The role of a conserved tyrosine residue in high-potential iron sulfur proteins.

Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.     

Expression, purification and characterization of a high potential iron–sulfur protein from Acidithiobacillus ferrooxidans

The high potential iron–sulfur protein (HiPIP) is involved in the iron respiratory electron transport chain of Acidithiobacillus ferrooxidans but its exact role is unclear. The gene of HiPIP from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, and the protein then purified by one-step affinity chromatography to homogeneity. The molecular mass of the HiPIP monomer was 7250.43 Da by MALDI-TOF MS, indicating the presence of the [Fe₄S₄] cluster. The optical and EPR spectra results of the recombinant protein confirmed that the iron–sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys25, Cys28, Cys37 and Cys50 were involved in ligating to the iron–sulfur cluster.     

An Electrochemical Study of the Factors Responsible for Modulating the Reduction Potential of Putidaredoxin

 The gene coding for putidaredoxin has been synthesized using a combination of chemical and enzymatic methods and subsequently expressed in Escherichia coli. The recombinant protein characterized by electronic spectroscopy, mass spectrometry, and electrochemistry was found to be identical to putidaredoxin obtained from Pseudomonas putida. Polylysine was found to promote the fast and reversible electrochemistry of putidaredoxin at negatively charged electrodes such as indium-doped tin oxide or gold surfaces modified with mercaptoalkanoate groups. The value of the heterogeneous electron transfer rate constant obtained from solutions containing a mixture of putidaredoxin and polylysine (k _s =1.3×10^–3 cm/s) is one order of magnitude larger than the values reported previously at gold electrodes modified with mercaptoethylamine or at antimony-doped tin oxide semiconductor electrodes. It was observed that when the reduction potential of putidaredoxin is measured by cyclic voltammetry, the resultant value is consistently more positive (64 mV) than the reduction potential measured with potentiometric titrations. A comparison between the electrochemical responses of putidaredoxin and spinach ferredoxin, combined with the examination of their corresponding three-dimensional structures, indicates that the positive shift in the reduction potential of putidaredoxin originates from the formation of a transient complex between putidaredoxin and polylysine at the electrode surface. The formation of this transient complex modulates the reduction potential of putidaredoxin by lowering the value of the dielectric constant around its iron-sulfur cluster microenvironment, specifically by neutralizing negative charges surrounding the active site and by excluding water from the solvent exposed iron sulfur cluster. The observed positive shift in E°′, which is induced by complexation with polylysine at the electrode-surface, suggests that similar factors are likely to contribute to the anodic shift in the E°′ of cytochrome P450_cam-bound putidaredoxin (+44 mV) with respect to the E°′ measured for free putidaredoxin. Received: 14 June 1999 / Accepted: 6 August 1999     

A cephalosporin C acetylhydrolase is present in the cultures of Nocardia lactamdurans

Two protein bands with strong esterase activity are present in broths of Nocardia lactamdurans MA4213 cultures. One of them shows cephalosporin C acetylhydrolase (CAH) activity. This activity is maximal at 48 h of growth and shows a pattern of regulation slightly different from that of cephamycin production in medium supplemented with glucose (166 mM), glycerol (326 mM) or ammonium chloride (60 mM). The CAH activity was purified to homogeneity by DEAE-Sepharose ion-exchange, Sephadex G-75 gel filtration, and phenyl-Sepharose hydrophobic interaction chromatography. It showed a molecular mass of 72,100 Da. The N-terminus of the protein was determined and showed the amino acid sequence GGAAPGGPGAHPLWLPAGKD. The enzyme showed K _m values of 7.0 mM and 8.3 mM for cephalosporin C and 7-aminocephalosporanic acid respectively but was not active on cephamycin C. Received: 17 December 1999 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c

 The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50  °C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E°′ observed with increasing ionic strength [ΔE°′_IS =(E°′) _{I =0.1 M}–(E°′) _{I =0 M}=–0.035 V at 25  °C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK _a for a residue deprotonation is the key event of this conformational change. Received: 7 April 1999 / Accepted: 19 July 1999     

