Relationship between Nitrite Reduction and Active Phosphate Uptake in the Phosphate-Accumulating Denitrifier Pseudomonas sp. Strain JR 12

Phosphate uptake by the phosphate-accumulating denitrifier Pseudomonas sp. JR12 was examined with different combinations of electron and carbon donors and electron acceptors. Phosphate uptake in acetate-supplemented cells took place with either oxygen or nitrate but did not take place when nitrite served as the final electron acceptor. Furthermore, nitrite reduction rates by this denitrifier were shown to be significantly reduced in the presence of phosphate. Phosphate uptake assays in the presence of the H⁺-ATPase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), in the presence of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or with osmotic shock-treated cells indicated that phosphate transport over the cytoplasmic membrane of this bacterium was mediated by primary and secondary transport systems. By examining the redox transitions of whole cells at 553 nm we found that phosphate addition caused a significant oxidation of a c-type cytochrome. Based on these findings, we propose that this c-type cytochrome serves as an intermediate in the electron transfer to both nitrite reductase and the site responsible for active phosphate transport. In previous studies with this bacterium we found that the oxidation state of this c-type cytochrome was significantly higher in acetate-supplemented, nitrite-respiring cells (incapable of phosphate uptake) than in phosphate-accumulating cells incubated with different combinations of electron donors and acceptors. Based on the latter finding and results obtained in the present study it is suggested that phosphate uptake in this bacterium is subjected to a redox control of the active phosphate transport site. By means of this mechanism an explanation is provided for the observed absence of phosphate uptake in the presence of nitrite and inhibition of nitrite reduction by phosphate in this organism. The implications of these findings regarding denitrifying, phosphate removal wastewater plants is discussed.     

Periplasmic cytochrome c3 of Desulfovibrio vulgaris is directly involved in H2-mediated metal but not sulfate reduction

Kinetic parameters and the role of cytochrome c(3) in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (K(m) = 220 micro M), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H(2) and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H(2) and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H(2), lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate- or pyruvate-reduced, but not H(2)-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H(2) was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H(2) was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c(3) is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.     

Novel domain packing in the crystal structure of a thiosulphate-oxidizing enzyme

A key component of the oxidative biogeochemical sulphur cycle involves the utilization by bacteria of reduced inorganic sulphur compounds as electron donors to photosynthetic or respiratory electron transport chains. The SoxAX protein of the photosynthetic bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that is involved in the oxidation of thiosulphate and sulphide. The recently solved crystal structure of the SoxAX complex represents the first structurally characterized example of a productive electron transfer complex between haemoproteins where both partners adopt the c-type cytochrome fold. The packing of c-type cytochrome domains both within SoxA and at the interface between the subunits of the complex has been compared with other examples and found to be unique.     

The role of blood platelets in tumor angiogenesis

The purple photosynthetic bacterium Rubrivivax gelatinosus has, at least, four periplasmic electron carriers, i.e., HiPIP, two cytochromes c?with low- and high-midpoint potentials, and cytochrome c? as electron donors to the photochemical reaction center. The quadruple mutant lacking all four electron carrier proteins showed extremely slow photosynthetic growth. During the long-term cultivation of this mutant under photosynthetic conditions, a suppressor strain recovering the wild-type growth level appeared. In the cells of the suppressor strain, we found significant accumulation of a soluble c-type cytochrome that has not been detected in wild-type cells. This cytochrome c has a redox midpoint potential of about +280 mV and could function as an electron donor to the photochemical reaction center in vitro. The amino acid sequence of this cytochrome c was 65% identical to that of the high-potential cytochrome c?of this bacterium. The gene for this cytochrome c was identified as nirM on the basis of its location in the newly identified nir operon, which includes a gene coding cytochrome cd?-type nitrite reductase. Phylogenetic analysis and the well-conserved nir operon gene arrangement suggest that the origin of the three cytochromes c? in this bacterium is NirM. The two other cytochromes c?, of high and low potentials, proposed to be generated by gene duplication from NirM, have evolved to function in distinct pathways.     

