Fermentative H2 production in an upflow anaerobic sludge blanket reactor at various pH values

Fermentative H(2) production in an upflow anaerobic sludge blanket reactor (UASB) at various pH values was investigated in this study. Experimental results show that the H(2) partial pressure in biogas, H(2) production rate and H(2) yield were all pH-dependent, in the range of 0.25-0.52 atm, 42-145 ml-H(2) l(-1) h(-1) and 0.47 to 1.61 mol-H(2)mol-glucose(-1), respectively. The maximum pH for the H(2) partial pressure was observed at pH 7.50. However, the optimum H(2) production rate and H(2) yield were observed at pH 6.50-7.50. In this UASB reactor, acetate, propionate, butyrate, i-butyrate, valerate, caporate and ethanol were present in the effluent as main aqueous products, and the dominant fermentation was butyrate-type at various pHs. The metabolic pathways and thermodynamics of H(2) production were also analyzed. Both H(2) production performance and fermentation pathways in this H(2)-producing UASB reactor were significantly affected by the pH value.     

The effect of hydrogen peroxide on isolated body wall of the lugworm Arenicola marina (L.) at different extracellular pH levels

The effect of hydrogen peroxide on the rate of tissue oxygen consumption, on intracellular pH (pH(i)) and on malondialdehyde (MDA) accumulation was studied in isolated body wall tissue of the lugworm Arenicola marina (L.). H2O2 effects were investigated at various levels of pH(i) by changing medium pH (pH(e)). The largest decrease of tissue oxygen consumption (by 17% below controls), as well as the highest degree of MDA accumulation (four-fold compared to control values) after H2O2 exposure were found at acidic pH(e) of 6.4. This was attributed to the higher redox potential of H2O2 in acidic solutions. Oxygen consumption at alkaline pH(e) (8.5) was not affected by H2O2. MDA accumulation in the tissue was considerably lower than at pH(e) 7.4 or 6.4. Despite pH dependent alterations of H2O2 redox potential, we observed more or less constant pH(e) independent acidification of the tissue upon exposure to H2O2. We attributed the acidification to an inhibition of ATP consuming proton equivalent ion transport across the cellular membrane. Inactivation of carrier proteins is discussed to be responsible for the decrease in tissue oxygen consumption. However, with a larger effect on oxygen consumption at acidic pH(e) values, the latter may not be the only explanation, but additional impairment of other energy demanding processes may be involved.     

Participation of hyf-encoded hydrogenase 4 in molecular hydrogen release coupled with proton-potassium exchange in Escherichia coli

In a previous work (Trchounian et al., Biol. Membrany 16:416-428 (1999) (in Russian)) we reported the interrelations between production of H2 and H+-K+ exchange in fermenting Escherichia coli grown under anaerobic conditions at pH 7.5. The ion fluxes had stable stoichiometry 2H+/K+ and were N,N'-dicyclohexylcarbodiimide (DCC)-inhibitable at different external pH and K+ activity. In the present study, the H2 production was further studied in fermenting bacteria grown at pH 7.5 or 6.5. The H2 production was inhibited by DCC and did not occur if bacteria were grown at pH 7.5 in a medium containing formate or upon hypoosmotic stress. The H2 production was not sensitive to osmotic stress when bacteria were grown at pH 6.5. Formation of H2 and 2H+/K+ exchange were not observed in mutants with deletions of the hyfoperon genes, encoding membrane-associated hydrogenase 4. K+ influx in these mutants was not sensitive to valinomycin, in contrast to the K+ influx in the parental strain. If grown at pH 6.5, the mutants produced H2 and carried out 2H+/K+ exchange, when subjected to the hyperosmotic stress. The results suggest a participation of hydrogenase 4 in the production of H2 and proton-potassium exchange in fermenting E. coli grown at pH 7.5. In bacteria grown at pH 6.5 or in a medium containing formate, another membrane-bound hydrogenase, namely hydrogenase 3, may be responsible for the H2 production.     