FTIR-monitored thermal titration reveals different mechanisms for the alkaline isomerization of tuna compared to horse and bovine cytochromes c

 Fourier transform infrared (FTIR) spectroscopy is used to compare the thermally induced conformational changes in horse, bovine and tuna ferricytochromes c in 50 mM phosphate/0.2 M KCl. Thermal titration in D₂O at pD 7.0 of the amide II intensity of the buried peptide NH protons reveals tertiary structural transitions at 54  °C in horse and at 57  °C in bovine c. These transitions, which occur well before loss of secondary structure, are associated with the alkaline isomerization involving Met80 heme-ligand exchange. In tuna c, the amide-II-monitored alkaline isomerization occurs at 35  °C, followed by a second amide II transition at 50  °C revealing a hitherto unreported conformational change in this cytochrome. Amide II transitions at 50  °C (tuna) and 54  °C (horse) are also observed during the thermal titration of the CN^–-ligated cytochromes (where CN^– displaces the Met80 ligand), but a well-defined 35  °C amide II transition is absent from the titration curve of the CN^–adduct of tuna c. The different mechanisms suggested by the FTIR data for the alkaline isomerization of tuna and the mammalian cytochromes c are discussed. After the alkaline isomerization, loss of secondary structure and protein aggregation occur within a 5  °C range with T _m values at 74  °C (bovine c), 70  °C (horse c) and 65  °C (tuna c), as monitored by changes in the amide I′ bands. The FTIR spectra were also used to compare the secondary structures of the ferricytochromes c at 25  °C. Curve fitting of the amide I (H₂O) and amide I′ (D₂O) bands reveals essentially identical secondary structure in horse and bovine c, whereas splitting of the α-helical absorption of tuna c indicates the presence of less-stable helical structures. CN^– adduct formation results in no FTIR-detectable changes in the secondary structures of either tuna or horse c, indicating that Met80 ligation does not influence the secondary structural elements in these cytochromes. The data provided here demonstrate for the first time that the selective thermal titration of the amide II intensity of buried peptide NH protons in D₂O is a powerful tool in protein conformational analysis. Received: 1 April 1999 / Accepted: 24 August 1999     

Exploitation of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid)

Active butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) were purified in three steps: ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on Matrex Red A from recombinant Escherichia coli K2006 (pJC7). They were then successfully exploited for in vitro synthesis of 3-hydroxybutyryl-CoA (3HBCoA), 4-hydroxybutyryl-CoA (4HBCoA), 4-hydroxyvaleryl-CoA (4HVCoA) and poly(hydroxyalkanoic acid) (PHA). In addition, the ability of the PHA synthase of Chromatium vinosum, PhaEC_Cv, to use these CoA thioesters was evaluated. Combination of Buk and Ptb with PhaEC_Cv established a new system for in vitro synthesis of poly(3-hydroxybutyric acid) [poly(3HB)]. In this system, 3-hydroxybutyric acid was converted to 3HBCoA by Buk and Ptb at the expense of ATP. Formation of 3HBCoA was further driven by the polymerization of 3HBCoA molecules to poly(3HB) by PHA synthase, and the released CoA was recycled by Ptb. This system therefore also ensured the regeneration of CoA. With ATP as the energy supply, which was hydrolyzed to ADP and phosphate, 2.6 mg poly(3HB) was obtained from a 1-ml reaction mixture containing 7.6 mg 3-hydroxybutyrate at the beginning. Studies showed that Ptb and PHA synthase were the rate-limiting steps in this system, and initial CoA concentrations ranging from 1 to 7 mM did not inhibit poly(3HB) synthesis. Synthesis of various polyesters of 3HB and 4HB with this system was also tested, and copolyesters containing 4HB of 1–46 mol % were obtained. Received: 17 September 1999 / Accepted: 1 November 1999     

