at potentials below –170 mV at 30 °C, even in the presence of 2 % O2. This behavior is consistent with the HoxHY moiety of the SH remaining active in the presence of O2 at the potential of the NAD+/NADH pool in vivo.

Nitrate reductase whole-cell assay: side effects associated with the use of benzyl viologen

A comparative examination of reduced methyl [MV·](+) and benzyl [BV·](+) viologens (as artificial electron donors for quantitative estimation of the respiratory periplasmic (Nap) and membrane-embedded (Nar) nitrate reductases) using a newly constructed nap mutant strain of Paracocccus denitrificans was done. The activity with [MV·](+) was high in whole-cell assays, confirming that this compound donates electrons to Nar. Initial rates of the more lipophilic [BV·](+) were considerably lower, which was interpreted to be caused by an inhibition of the active transport of nitrate into the cells. Anionophoric activity of [BV·](+) was detectable but too low to effectively circumvent the inhibition of nitrate transporter.

Stabilization of Hypoxia-inducible Factor-1α Protein in Hypoxia Occurs Independently of Mitochondrial Reactive Oxygen Species Production

The transcription factor hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the cellular response to low oxygen. HIF-1α protein accumulates in hypoxia due to inhibition of prolyl hydroxylase enzymes, which under normoxic conditions use molecular oxygen to hydroxylate HIF-1α on two conserved proline residues (Pro(402) and Pro(564)), thus targeting the protein for 26 S proteasome-dependent degradation. A functional mitochondrial electron transport chain is known to be necessary for HIF-1α stabilization in hypoxia. It has been reported that reactive oxygen species (ROS), produced under hypoxia by complex III of the mitochondrial electron transport chain, play a critical role in the stabilization of the HIF-1α protein, possibly by directly inhibiting prolyl hydroxylase enzymes. In contrast, we found that ROS production by complex III is not required for hypoxia-induced HIF-1α stabilization. Thus, reestablishing mitochondrial oxygen consumption in the presence of a complex III inhibitor by using an artificial electron donor to complex IV or by overexpressing Ciona intestinalis alternative oxidase results in HIF-1α protein stabilization in hypoxia. Furthermore, five inhibitors that target different sites of the mitochondrial electron transport chain have similar effects on the HIF-1α protein half-life in hypoxia but vary in their effects on mitochondrial ROS production. Finally, ROS do not regulate prolyl hydroxylase activity directly. We conclude that HIF-1α protein stabilization in hypoxia occurs independently of mitochondrial ROS production. However, mitochondria can modulate the cellular hypoxic response through altered respiratory activity, likely by regulating the cellular oxygen availability.

Electron transfer between exogenous electron donors and reaction center of photosystem 2

Transfer of electrons between artificial electron donors diphenylcarbazide (DPC) and hydroxylamine (NH2OH) and reaction center of manganese-depleted photosystem 2 (PS2) complexes was studied using the direct electrometrical method. For the first time it was shown that reduction of redox-active amino acid tyrosine Y(Z)(.) by DPC is coupled with generation of transmembrane electric potential difference (DeltaPsi). The amplitude of this phase comprised ~17% of that of the DeltaPsi phase due to electron transfer between Y(Z) and the primary quinone acceptor Q(A). This phase is associated with vectorial intraprotein electron transfer between the DPC binding site on the protein-water interface and the tyrosine Y(Z)(.). The slowing of DeltaPsi decay in the presence of NH2OH indicates effective electron transfer between the artificial electron donor and reaction center of PS2. It is suggested that NH2OH is able to diffuse through channels with diameter of 2.0-3.0 A visible in PS2 structure and leading from the protein-water interface to the Mn(4)Ca cluster binding site with the concomitant electron donation to Y(Z)(.). Because the dielectrically-weighted distance between the NH2OH binding site and Y(Z)(.) is not determined, the transfer of electrons from NH2OH to Y(Z)(.) could be either electrically silent or contribute negligibly to the observed electrogenicity in comparison with hydrophobic donors.