Hematoporphyrin-promoted photoinactivation of mitochondrial ubiquinol-cytochrome c reductase: selective destruction of the histidine ligands of the iron-sulfur cluster and protective effect of ubiquinone.

Purified ubiquinol-cytochrome c reductase of beef heart mitochondria is very stable in aqueous solution; it suffers little damage upon illumination with visible light under aerobic or anaerobic conditions. However, it is rapidly inactivated when the photosensitizer hematoporphyrin is present during illumination. The hematoporphyrin-promoted photoactivation is dependent on sensitizer dose, illumination time, and oxygen. Singlet oxygen is shown to be the destructive agent in this system. The photoinactivation of ubiquinol-cytochrome c reductase is prevented by excess exogenous ubiquinone, regardless of its redox state. This protective effect is not due to protein-ubiquinone interactions but to the singlet oxygen scavenger property of ubiquinone. Ubiquinone also protects against hematoporphyrin-promoted photoinactivation of succinate-ubiquinone reductase and cytochrome c oxidase. The photoinactivation site in ubiquinol-cytochrome c reductase is the iron-sulfur cluster of Rieske's protein. Two histidine residues, presumably serving as two ligands for the iron-sulfur cluster of Rieske's protein, are destroyed. No polypeptide bond cleavage is detected. Photoinactivation has little effect on the spectral properties of cytochromes b and c1 but alters their reduction rates substantially. this photoinactivation also causes the formation of proton-leaking channels in the complex. When the photoinactivated reductase is co-inlaid with intact ubiquinol-cytochrome c reductase or cytochrome c oxidase in a phospholipid vesicle, no proton ejection can be detected during the oxidation of their corresponding substrates.     

Studies on the oxidation of ammonia by Nitrosomonas

1. Free-energy calculations for pH7 showed that the oxidation of ammonia to hydroxylamine is endergonic and that the oxidations of hydroxylamine to nitrite and hydrazine to nitrogen are exergonic. It is suggested that the oxidation of ammonia requires the expenditure of energy. 2. The anaerobic dehydrogenation of hydrazine to nitrogen by extracts of the autotrophic nitrifying micro-organism, Nitrosomonas, in the presence of methylene blue as electron acceptor, was less rapid than the anaerobic dehydrogenation of hydroxylamine to nitric oxide. The inhibition by hydrazine of the dehydrogenation of hydroxylamine was attributed to substrate competition. 3. Whole cells in air did not produce nitrite from hydrazine. They produced nitrite from low concentrations of hydroxylamine more rapidly than from equimolar concentrations of ammonia; this result is consistent if hydroxylamine is an intermediate of the oxidation of ammonia. 4. The production of nitrite from hydroxylamine by whole cells was slightly inhibited by hydrazine, but the production of nitrite from ammonia was greatly inhibited and small amounts of hydroxylamine were formed. These results suggested that the dehydrogenation of hydroxylamine supplied energy required for the oxidation of ammonia and that hydroxylamine appeared because the energy production was replaced by that of the dehydrogenation of hydrazine. 5. The oxidation of hydroxylamine by whole cells was not inhibited by thiourea, but micromolar concentrations of the metal-binding agent markedly inhibited the oxidation of ammonia to hydroxylamine, suggesting that the oxidation of ammonia involved copper. A possible mechanism for the activation of ammonia is suggested.     

Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea

1. Cells of Nitrosomonas europaea produced N(2)O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N(2)O in cells. 3. Cells also produced N(2)O, but not N(2) gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N(2)O aerobically, but to yield N(2)O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N(2)O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N(2)O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.     

A partial resolution and reconstitution of the ammonia-oxidizing system of Nitrosomonas europaea: role of cytochrome c554

The cell-free ammonia-oxidizing system of Nitrosomonas europaea was resolved into three major fractions: a membrane fraction containing cytochrome a1 and c-type cytochromes, a fraction with hydroxylamine-cytochrome c reductase and a cytochrome c fraction. The ammonia-oxidizing activity was reconstituted by the combination of these three fractions. The activity was more consistently reconstituted by adding Nitrosomonas cytochrome c554 to the membrane fraction. The hydroxylamine-cytochrome c reductase activity of the membrane fraction increased with the addition of cytochrome c554, but the oxidation of hydroxylamine to nitrite required a further addition of cytochrome c552. The ammonia oxidation by the membrane plus cytochrome c554 was affected by the concentration of phosphate and the addition of bovine serum albumin, spermine, or MgCl2.     