In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum

Purified recombinant poly(hydroxyalkanoic acid) (PHA) synthase from Chromatium vinosum (PhaEC_Cv) was used to examine in vitro the specific synthase activity, turnover of R-(−)-3-hydroxybutyryl coenzyme A (3HB-CoA) and poly(3-hydroxybutyric acid) formation under various conditions. The 3HB-CoA consumption was terminated by a reaction-dependent inactivation of the PHA synthase. Salts (MgCl₂, CaCl₂, NaCl), proteins (bovine serum albumin, lysozyme, phasine) or detergent (Tween 20) increased the 3HB-CoA turnover to 2.5-fold. Specific PHA synthase activity was only partially affected by the added components. In general, a higher concentration of salt often inhibited the activity of PhaEC_Cv without affecting the yield according to 3HB-CoA turnover. NAD⁺ and NADP⁺ (2 mM) inhibited PhaEC_Cv completely, where-as NADH and NADPH did not. Macroscopic poly(3HB) granules were formed in vitro if PhaEC_Cv was incubated in the presence of sufficient amounts of 3HB-CoA and if MgCl₂ was present. The form and size of the granules synthesized in vitro were affected by the concentration of the PHA synthase protein as well as by bovine serum albumin and the GA24 protein, a poly(3HB)-granule-associated protein of Alcaligenes eutrophus. Scanning electron micrographs from the synthesized granules were obtained. The granules consisted of poly(3HB) that had a molar mass in the range (1–2) × 10⁶ g/mol. Received: 12 September 1997 / Received revision: 24 October 1997 / Accepted: 31 October 1997     

Kinetics and characteristics of 70 °C, VFA-grown, UASB granular sludge

We studied in batch reactors the kinetics and characterization of 70 °C, volatile fatty acids (VFAs)-grown, upflow anaerobic sludge blanket granular sludge with 55 and 35 °C sludge as reference. The half-saturation constant (K _s), the inhibition constant (K _i), the maximum specific methane production rate (μ_CH4max), and the inhibition response coefficient (n) of the 70 °C sludge were 6.15 mM, 48.2 mM, 0.132 h⁻¹, and 2.48, respectively, while no inhibition occurred at 55 and 35 °C, where the K _s was 3.67 and 3.82 mM, respectively. At 70 °C, the highest initial specific methanogenic activity (ISMA, 0.311 gCH₄-COD per gram volatile solids per day) on VFAs was about 12–15% lower than that on acetate and three to four times less than the ISMA for the 55 and 35 °C sludge. In the acetate conversion study, residual acetate (79 mg l⁻¹) at 70 °C was three to five times higher than that at 55 and 35 °C. Further, the methane produced as percentage of the acetate consumed at 70 °C (89%) was lower than that at 55 (95%) and 35 °C (97%). At 70 °C, 10% of the ISMA remained after 15 days of starvation as compared to 26% (55 °C) and 92% (35 °C) after 30 days of starvation. Thus, the kinetics of the 70 °C granular sludge seem to differ from those at 55 and 35 °C. Received: 1 February 1999 / Accepted: 20 March 1999     

Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture

In this work, a BHK21 clone producing a recombinant antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on glucose and glutamine concentrations respectively. A similar dependence is also observed for lactate and ammonia productions. The estimated value of the Michaelis constant for the dependence of lactate production on glucose (K ^Glc _Lac) was 1.4 ± 0.1 mM and for the dependence of ammonia production on glutamine (K ^Gln _Amm) was 0.25 ± 0.11 mM and 0.10 ± 0.03 mM, at glucose concentrations of 0.28 mM and 5.6 mM respectively. At very low glucose concentrations, the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower lactate production. This␣metabolic shift was also confirmed by the significant increase in the specific oxygen consumption rate also observed at low glucose concentrations. Although it was␣highly dependent on glucose concentration, the oxygen consumption also increased with the increase in␣glutamine concentration. At very low glutamine concentrations, the glutamine to ammonia yield increased, showing a more efficient glutamine metabolism. Received: 21 August 1998 / Received revision: 11 November 1998 / Accepted: 17 January 1999     

Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. UTEX LB1999

Limited feeding of nitrate during culture of Nannochloris sp. UTEX LB1999 for intracellular lipid and triglyceride accumulation was investigated with the aim of obtaining cells superior for liquefaction into a fuel oil. The intracellular lipid contents and the percentage of triglycerides in the lipids of cells grown in a nitrogen-limited medium (0.9 mM KNO₃) were 1.3 times as high as those grown in a modified NORO medium containing 2.0–9.9 mM KNO₃. However, the cell concentration was too low for the practical production of fuel oil by high-pressure liquefaction of the cell mass. A single feeding of 0.9 mM nitrate after nitrate depletion during cultivation in a nitrate-limited medium increased the cell concentration to twice that obtained without such feeding, and the lipid content was maintained at a high level. The timing of nitrate feeding, i.e., whether it was given during the log phase (before nitrate depletion), the constant growth phase (just after the depletion), or the stationary phase (after the depletion), had negligible effect on the intracellular lipid content and percentage of triglycerides in the lipids. When 0.9 mM nitrate was intermittently fed ten times during the log phase in addition to the initial nitrate feed (0.9 mM), the cell concentration reached almost the same (2.16 g/l) and the intracellular lipid content and the percentage of triglycerides in the lipids increased from 31.0 to 50.9% and 26.0 to 47.6%, respectively, compared with those of cells cultured in a modified NORO medium containing 9.9 mM KNO₃ without additional nitrate feeding. Received: 27 October 1999 / Received revision: 26 November 1999 / Accepted: 24 December 1999     

Screening of mannanases in actinomycetes and their potential application in the biobleaching of pine kraft pulps

Fifty actinomycete strains were screened for the production of mannanase activity during growth in both liquid and solid media. Streptomyces scabies CECT 3340 and Streptomyces ipomoea CECT 3341 were selected for their ability to produce high levels of mannanase (294.3 U/l and 242.9 U/l, respectively) during growth in liquid culture. β-Mannosidase (15.3 U/l) and α-galactosidase (7.7 U/l) activities were also detected in culture filtrate from S. scabies CECT 3340. Highest levels of mannanase activity for S. scabies CECT 3340 were achieved in media containing locust bean gum and asparagine (4.8 U mg⁻¹ protein) whilst in S. ipomoea CECT 3341 greatest activity was detected in media containing locust bean gum and yeast extract (13.2 U mg⁻¹ protein). No carboxymethylcellulase activity was detected. In biobleaching experiments, enzyme treatment, carried out with mannanase activity produced by S. ipomoea CECT 3341, followed by alkaline extraction of pine kraft pulp resulted in the release of colour (A ₄₆₅, 0.69) and chromophoric material from the pulp (A ₂₃₇, 12.9; A ₂₅₄, 6.9 and A ₂₈₀, 6.7). The ability of this enzyme complex to improve the bleaching of pine kraft pulps was also shown by a pulp brightness increase (2.4 units ISO) and a reduction in kappa number (from 21.4 units to 20.1 units) with the absence of variations on the viscosity values. Received: 23 February 1999 / Received revision: 1 April 1999 / Accepted: 6 April 1999     

Osmotic water permeability of isolated vacuoles

We measured the osmotic water permeability (P _os) of vacuoles isolated from onion (Allium cepa L.), rape (Brassica napus L.), petunia (Petunia hybrida Hook.) and red beet (Beta vulgaris L.). For all the vacuolar types investigated, P _os values were in the range 200–1000 μm s⁻¹. The change in membrane surface area induced by an osmotic gradient was smaller than 2–6%. The vacuolar P _os values for red beet and onion were reduced by 1 mM HgCl₂, to 14% and 30% of the control values, respectively, but were partially restored to 51% and 76% by 5 mM β-mercaptoethanol. These results suggest that aquaporins were present in all the vacuoles tested. In HgCl₂-treated onion vacuoles, the reduced P _os (56 μm s⁻¹) had a low activation energy (approx. 6 kJ mol⁻¹), indicating that water permeation was still occurring mainly via aquaporins, and that the water permeability of the lipid part of the vacuolar membrane is probably very low. Received: 18 February 1999 / Accepted: 21 June 1999     