Proton transfer reactions associated with the reaction of the fully reduced,purified cytochrome C oxidase with molecular oxygen and ferricyanide

A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from approximately 3.0 H(+)/COX at pH 6.2-6.3 to approximately 1.2 H(+)/COX at pH 8.0-8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from approximately 1.0 H(+)/COX at pH 6.2-6.3 to approximately 2.8 H(+)/COX at pH 8.0-8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3-1.6 H(+)/COX in the pH range 6.2-8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O(2) to 2H(2)O. The results show that the expected stoichiometric proton consumption of 4H(+) in the reduction of O(2) to 2H(2)O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H(+)/COX are initially taken up in the reduction of O(2) to two OH(-) groups bound to the binuclear Fe a(3)-Cu(B) center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H(2)O. At alkaline pHs the third and fourth protons are taken up with formation of 2H(2)O only upon rereduction of COX.     

Behavior of hydrogen peroxide in electrolyzed anode water

Oxygen electrodes and spectrophotometric analysis have been used to evaluate the contribution of H2O2, in addition to available chlorine, to the high redox potential of electrolyzed anode water (EAW) with potassium chloride as an electrolyte. H2O2 was added externally to EAW, and the reaction between H2O2 and the available chlorine in the water was examined. EAW has a low pH (2.5), a high concentration of dissolved oxygen, and extremely high redox potentials (19 mg/l and 1,319 mV) when the available chlorine is at the concentration of about 580 microM. The addition of H2O2 to EAW led to H2O2 decomposition, and the amount of oxygen produced was equivalent to the amount of available chlorine. Oxygen production was reduced by ascorbic acid, and completely inhibited by 600 microM ascorbate. The rate of oxygen production was much affected by pH, and was slowest at or near pH 5.0. Rates were particularly high in alkaline solution. Absorbance at 235 nm (pH 3.0 and 5.0) and 292 nm (pH 10.0) decreased when H2O2 was added to the EAW at these pHs, and the extent of decrease was similar pH dependency to that of the oxygen production rate. Oxygen was not produced after H2O2 was added to EAW at pH 2.6 when available chlorine was absent, but oxygen was produced after potassium hypochlorite was added to such EAW. The oxygen production rates in EAW without available chlorine at pH 5.0 and 2.0, pH adjustment with KOH and HCl, respectively, were faster than the rate at pH 2.6, and fastest at pH 2.0. These results suggest that H2O2 or hydroxyl radicals derived from Fenton's reaction did not contribute to the high redox potential of EAW prepared with chlorine compounds as an electrolyte, so that the decomposition of H2O2 occurred rapidly with the reactions of chlorine and hypochlorite ions in EAW.     

Top and root growth and nutrient absorption of Prunus avium L. at two soil pH and P levels

One-year-old Prunus avium L. were grown under greenhouse conditions in a Countesswells soil in all combinations of 2 pH and 2 P levels. The soil, obtained from a long-term liming and fertilizer experiment, provided pH values throughout the experiment of 3.75–3.99 (pH 1) and 4.81–5.41 (pH 2). The P treatments had 0.43% acetic acid extractable P of 31–44 g g^-1 (P1) and 145–173 g g^-1 (P2). The trees were harvested 92 (H1), 134 (H2), and 168 (H3) days after initiation of growth.Top (leaf+new stem) dry weight was significantly increased for pH 2 and P2 at H2 and H3. P2 also increased leaf weight (H1), the weight of the original stem-root (H2 and H3), and root length but decreased root diameter at both soil pHs (H2 and H3). Total tree uptake of N, P, K, Ca, and Mg was also increased by pH-P combinations which had significantly greater dry matter production and root length. Total Mn uptake decreased at pH2. Root nutrient inflows (uM m^-1 day^-1) were increased for Ca at pH2 and for P at P2. Mn inflow decreased at pH2 and at pH1 P2 although the increased root length associated with the latter treatmen resulted in increased total tree Mn uptake. In general, high nutrient inflows occurred in all trees at H1 and in severely stunted trees at pH1 P1; both had larger than average root diameters.     

Structure of the histone tetramer (H3-H4)2: 1. Peculiarities of optical absorption,fluorescence and light scattering in response to changes in ionic strength and pH of the medium

Using the methods of spectrophotometry, spectrofluorimetry, light scattering and gel filtration, it was shown that, at pH 5.6 and 7.4 and various ionic strengths, the histone tetramer (H3-H4)₂ may have several structural states with different packing of the polypeptide chains of histones H3 and H4. Two structural changes of the tetramer (H3-H4)₂ at pH 7.4 in the ranges 0.1–0.3 m and 0.7–0.9 m NaCl were observed. In the high ionic strength solution, the tetramer (H3-H4)₂ had a more compact structure at pH 7.4 than at pH 5.6. At pH 3.0 destruction of the histone tetramer (H3-H4)₂ and formation of non-specific aggregates took place.     