The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria

Studies were conducted to elucidate the mechanism of action of 2-chloro-6-(trichloromethyl)pyridine or Technical N-SERVE on the nitrification process brought about byNitrosomonas europaea. The growth ofNitrosomonas was completely inhibited in the presence of 0.2 ppm N-SERVE while 1.0 ppm of the chemical was effective in the complete inhibition of ammonia oxidation by fresh cell suspensions. Cells stored at 4 C for a period of three days required somewhat higher concentrations (1.5 ppm) of N-SERVE for the complete inhibition of their ammonia oxidizing ability while the cytochrome oxidase of these cells was inhibited to the extent of 65 to 70 percent in the presence of a corresponding amount of N-SERVE. A 45 – 70 percent reversal of the inhibition of ammonia oxidation caused by N-SERVE was obtained by the addition of 6×10^–4 M Cu⁺⁺. An equivalent concentration of Cu⁺⁺ was also effective for the complete reversal of the inhibition of cytochrome oxidase present in whole cells.Hydroxylamine oxidation by intactNitrosomonas cells was not affected by levels of N-SERVE ranging from 1 – 3 ppm. The cytochrome oxidase effective in hydroxylamine oxidation and present in cell-free extracts was not inhibited by even 100 ppm N-SERVE. Likewise, the hydroxylamine activating enzyme hydroxylamine cytochromec reductase was also not inhibited by such levels of the chemical. Raising the concentration to 170 ppm N-SERVE, however, caused a 90 percent inhibition of the enzyme.Although a 5×10^–6 M concentration of allylthiourea completely inhibited ammonia oxidation byNitrosomonas cells, concentrations up to 10^–3 M of this compound did not affect the cytochrome oxidase activity of whole cells or cell-free extracts. The inhibition of ammonia oxidation caused by 5×10^–6 M allythiourea, unlike the inhibition by N-SERVE, could not be reversed by the addition of 6×10^–4 M Cu⁺⁺.Evidence is presented that the action of N-SERVE is on that component of cytochrome oxidase which is involved in ammonia oxidation.     

Photorespiration is More Effective than the Mehler Reaction in Protecting the Photosynthetic Apparatus against Photoinhibition

I. Isolated intact chloroplasts: Photosystem II, but not photosystem I, of the electron transport chain is rapidly photoinactivated even by very low intensities of red light when no large proton gradient can be formed and the electron transport chain becomes over-reduced in the absence of oxygen and other reducable substrates. Electron acceptors including oxygen provide protection against photoinactivation. Nevertheless, photosystem II is rapidly, and photosystem I more slowly, photoinactivated by high intensities of red light when oxygen is the only electron acceptor available. Increased damage is observed at increased oxygen concentrations although catalase is added to destroy H₂O₂ formed during oxygen reduction in the Mehler reaction. Photoinactivation can be decreased, but not prevented by ascorbate which reduces hydrogen peroxide inside the chloroplasts and increases coupled electron flow. II. Leaves: Simple measurements of chlorophyll fluorescence permit assessment of damage to photosystem II after exposure of leaves to high intensity illumination. In contrast to isolated chloroplasts, chloroplasts suffer more damage in situ at reduced than at elevated oxygen concentrations. The difference in the responses is due to photorespiration which is active in leaves, but not in isolated chloroplasts. After photosynthesis and photorespiration are inhibited by feeding glyceraldehyde to leaves, photoinactivation is markedly increased, although oxygen reduction in the Mehler reaction is not affected by glyceraldehyde. In the presence of reduced CO₂ levels, photorespiratory reactions, but not the Mehler reaction, can prevent the overreduction of the electron transport chain. Over-reduction indicates ineffective control of photosystem II activity. Effective control is needed for protection of the electron transport chain against photoinactivation. It is suggested to be made possible by coupled cyclic electron flow around photosystem I which is facilitated by the redox poising resulting from the interplay between photorespiratory carbohydrate oxidation and the refixation of evolved CO₂.     