Influence of Volatile Fatty Acids on Nitrite Accumulation by a Pseudomonas stutzeri Strain Isolated from a Denitrifying Fluidized Bed Reactor

Intermediate nitrite accumulation during denitrification by Pseudomonas stutzeri isolated from a denitrifying fluidized bed reactor was examined in the presence of different volatile fatty acids. Nitrite accumulated when acetate or propionate served as the carbon and electron source but did not accumulate in the presence of butyrate, valerate, or caproate. Nitrite accumulation in the presence of acetate was caused by differences in the rates of nitrate and nitrite reduction and, in addition, by competition between nitrate and nitrite reduction pathways for electrons. Incubation of the cells with butyrate resulted in a slower nitrate reduction rate and a faster nitrite reduction rate than incubation with acetate. Whereas nitrate inhibited the nitrite reduction rate in the presence of acetate, no such inhibition was found in butyrate-supplemented cells. Cytochromes b and c were found to mediate electron transport during nitrate reduction by the cells. Cytochrome c was reduced via a different pathway when nitrite-reducing cells were incubated with acetate than when they were incubated with butyrate. Furthermore, addition of antimycin A to nitrite-reducing cells resulted in partial inhibition of electron transport to cytochrome c in acetate-supplemented cells but not in butyrate-supplemented cells. On the basis of these findings, we propose that differences in intermediate nitrite accumulation are caused by differences in electron flow to nitrate and nitrite reductases during oxidation of either acetate or butyrate.     

Expression and characterisation of a major c-type cytochrome encoded by gene kustc0563 from Kuenenia stuttgartiensis as a recombinant protein in Escherichia coli

The purification of small quantities of a major small c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis has recently been reported. In order to characterise this protein further we have expressed the gene encoding this cytochrome in Escherichia coli and have purified the protein to homogeneity. The protein is directed to the E. coli periplasm using its native signal sequence suggesting that it may be translocated via a Sec-type system in K. stuttgartiensis. The cytochrome has the visible spectroscopic properties typical of a low-spin c-type cytochrome, but these spectroscopic features broaden in high salt solutions. The oxidised cytochrome was able to bind the ligands NO and cyanide. A redox potential of +230 mV suggests that the protein is suitable to act as an electron carrier protein that may be involved in the respiratory chain between hydrazine oxidation and the reduction of nitrite. The predicted protein sequence for the cytochrome suggests it to be a predominantly alpha-helical protein, and this is supported by circular dichroism.     

Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga.

The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water.     

Characterization of cytochromes from Methanosarcina strain Göl and their involvement in electron transport during growth on methanol.

Methanosarcina strain G?1 was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (c547 and c552) cytochromes in membranes from Methanosarcina strain G?1. The midpoint potentials determined were Em,7 = -135 +/- 5 and -240 +/- 11 mV (b-type cytochromes) and Em,7 = -140 +/- 10 and -230 +/- 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 mumol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide.     

The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c(552), is similar to a number of c-type cytochromes from the alpha-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c(552) revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

Thermostability of α-mannosidase in plasma from cystic fibrosis patients and carriers

The hypothesis presented is that the different classes of c-type cytochrome originated as proteins located in the bacterial periplasmic space, or on the periplasmic side of the cytoplasmic membrane. In these locations, covalent bonds between haem and protein prevented the haem from being lost to the surrounding medium. Subsequent evolution has led to internal location of c-type cytochromes in eucaryotes and cyanobacteria. The covalent links have been retained because of their structural role; a b-type cytochrome could be created with similar molecular properties, but its formation would require a large evolutionary jump. If this hypothesis is correct, it should be useful in unravelling electron transport chains with unconventional donors or acceptors. Apparent exceptions deserve further investigation.     

Light-Mediated Nitrite Accumulation during Denitrification by Pseudomonas sp. Strain JR12

The effect of light on the denitrifying characteristics of a nonphotosynthetic denitrifier, Pseudomonas sp. strain JR12, was examined. Already at low light intensities, nitrite accumulated as a result of light inhibition of nitrite but not of nitrate reduction rates. Exposure of this bacterium to light caused a photooxidation of cytochrome c, an intermediate electron carrier in its respiratory pathway. Photoinhibition of nitrite reduction was reversible, as nitrite reduction rates returned to preillumination levels when light-exposed cells were returned to dark conditions. Antimycin A reversed the inhibitory effect of light on nitrite reduction by preventing a reversed electron flow. Aerobic respiration by this bacterium was not affected by light.     

Spectrophotometric and Kinetic Study of Nitrite and Formate Oxidation in Nitrobacter winogradskyi

The reduction levels of cytochrome c and a(1) in intact Nitrobacter cells and cell-free extracts, during and after nitrite or formate oxidation, were examined in combination with the amperometric measurement of oxygen uptake. Quite different reduction patterns were observed when comparing nitrite oxidation by intact cells and cell-free extracts. An inverse relationship was observed between the rate of electron flow and the steady-state reduction level of cytochrome a(1). Parallel observations on nitrite oxidation, by use of formate and reduced nicotinamide adenine dinucleotide as electron donors, showed the influence of the high oxidation-reduction potential of the nitrite-nitrate system on cytochrome reduction. A value for the apparent activation energy of the overall nitrite oxidation process, amounting to 15 kcal, was found in a study of the temperature dependence of cytochrome reduction.     