Lignin peroxidase H2 from Phanerochaete chrysosporium: purification, characterization and stability to temperature and pH

The wood-destroying fungus Phanerochaete chrysosporium secretes extracellular enzymes known as lignin peroxidases that are involved in the biodegradation of lignin and a number of environmental pollutants. Several lignin peroxidases are produced in liquid cultures of this fungus. However, only lignin peroxidase isozyme H8 has been extensively characterized. In agitated nutrient nitrogen-limited culture, P. chrysosporium produces two lignin peroxidases in about equal proportions. The molecular weights of these two major proteins (H2 and H8) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 38,500 (H2) and 42,000 (H8). The isoelectric points of these enzymes were 4.3 for H2 and 3.65 for H8. All subsequent experiments in this study were performed with H2 as it contributed the most (42%) to total activity and had the highest specific activity (57.3 U/mg). The Km values of lignin peroxidase H2 for H2O2 and veratryl alcohol were calculated to be 47 microM and 167 microM at pH 3.5, respectively. The pH optima for veratryl alcohol oxidase activity were pH 2.5 at 25 degrees C, pH 3.0 at 35 degrees C, and pH 3.5 at 45 degrees C. In the same manner the temperature optimum shifted from 25 degrees C at pH 2.5 to 45 degrees C at pH 3.5 and approximately 45-60 degrees C at pH 4.5. During storage the resting enzyme was relatively stable for 48 h up to 50 degrees C. Above this temperature the enzyme lost all activity within 6 h at 60 degrees C. At 70 degrees C all activity was lost within 10 min. The resting enzyme retained approximately 80% of its initial activity when stored at 40 degrees C for 21 h at a pH range of 4.0-6.5. Above pH 7.5 and below 4.0, the enzyme lost all activity in less than 5 h. During turnover the enzyme remained active at pH 5.5 for over 2 h whereas the enzyme activity was lost after 45 min at pH 2.5. The oxidation of veratryl alcohol was inhibited by EDTA, azide, cyanide, and by the catalase inhibitor 3-amino-1,2,4-triazole, but not by chloride. In the absence of another reducing substrate incubation of lignin peroxidase H2 with excess H2O2 resulted in partial and irreversible inactivation of the enzyme. The spectral characteristics of lignin peroxidase H2 are similar to those of other peroxidases. The suitability of lignin peroxidases for industrial applications is discussed.     

Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: equilibrium and kinetics studies

The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) and other divalent cations

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl(2) reduced the H(+) current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H(+) current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn(2+) were inconsistent with classical voltage-dependent block in which Zn(2+) binds within the membrane voltage field. Instead, Zn(2+) binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn(2+) were strongly pH(o) dependent but were insensitive to pH(i), suggesting that protons and Zn(2+) compete for external sites on H(+) channels. The apparent potency of Zn(2+) in slowing activation was approximately 10x greater at pH(o) 7 than at pH(o) 6, and approximately 100x greater at pH(o) 6 than at pH(o) 5. The pH(o) dependence suggests that Zn(2+), not ZnOH(+), is the active species. Evidently, the Zn(2+) receptor is formed by multiple groups, protonation of any of which inhibits Zn(2+) binding. The external receptor bound H(+) and Zn(2+) with pK(a) 6.2-6.6 and pK(M) 6.5, as described by several models. Zn(2+) effects on the proton chord conductance-voltage (g(H)-V) relationship indicated higher affinities, pK(a) 7 and pK(M) 8. CdCl(2) had similar effects as ZnCl(2) and competed with H(+), but had lower affinity. Zn(2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(+) currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn(2+) receptor may be the same modulatory protonation site(s) at which pH(o) regulates H(+) channel gating.     

Kinetics and equilibrium studies on autologous and heterologous recombinations of heavy and light chains of myeloma proteins.