Growth of Nitrosomonas europaea on hydroxylamine

Abstract Hydroxylamine is an intermediate in the oxidation of ammonia to nitrite, but until now it has not been possible to grow Nitrosomonas europaea on hydroxylamine. This study demonstrates that cells of N. europaea are capable of growing mixotrophically on ammonia and hydroxylamine. The molar growth yield on hydroxylamine (4.74 g mol⁻¹ at a growth rate of 0.03 h⁻¹) was higher than expected. Aerobically growing cells of N. europaea oxidized ammonia to nitrite with little loss of inorganic nitrogen, while significant inorganic nitrogen losses occurred when cells were growing mixotrophically on ammonia and hydroxylamine. In the absence of oxygen, hydroxylamine was oxidized with nitrite as electron acceptor, while nitrous oxide was produced. Anaerobic growth of N. europaea on ammonium, hydroxylamine and nitrite could not be observed at growth rates of 0.03 h⁻¹ and 0.01 h⁻¹.     

The role of inactive photosystem-II-mediated quenching in a last-ditch community defence against high light stress in vivo

Photoinactivation of photosystem II (PSII), the light-induced loss of ability to evolve oxygen, is an inevitable event during normal photosynthesis, exacerbated by saturating light but counteracted by repair via new protein synthesis. The photoinactivation of PSII is dependent on the dosage of light: in the absence of repair, typically one PSII is photoinactivated per 10(7) photons, although the exact quantum yield of photoinactivation is modulated by a number of factors, and decreases as fewer active PSII targets are available. PSII complexes initially appear to be photoinactivated independently; however, when less than 30% functional PSII complexes remain, they seem to be protected by strongly dissipative PSII reaction centres in several plant species examined so far, a mechanism which we term 'inactive PSII-mediated quenching'. This mechanism appears to require a pH gradient across the photosynthetic membrane for its optimal operation. The residual fraction of functional PSII complexes may, in turn, aid in the recovery of photoinactivated PSII complexes when conditions become less severe. This mechanism may be important for the photosynthetic apparatus in extreme environments such as those experienced by over-wintering evergreen plants, desert plants exposed to drought and full sunlight and shade plants in sustained sunlight.     

Change in the ratio of cytochrome oxidase activity to nitrite reductase activity of Pseudomonas aeruginosa nitrite reductase with the kind of C-type cytochrome used as an electron donor.

The ratio between the nitrite reductase and cytochrome oxidase activities of Pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2.] varies with kind of C-type cytochrome used as the electron donor. Withe cytochrome c-548, 554 (Micrococcus sp.), the nitrite reductase activity is greater than the cytochrome oxidase activity, while the former is smaller than the latter with cytochrome c-554 (Navicula pelliculosa). The aerobic oxidation catalyzed by this enzyme of denitrifying bacterial ferrocytochrome c is greatly accelerated on addition of nitrite, while that of the algal ferrocytochrome c is not affected or is even depressed by the salt. An accelerative effect of nitrite is generally observed with many kinds of C-type cytochromes which react with the enzyme very or fairly rapidly. The difference in the ratio of the two activities of the enzyme seems to arise according to whether or not nitrite affects the interaction of C-type cytochrome with the enzyme.     

Hydroxylamine metabolism in Pseudomonas PB16: involvement of a novel hydroxylamine oxidoreductase

Pseudomonas strain PB16, a Gram-negative heterotrophic nitrifying bacterium closely related to Pseudomonas azalaica on the basis of 16 S rDNA analysis, was able to use hydroxylamine as an additional energy source during growth in acetate limited chemostat cultures giving an increased biomass yield. In aerobically growing cells of Pseudomonas PB16 only 50% of supplemented hydroxylamine could be recovered as nitrite. In addition to nitrite, N2O could be detected in the chemostat off-gas, indicating combined heterotrophic nitrification and aerobic denitrification. The maximum specific hydroxylamine oxidizing activity observed was 450 nmol per min per mg dry weight, with a Ks of approximately 40 µm. Upon addition of hydroxylamine to the medium, Pseudomonas PB16 induced a soluble 132 KDa dimeric hydroxylamine oxidoreductase. The enzyme had a pH optimum of 9, and did not contain spectroscopic features typical for cytochromes, which is in contrast to hydroxylamine oxidoreductases fou nd in autotrophic bacteria.     

Interrelationship between hydrogen peroxide,ammonia, glutamine,hydroxylamine and nitrite metabolism in the cyanobacterium Phormidium uncinatum

On transition from nitrogen starvation to ammonia or ammonia/glutamine sufficiency Phormidium uncinatum produces high amounts of H₂O₂, which is consumed by several oxidative reactions catalyzed by thylakoid membrane bound enzymes. These include: oxidation of glutamine to free hydroxylamine, of ammonia to nitrite, of bound hydroxylamine to nitrite, and dismutation of free hydroxylamine to ammonia and nitrite. A possible role of these transformations for detoxification is discussed.Non-standard abbreviations FCCP p-trifluormethoxy carbonylcyanide phenylhydrazone - DCMU dichloromethyl urea     

Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.     