Purification and properties of a low-redox-potential tetraheme cytochrome c3 from Shewanella putrefaciens.

Shewanella putrefaciens is a facultatively anaerobic bacterium in the gamma group of the proteobacteria, capable of utilizing a wide variety of anaerobic electron acceptors. An examination of its cytochrome content revealed the presence of a tetraheme, low-redox-potential (E'o = -233 mV), cytochrome c-type cytochrome with a molecular mass of 12,120 Da and a pI of 5.8. The electron spin resonance data indicate a bis-histidine coordination of heme groups. Reduction of ferric citrate was accompanied by oxidation of the cytochrome. The biochemical properties suggested that this protein was in the cytochrome c3 group, which is supported by N-terminal sequence data up to the first heme binding site.     

H2 consumption by Escherichia coli coupled via hydrogenase 1 or hydrogenase 2 to different terminal electron acceptors

Hydrogen uptake in the presence of various terminal electron acceptors was examined in Escherichia coli mutants synthesizing either hydrogenase 1 or hydrogenase 2. Both hydrogenases mediated nitrate-dependent H2 consumption but neither of them was coupled with nitrite. Unlike hydrogenase 2, hydrogenase 1 demonstrated poor activity with electron acceptors of low midpoint redox potential. Oxygen-linked H2 uptake via hydrogenase 1 was observed over a wide range of air concentrations. Hydrogenase 2 catalyzed this reaction only at low air concentrations. Thus, hydrogenase 1 works in cells at higher redox potential, being more tolerant to oxygen than hydrogenase 2.     

Energetics of photosynthesis in Zea mays. I. Studies of the flash-induced electrochromic shift and fluorescence induction in bundle sheath cells

Bundle sheath strands free of mesophyll contamination were isolated from 3–4-week-old leaves of maize (Zea mays L.). Patterns of electron flow in the preparations were studied in the presence of physiological substrates. Relative electron flow rates were estimated from the flash-induced electrochromic band shift changes (P-518) and cytochrome f turnover. Induction of chlorophyll fluorescence was also measured. Little Photosystem II activity was found to be present, the principal pathway of electron flow being Photosystem I-driven cyclic electron transfer. The latter was activated through reductive poising by NADPH, generated via malate decarboxylation or (less efficiently) from dihydroxyacetone phosphate. The actions of these electron donors and of oxygen, nitrite and methyl viologen as electron acceptors in redox poising the Photosystem I-driven cycle were investigated and are discussed in relation to the regulation of photosynthesis in the bundle sheath.     

Reduction of nitrite to nitrous oxide by a cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus.

A cytoplasmic membrane fraction from the marine denitrifier Pseudomonas perfectomarinus reduced nitrite to nitrous oxide in a stoichiometric reaction without nitric oxide as free intermediate. The membrane system had a specific requirement for FMN with NAD(P)H as electron donors. Other electron donors were ascorbate-reduced cytochrome c-551 or phenazine methosulfate. The membrane fraction contained tightly bound cytochrome cd which represented only a small portion of the total cytochrome cd of the cell. As further terminal oxidase cytochrome o was identified. The membrane fraction produced also nitrous oxide from nitric oxide, however, at a substantially lower rate than from nitrite when using ascorbate-reduced phenazine methosulfate as electron donor.     

The bioenergetics of denitrification

In anaerobically grown Paracoccus denitrificans the dissimilatory nitrate reductase is linked to the respiratory chain at the level of cytochromes b. Electron transport to nitrite and nitrous oxide involves c-type cytochromes. During electron transport from NADH to nitrate one phosphorylation site is passed, whereas two sites are passed during electron transport from NADH to oxygen, nitrite and nitrous oxide. The presentation of a respiratory chain as a linear array of electron carriers gives a misleading picture of the efficiency of energy conservation since the location of the reductases is not taken into account. For the reduction of nitrite and nitrous oxide, protons are utilized from the periplasmic space, whereas for the reduction of oxygen and nitrate, protons are utilized from the cytoplasmic side of the inner membrane. Evidence for two transport systems for nitrate was obtained. One is driven by the proton motive force; this system is used to initiate nitrate reduction. The second system is a nitrate-nitrite antiport system. A scheme for proton translocation and electron transport to nitrate, nitrite, nitrous oxide and oxygen is presented. The number of charges translocated across the membrane during flow of two electrons from NADH is the same for all nitrogenous oxides and is 67-71% of that during electron transfer to oxygen via cytochrome o. These findings are in accordance with growth yield studies. YMAX electron values determined in chemostat cultures for growth with various substrates and hydrogen acceptors are proportional to the number of charges translocated to these hydrogen acceptors during electron transport.     