1. The kinetics of the heterologous recombination reaction of alkylated H chains of a myeloma protein (Jo) with alkylated L chains of another myeloma protein (Ita) were studied by following changes with time in the circular dichroism at 235 nm and the results were compared with those for the autologous recombination of Jo-H chains with Jo-H chains reported previously (T. Azuma et al.(1975) J. Biochem. 77, 473-479 and the preceding paper). The heterologous reaction also followed second-order kinetics. The second-order rate constant (kapp) for heterologous recombination was about seven times smaller than that for autologous recombination at pH 5.5, while they were similar between pH 4.2 and 4.7. 2. The apparent association constants (Kapp) for the reaction, H2+L2=H2L2, were determined by measuring the ellipticities at 235 nm of mixtures of H and L chains in various ratios. The values of Kapp for the autologous and heterologous recombinations were both pH-dependent and changed from 10(6) M-1 at pH 3.9 to 108 M-1 at pH 4.3. Using these values of kapp and Kapp, the half-time for the dissociation of autologous H2L2 to H2 and L2 at pH 4.3 was estimated to be 80 hr.     

Influence of pH on Ca²⁺ current and its control of electrical and Ca²⁺ signaling in ventricular myocytes

Modulation of L-type Ca(2+) current (I(Ca,L)) by H(+) ions in cardiac myocytes is controversial, with widely discrepant responses reported. The pH sensitivity of I(Ca,L) was investigated (whole cell voltage clamp) while measuring intracellular Ca(2+) (Ca(2+)(i)) or pH(i) (epifluorescence microscopy) in rabbit and guinea pig ventricular myocytes. Selectively reducing extracellular or intracellular pH (pH(o) 6.5 and pH(i) 6.7) had opposite effects on I(Ca,L) gating, shifting the steady-state activation and inactivation curves to the right and left, respectively, along the voltage axis. At low pH(o), this decreased I(Ca,L), whereas at low pH(i), it increased I(Ca,L) at clamp potentials negative to 0 mV, although the current decreased at more positive potentials. When Ca(2+)(i) was buffered with BAPTA, the stimulatory effect of low pH(i) was even more marked, with essentially no inhibition. We conclude that extracellular H(+) ions inhibit whereas intracellular H(+) ions can stimulate I(Ca,L). Low pH(i) and pH(o) effects on I(Ca,L) were additive, tending to cancel when appropriately combined. They persisted after inhibition of calmodulin kinase II (with KN-93). Effects are consistent with H(+) ion screening of fixed negative charge at the sarcolemma, with additional channel block by H(+)(o) and Ca(2+)(i). Action potential duration (APD) was also strongly H(+) sensitive, being shortened by low pH(o), but lengthened by low pH(i), caused mainly by H(+)-induced changes in late Ca(2+) entry through the L-type Ca(2+) channel. Kinetic analyses of pH-sensitive channel gating, when combined with whole cell modeling, successfully predicted the APD changes, plus many of the accompanying changes in Ca(2+) signaling. We conclude that the pH(i)-versus-pH(o) control of I(Ca,L) will exert a major influence on electrical and Ca(2+)-dependent signaling during acid-base disturbances in the heart.     

Bioreduction and biocrystallization of palladium by Desulfovibrio desulfuricans NCIMB 8307

The reduction of Pd(II) to Pd(0) was accelerated by using the sulfate-reducing bacterium Desulfovibrio desulfuricans NCIMB 8307 at the expense of formate or H(2) as electron donors at pH 2-7. With formate no reduction occurred at pH 2, but with H(2) 50% of the activity was retained at pH 2, with the maximum rate (1.3-1.4 micromol min(-1) mg dry cells(-1)) seen at pH 3-7, which was similar to the rate with formate at neutral pH. Excess nitrate was inhibitory to Pd(II) reduction using formate, but not H(2). Chloride ion was inhibitory as low as 100 mM using formate but with H(2) only ca. 25% inhibition was observed at 500 mM Cl(-) and H(2) was concluded to be the electron donor of choice for the potential remediation of industrial wastes. Deposited Pd was visible on the cells using transmission and scanning electron microscopy and analysis by energy dispersive X-ray microanalysis (EDAX) identified the deposit as Pd, confirmed as Pd(0) by X-ray powder diffraction analysis (XRD). The crystal size of the biodeposited Pd(0) was determined to be only 50% of the size of Pd(0) crystals manufactured chemically from Pd(II) at the expense of H(2) and, unlike the chemically manufactured material, the biocrystal size was independent of the pH. The "biological" Pd(0) functioned as a superior chemical catalyst in a test reaction which liberated hydrogen from hypophosphite. Pd, and also Pt and Rh, could be recovered by resting cell suspensions under H(2) from an industrial processing wastewater, suggesting a possible future application of bioprocessing technology for precious metals.     