Electron transfer during the oxidation of ammonia by the chemolithotrophic bacterium Nitrosomonas europaea

The combined action of ammonia monooxygenase, AMO, (NH(3)+2e(-)+O(2)-->NH(2)OH) and hydroxylamine oxidoreductase, HAO, (NH(2)OH+H(2)O-->HNO(2)+4e(-)+4H(+)) accounts for ammonia oxidation in Nitrosomonas europaea. Pathways for electrons from HAO to O(2), nitrite, NO, H(2)O(2) or AMO are reviewed and some recent advances described. The membrane cytochrome c(M)552 is proposed to participate in the path between HAO and ubiquinone. A bc(1) complex is shown to mediate between ubiquinol and the terminal oxidase and is shown to be downstream of HAO. A novel, red, low-potential, periplasmic copper protein, nitrosocyanin, is introduced. Possible mechanisms for the inhibition of ammonia oxidation in cells by protonophores are summarized. Genes for nitrite- and NO-reductase but not N(2)O or nitrate reductase are present in the genome of Nitrosomonas. Nitrite reductase is not repressed by growth on O(2); the flux of nitrite reduction is controlled at the substrate level.     

Pentahaem cytochrome c nitrite reductase: reaction with hydroxylamine,a potential reaction intermediate and substrate

The pentahaem enzyme cytochrome c nitrite reductase catalyses the reduction of nitrite to ammonia, a key reaction in the biological nitrogen cycle. The enzyme can also transform nitrogen monoxide and hydroxylamine, two potential bound reaction intermediates, into ammonia. Structural and mechanistic aspects of the multihaem enzyme are discussed in comparison with hydroxylamine oxidoreductase, a trimeric protein with eight haem molecules per subunit.     

Electron transport in a Park-Williams strain of Corynebacterium diphtheriae

1. The electron-transport mechanism was examined in the ;particulate' and ;supernatant' fractions of disintegrated cells of a Park-Williams strain of Corynebacterium diphtheriae. 2. Succinate-oxidase activity was found mainly in the ;particulate' fraction, and NADH(2) oxidase mainly in the ;supernatant', which was devoid of cytochromes and menaquinone. 3. The sum of the activities of particles and supernatant fractions, with respect to both succinate oxidase and NADH(2) oxidase, was substantially less than that of the crude cell extract from which they were obtained. Full activity was restored on recombining ;particles' and ;supernatant'. The characteristics of this reassembled system were investigated. 4. The strain of organism (CN2000) examined contained cytochromes corresponding spectroscopically to ;a', ;b' and ;c' types. All three were reduced by succinate, lactate or NADH(2); but a portion of the cytochrome b, susceptible to reduction by dithionite, could not be reduced by the substrates. 5. Triton X-100 inhibits oxidation of succinate by particulate fraction; on adding succinate, the reduction of cytochrome b is not affected but that of cytochromes a and c is delayed. 6. Irradiation at 360mmu completely destroys menaquinone in the particle fraction. Succinate oxidation is severely decreased; succinate dehydrogenase and NADH(2) oxidation are little affected. Certain menaquinones will restore succinate oxidation in the irradiated material. 7. On adding succinate to irradiated particulate material cytochrome b is partially reduced at once, but reduction of cytochromes a and c is much delayed. A portion of the cytochrome b remains not reduced, but reduction occurs rapidly on the addition of menaquinone (MK-2).     

The partial characterization of purified nitrite reductase and hydroxylamine oxidase from Nitrosomonas europaea

Nitrite reductase has been separated from cell-free extracts of Nitrosomonas and partially purified from hydroxylamine oxidase by polyacrylamide-gel electrophoresis. In its oxidized state the enzyme, which did not contain haem, had an extinction maximum at 590nm, which was abolished on reduction. Sodium diethyldithiocarbamate was a potent inhibitor of nitrite reductase. Enzyme activity was stimulated 2.5-fold when remixed with hydroxylamine oxidase, but was unaffected by mammalian cytochrome c. The enzyme also exhibited a low hydroxylamine-dependent nitrite reductase activity. The results suggest that this enzyme is similar to the copper-containing ;denitrifying enzyme' of Pseudomonas denitrificans. A dithionite-reduced, 465nm-absorbing haemoprotein was associated with homogeneous preparations of hydroxylamine oxidase. The band at 465nm maximum was not reduced during the oxidation of hydroxylamine although the extinction was abolished on addition of hydroxylamine, NO(2) (-) or CO. These last-named compounds when added to the oxidized enzyme precluded the appearance of the 465nm-absorption band on addition of dithionite. Several properties of 465nm-absorbing haemoprotein are described.     