The effect of growth conditions on the biodegradation of tributyl phosphate and potential for the remediation of acid mine drainage waters by a naturally-occurring mixed microbial culture

The biodegradation of tributyl phosphate (Bu₃-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu₃-P biodegradation by growing cells is described. Growth at the expense of Bu₃-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu₃-P biodegradation was optimal at 30 °C, reduced at 20 °C and negligible at 4 °C and 37 °C. Incorporation of Cu or Cd inhibited, and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu₃-P biodegradation significantly, but nitrate had no effect. Sulphate (10–100 mM) was inhibitory. When pregrown biomass was used the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 μmol h⁻¹ mg protein⁻¹ and 37 μmol h⁻¹ mg protein⁻¹ respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate precipitation at the expense of Bu₃-P hydrolysis in the presence of 35 mM SO₄ ²⁻. At pH 4.5, 79% of the UO₂ ²⁺ was removed at a flow rate of 1.4 ml/h on a 7-ml test column. Received: 2 June 1997 / Received revision: 15 September 1997 / Accepted: 19 September 1997     

Orientation-selected ENDOR of the active center in Chromatium vinosum [NiFe] hydrogenase in the oxidized "ready" state

 Electron nuclear double resonance (ENDOR) was applied to study the active site of the oxidized "ready" state, Ni_r, in the [NiFe] hydrogenase of Chromatium vinosum. The magnetic field dependence of the EPR was used to select specific subsets of molecules contributing to the ENDOR response by stepping through the EPR envelope. Three hyperfine couplings could be clearly followed over the complete field range. Two protons, H1 and H2, display a very similar large isotropic coupling of 12.5 and 12.6 MHz, respectively. Their dipolar coupling is small (2.1 and 1.4 MHz, respectively). A third proton, H3, exhibits a small isotropic coupling of 0.5 MHz and a larger anisotropic contribution of 3.5 MHz. Based on a comparison with structural data obtained from X-ray crystallography of single crystals of hydrogenases from Desulfovibrio gigas and D. vulgaris and the known g-tensor orientation of Ni_r, an assignment of the ¹H hyperfine couplings could be achieved. H1 and H2 were assigned to the β-CH₂ protons of the bridging cysteine Cys533 and H3 could belong to a β-CH₂ proton of Cys68 or to a protonated cysteine (-SH) of Cys68 or Cys530. Received: 26 November 1998 / Accepted: 1 April 1999     

Purification and characterization of a fix ABCX-linked 2[4Fe-4S] ferredoxin from Azotobacter vinelandii

 Ferredoxins that contain 2[4Fe-4S]^2+/+ clusters can be divided into two classes. The "clostridial-type" ferredoxins have two CysXXCysXXCysXXXCysPro motifs. The "photosynthetic bacterial and nif-related" ferredoxins have one motif of that type and one more unusual CysXXCysX_7–9CysXXXCysPro motif. In Azotobacter vinelandii three gene sequences have been reported that contain the latter motif, but until now none of the gene products has been purified. Here we report the purification of a small anionic [Fe-S] protein with yields of ∼3 mg per 500 g cell paste. NH₂-terminal sequence analysis shows that this protein is the product of a previously sequenced A. vinelandii gene that is found upstream of fixA and is cotranscribed with fixABCX. That gene was originally named fixP, but since that gene designation is now commonly used for a very different cb-type cytochrome oxidase we have renamed the gene fixFd and its product Fix Fd. Its sequence places Fix Fd in the class of "photosynthetic bacterial and nif-related" 2[4Fe-4S]^2+/1+ ferredoxins that includes Chromatium vinosum ferredoxin. Studies of the purified protein by Fe analysis, absorption, CD and EPR spectroscopies and electrochemistry confirm this characterization; the reduction potentials of the two clusters are –440 mV vs SHE. The fact that A. vinelandii synthesizes three different proteins with the same sequence motif, each of which is likely to have a different function, shows that although sequence motifs may be used reliably to classify ferredoxins by cluster type they cannot yet be used reliably for classifying ferredoxins by function. Received: 31 January 1997 / Accepted: 9 June 1997     