Effects of pH on allosteric and catalytic properties of the guanosine cyclic 3',5'-phosphate stimulated cyclic nucleotide phosphodiesterase from calf liver

We have investigated effects of pH on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver. In the "activated" state, i.e., with 0.5 microM [3H]cAMP plus 1 microM cGMP or at saturating substrate concentrations (250 microM [3H]cAMP or [3H]cGMP), hydrolysis was maximal at pH 7.5-8.0 in assays of different pH. Hydrolysis of concentrations of substrate not sufficient to saturate regulatory sites and below the apparent Michaelis constant (Kmapp), i.e., 0.5 microM [3H]cAMP or 0.01 microM [3H]cGMP, was maximal at pH 9.5. Although hydrolysis of 0.5 microM [3H]cAMP increased with pH from 7.5 to 9.5, cGMP stimulation of cAMP hydrolysis decreased. As pH increased or decreased from 7.5, Hill coefficients (napp) and Vmax for cAMP decreased. Thus, assay pH affects both catalytic (Vmax) and allosteric (napp) properties. Enzyme was therefore incubated for 5 min at 30 degrees C in the presence of MgCl2 at various pHs before assay at pH 7.5. Prior exposure to different pHs from pH 6.5 to 10.0 did not alter the Vmax or cGMP-stimulated activity (assayed at pH 7.5). Incubation at high (9.0-10.0) pH did, in assays at pH 7.5, markedly increase hydrolysis of 0.5 microM [3H]cAMP and reduce Kmapp and napp. After incubation at pH 10, hydrolysis of 0.5 microM [3H]cAMP was maximally increased and was similar in the presence or absence of cGMP. Thus, after incubation at high pH, the phosphodiesterase acquires characteristics of the cGMP-stimulated form. Activation at high pH occurs at 30 degrees C but not 5 degrees C, requires MgCl2, and is prevented but not reversed by ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of metabolism using stoichiometry in fermentative biohydrogen

We first constructed full stoichiometry, including cell synthesis, for glucose mixed-acid fermentation at different initial substrate concentrations (0.8-6 g-glucose/L) and pH conditions (final pH 4.0-8.6), based on experimentally determined electron-equivalent balances. The fermentative bioH2 reactions had good electron closure (-9.8 to +12.7% for variations in glucose concentration and -3 to +2% for variations in pH), and C, H, and O errors were below 1%. From the stoichiometry, we computed the ATP yield based on known fermentation pathways. Glucose-variation tests (final pH 4.2-5.1) gave a consistent fermentation pattern of acetate + butyrate + large H2, while pH significantly shifted the catabolic pattern: acetate + butyrate + large H2 at final pH 4.0, acetate + ethanol + modest H2 at final pH 6.8, and acetate + lactate + trivial H2 at final pH 8.6. When lactate or propionate was a dominant soluble end product, the H2 yield was very low, which is in agreement with the theory that reduced ferredoxin (Fd(red)) formation is required for proton reduction to H2. Also consistent with this hypothesis is that high H2 production correlated with a high ratio of butyrate to acetate. Biomass was not a dominant sink for electron equivalents in H2 formation, but became significant (12%) for the lowest glucose concentration (i.e., the most oligotrophic condition). The fermenting bacteria conserved energy similarly at approximately 3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had approximately 3.5 mol ATP/mol glucose) over a wide range of H2 production. The observed biomass yield did not correlate with ATP conservation; low observed biomass yields probably were caused by accelerated rates of decay or production of soluble microbial products.     

pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase. The role of H2O produced at the oxygen-reduction site

A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R --> O transition) decreased from approximately 2.4 at pH 6.5 to approximately 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O --> R transition), the H+/COX ratio for proton release from COV increased from approximately 0.3 at pH 6.5 to approximately 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH < or = 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH > or = 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R --> O --> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.     

Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells

In an effort to determine whether the Na+-dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4- or HPO4(-2), Pi fluxes were determined by measuring 32Pi-Pi self-exchange. Three experimental approaches were employed. First, the effect of pH on steady-state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25-9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4-] (0.05-4.2 mM) at constant [HPO4(-2)] (0.5 mM), and the converse, [HPO4(-2)] (0.06-4.5 mM) at constant [H2PO4-] (0.5 mM), was evaluated. Ks (apparent half-saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4- comprises 93% of the total Pi present, and the calculated Ks is 0.055 +/- 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4-] relationship (0.056 +/- 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4(-2)), the measured Ks is 0.58 +/- 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4(-20)] curve (0.106 +/- 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4- is the substrate for the Na+-dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.     

Optimization of glutamate concentration and pH for H production from volatile fatty acids by Rhodopseudomonas capsulata

AIMS: This study attempted to employ response surface methodology (RSM) to evaluate the effects of glutamate concentration and pH on H(2) production from volatile fatty acids by Rhodopseudomonas capsulata. METHODS AND RESULTS: A mixture of acetate, propionate and butyrate was used as a carbon source for the H(2) production by R. capsulata. The H(2) yield and H(2) production rate were strongly affected by the glutamate concentration, pH and their interaction. The predicted maximum H(2) yield of 0.534 was obtained when glutamate concentration and pH were 6.56 mmol l(-1) and 7.29 respectively. On the contrary, the maximum H(2) production rate of 18.72 ml l(-1) h(-1) was achieved at a glutamate concentration of 7.01 mmol l(-1) and pH 7.31. CONCLUSIONS: Taking H(2) yield and H(2) production rate together into account, a glutamate concentration of 6.56-7.01 mmol l(-1) and pH of 7.29-7.31 should be selected for H(2) production from a mixture of acetate, propionate and butyrate by R. capsulata. SIGNIFICANCE AND IMPACT OF THE STUDY: The RSM was a useful tool for maximizing H(2) production by photosynthetic bacteria (PSB).     

The roles of hydrogenases 3 and 4, and the F0F1-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH

The hyc operon of Escherichia coli encodes the H2-evolving hydrogenase 3 (Hyd-3) complex that, in conjunction with formate dehydrogenase H (Fdh-H), constitutes a membrane-associated formate hydrogenlyase (FHL) catalyzing the disproportionation of formate to CO2 and H2 during fermentative growth at low pH. Recently, an operon (hyf) encoding a potential second H2-evolving hydrogenase (Hyd-4) was identified in E. coli. In this study the roles of the hyc- and hyf-encoded systems in formate-dependent H2 production and Fdh-H activity have been investigated. In cells grown on glucose under fermentative conditions at slightly acidic pH the production of H2 was mostly Hyd-3- and Fdh-H-dependent, and Fdh-H activity was also mainly Hyd-3-dependent. However, at slightly alkaline pH, H2 production was found to be largely Hyd-4, Fdh-H and F0F1-ATPase-dependent, and Fdh-H activity was partially dependent on Hyd-4 and F0F1-ATPase. These results suggest that, at slightly alkaline pH, H2 production and Fdh-H activity are dependent on both the F0F1-ATPase and a novel FHL, designated FHL-2, which is composed of Hyd-4 and Fdh-H, and is driven by a proton gradient established by the F0F1-ATPase.     

Hydroxyl free radical production by the reaction of N-methyl-N'-nitro-N-nitrosoguanidine with hydrogen peroxide without exposure to light.

We examined hydroxyl free radical (.OH) production in the mixture of H2O2 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) without exposure to light using the electron spin resonance spin-trapping technique. When the mixtures were protected from exposure to light, .OH was formed at pH 6.5 and above; it was not formed at pH 5.0 and below, consistent with our previous report. The amount of .OH trapped depended on the concentrations of MNNG and H2O2 and the pH. Nitrite ion was also detected colorimetrically at pH 6.5 and above, but not detected at pH 5.0 and below in the mixtures without exposure to light. Moreover, its production depended on the concentrations of MNNG and H2O2 and the pH. The formation of N-methyl-N'-nitroguanidine in the mixture at pH 7.8 was confirmed by thin-layer chromatography and melting point. These results suggest that nucleophilic attack by H2O2 on the nitroso nitrogen of MNNG results in the formation of N-methyl-N'-nitroguanidine and peroxynitrous acid, which degrades homolytically to yield .OH and nitrogen dioxide, resulting in the production of nitrite ion, at pH 6.5 and above without exposure to light.