Relationship between amino acid transport and electron transport by membrane vesicles of Micrococcus denitrificans

Membrane vesicles were prepared from Micrococcus denitrificans by osmotic shock of lysozyme spheroplasts. These vesicles concentrated 4 amino acids via two systems; one for glycine-alanine and the other for asparagine-glutamine. Amino acid transport was coupled to the membrane-bound electron transport system and involved interactions of the primary dehydrogenases, cytochromes, cytochrome oxidase and oxygen. After transport the amino acids were recovered unchanged from the vesicles. The substrates of the membrane-bound electron transport system d-lactate, l-lactate, formate, succinate, NADH, glucose-6-phosphate and α-glycerolphosphate all stimulated transport at least 2-fold. Both oxygen and nitrate could serve as terminal electron acceptors with vesicles prepared from cells grown anaerobically with nitrate. Anaerobic transport in the presence of nitrate was not inhibited by cyanide but was inhibited by nitrite. A system stimulated by substrates of the electron transport system but independent of added terminal electron acceptors was found also in the vesicles prepared from anaerobically grown cells. Addition of one combination of two substrates for electron transport produced an amino acid uptake 12 to 15% greater than the sum of the rates for each substrate added singly. Additions of other combinations gave rates of transport less than the sum of the rates of each added alone. Both the dehydrogenase activities and the coupling of electron transport to amino acid uptake were modified by changing the growth conditions and differences between the effectiveness of each substrate for each of the two transport systems could be detected. The efficiency of the vesicles per protoheme, the prosthetic group of the membrane-bound cytochrome b, with d-lactate as substrate was 27% for glutamine and 6% for glycine of the rates of transport of these two amino acids in intact cells when driven by endogenous respiration. Assuming one amino acid transported per electron, the transport of glycine utilized 1% of the respiratory capacity with glucose-6-phosphate as substrate. The coupling to the electron transport with the other substrates was less efficient. It appeared that a small portion of the total capacity of the electron transport system was coupled to amino acid transport and the coupling to respiration, as well as the primary dehydrogenase activities and terminal cytochrome oxidase, were modified in response to the conditions of growth.     

Phosphate uptake by phosphorus-starved cells of the cyanobacterium Phormidium laminosum

Phosphorus(P)-starved cells of the cyanobacterium Phormidium laminosum have been investigated in relation to their phosphate uptake characteristics. P-deficient cells showed much higher phosphate uptake rates from ultrapure water supplemented with this anion than P-sufficient ones. After 9 days of starvation in P-free medium, the total cellular P content of P-deficient cells was approximately five times lower than that of cells grown in the presence of phosphate. Phosphate uptake by P-deficient cells occurred in both light and dark under aerobic conditions. In anaerobiosis, light was required for uptake, suggesting that the necessary energy could be derived from the respiratory electron transport chain. Phosphate uptake in P-deficient cells was sensitive to vanadate, suggesting the involvement of a plasma membrane ATPase.     

Thermodynamic properties of triheme cytochrome PpcF from Geobacter metallireducens reveal unprecedented functional mechanism

The bacterium Geobacter metallireducens is highly efficient in long-range extracellular electron transfer, a process that relies on an efficient bridging between the cytoplasmic electron donors and the extracellular acceptors. The periplasmic triheme cytochromes are crucial players in these processes and thus the understanding of their functional mechanism is crucial to elucidate the extracellular electron transfer processes in this microorganism. The triheme cytochrome PpcF from G. metallireducens has the lowest amino acid sequence identity with the remaining cytochromes from the PpcA-family of G. sulfurreducens and G. metallireducens, making it an interesting target for structural and functional studies. In this work, we performed a detailed functional and thermodynamic characterization of cytochrome PpcF by the complementary usage of NMR and visible spectroscopic techniques. The results obtained show that the heme reduction potentials are negative, different from each other and are also modulated by the redox and redox-Bohr interactions that assure unprecedented mechanistic features to the protein. The results showed that the order of oxidation of the hemes in cytochrome PpcF is maintained in the entire physiological pH range. The considerable separation of the hemes' redox potential values facilitates a sequential transfer within the chain of redox centers in PpcF, thus assuring electron transfer directionality to the electron acceptors.