Reduction of the nitrogen and carbon content in swine waste with algae and bacteria

Animal waste causes environmental problems like eutrophication of ground and surface water or the pollution of the atmosphere because of its high NH₄ ⁺ content. The aim of our study was to fix the nitrogen of swine waste as biomass. Therefore, an isolated alga, Chlorella sp., and bacteria naturally living in liquid manure were grown in batch cultures (containing diluted swine waste supplied with a nutrient solution) and continuous cultures (undiluted liquid manure) to achieve reduction of NH₄ ⁺ and total organic carbon (TOC) contents. For continuous cultivation, a photobioreactor of our own design was used. The batch cultivation of Chlorella sp. and bacteria in swine waste resulted in good growth of both groups of organisms and in a reduction of 25% NH₄ ⁺ and 80% TOC. In the continuous cultivation a steady state was not achieved owing to a change in the composition of the bacterial population. NH₄ ⁺ was totally removed, but NO₂ ⁻ (up to 100 mM) was transiently released. NO₃ ⁻ was not detected. These effects might be explained by the presence of heterotrophic nitrifiers, which are able to oxidize NH₄ ⁺ to NO₂ ⁻ and to reduce NO₂ ⁻ to gaseous compounds. Received: 21 January 1999 / Received revision: 9 March 1999 / Accepted: 14 March 1999     

Interaction of nitric oxide with the oxygen evolving complex of photosystem II and manganese catalase: a comparative study

Thermus thermophilus catalase. Flash fluorescence studies indicate that the S₃ state of the OEC in the presence of ca. 0.6 mM NO is reduced to the S₁ with an apparent halftime of ca. 0.4 s at about 18 °C, compared with a biphasic decay, with approximate halftimes of 28 s for S₃ to S₂ and 140 s for S₂ to S₁ in the absence of NO. Under similar conditions the S₂ state is reduced by NO to the S₁ state with an approximate halftime of 2 s. These results extend a recent study indicating a slow reduction of the S₁ state at −30°C, via the S₀ and S₋₁ states, to a Mn(II)-Mn(III) state resembling the corresponding state in catalase. The reductive mode of action of NO is repeated with the di-Mn cluster of catalase: the Mn(III)-Mn(III) redox state is reduced to the Mn(II)-Mn(II) state via the intermediate Mn(II)-Mn(III) state. The kinetics of this reduction suggest a decreasing reduction potential with decreasing oxidation state, similar to what is observed with the active states of the OEC. What is unique about the OEC is the rapid interaction of NO with the S₃ state of the OEC, which is compatible with a metalloradical character of this state. Received: 16 June 1999 / Accepted: 28 February 2000     

Redox chemistry of biological tungsten: an EPR study of the aldehyde oxidoreductase from Pyrococcus furiosus

 Aldehyde:ferredoxin oxidoreductase (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus is a homodimeric protein. Each subunit carries one [4Fe-4S] cubane and a novel tungsten cofactor containing two pterins. A single iron atom bridges between the subunits. AOR has previously been studied with EPR spectroscopy in an inactive form known as the red tungsten protein (RTP): reduced RTP exhibits complex EPR interaction signals. We have now investigated the active enzyme AOR with EPR, and we have found an S = 1/2 plus S = 3/2 spin mixture from a non-interacting [4Fe-4S]¹⁺ cluster in the reduced enzyme. Oxidized AOR affords EPR signals typical for W(V) with g–values of 1.982, 1.953, and 1.885. The W(V) signals disappear at a reduction potential E _m,7.5 of +180 mV. This unexpectedly high value indicates that the active-site redox chemistry is based on the pterin part of the cofactor. Received: 18 December 1995 / Accepted: 26 March 1996     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.