Particulate formate oxidase from Nitrobacter agilis

1. The nitrite oxidase particles obtained by sonic oscillation of Nitrobacter agilis cells also possessed appreciable formate oxidase activity, ranging from about 25 to 50% of the nitrite oxidase activity depending upon the N. agilis strain. Both activities distributed themselves in the same pattern and proportions during differential centrifugation, and resided solely in the pellet resulting from high-speed centrifugation.

2. Difference spectra of formate-reduced particles or intact cells demonstrated the presence of cytochromes of the c- and a-types like those of the NO₂⁻-reduced material. Under anaerobic conditions NO₃⁻ or fumarate acted as an alternate electron acceptor in place of O₂ in formate oxidation. Under aerobic conditions increasing NO₃⁻ concentrations resulted in (a) an increased role of NO₃⁻ as a terminal electron acceptor compared to O₂, (b) a greater total enzymatic transfer of electrons from formate than if O₂ were the sole electron acceptor, and (c) a partial inhibition of O₂ uptake suggestive of a competition for electrons by the two acceptors. The formate oxidase system failed to catalyze consistently the transfer of electrons to either added mammalian cytochrome c or Fe(CN)₆³⁻. The marked sensitivity of the system to certain inhibitors implicated cytochrome oxidase as an integral part of the formate oxidase. The system was also inhibited significantly by a variety of chelating agents, indicating a metal component in the formate dehydrogenase or early portion of the electron transfer sequence.

3. The stoichiometry of the formate oxidase system was shown to approach the theoretical value of 2 moles of CO₂ evolved per mole of O₂ or per 2 moles of formate consumed.

4. To a limited extent, phosphorylation occurred concomittantly with the oxidation of formate in the presence of the cell-free particulate system.     

Metabolism of inorganic N compounds by ammonia-oxidizing bacteria

Ammonia oxidizing bacteria extract energy for growth from the oxidation of ammonia to nitrite. Ammonia monooxygenase, which initiates ammonia oxidation, remains enigmatic given the lack of purified preparations. Genetic and biochemical studies support a model for the enzyme consisting of three subunits and metal centers of copper and iron. Knowledge of hydroxylamine oxidoreductase, which oxidizes hydroxylamine formed by ammonia monooxygenase to nitrite, is informed by a crystal structure and detailed spectroscopic and catalytic studies. Other inorganic nitrogen compounds, including NO, N2O, NO2, and N2 can be consumed and/or produced by ammonia-oxidizing bacteria. NO and N2O can be produced as byproducts of hydroxylamine oxidation or through nitrite reduction. NO2 can serve as an alternative oxidant in place of O2 in some ammonia-oxidizing strains. Our knowledge of the diversity of inorganic N metabolism by ammonia-oxidizing bacteria continues to grow. Nonetheless, many questions remain regarding the enzymes and genes involved in these processes and the role of these pathways in ammonia oxidizers.     

Mechanism of photoinactivation in photosynthetic systems II. The occurrence and properties of two different types of photoinactivation

Photoinactivation of the activity of NADP photoreduction withreduced DPIP or with reduced TMPD as the electron donor wasinhibited by the absence of oxygen in the atmosphere or by thepresence of photosynthetic inhibitors (CMU, DCMU, o-phenanthroline)in the preillumination mixture. Photoinactivation of the photoreductionof NADP or DPIP with water as the electron donor was not affected,or even accelerated, by these conditions of preillumination.The concentrations of inhibitors required for maximum inhibitionin the former case corresponded to those required for inhibitionof photosynthetic electron transport. The results indicatedthe occurrence of 2 different types of photoinactivation, eachspecifically affecting photosystems I and II, and differingin behaviours; including their requirement for oxygen in theatmosphere and their responses toward the presence of photosyntheticinhibitors during the preillumination period. (Received July 30, 1969; )