人脑醛糖还原酶的纯化与性质

联合采用ＤＥＡＥ－纤维素层析、色谱聚焦、ＮＡＤＰ亲和层析与ＳｅｐｈａｄｅｘＧ－１００的凝胶过滤,对人脑醛糖还原酶（ＥＣ１．１．１．２１;ＡＬＲ）进行纯化．现测得该酶的等电点ｐＨ值为５．８５．经聚丙烯酰胺凝胶盘状电泳和Ｗｅｓｔｅｒｎ印迹证实,获得了满意的酶纯度．同葡萄糖,葡糖－６－磷酸与ＮＡＤＰＨ保温后,人脑ＡＬＲ纯品的活性与对照酶组相似,且不被糖酵解途径的一些磷酸化中间产物抑制．苯基硼酸琼脂糖柱层析洗脱谱峰和氢硼化钠还原反应提示,当同葡萄糖保温时,人脑ＡＬＲ（特别是其均一态）可能未被进一步糖基化．在糖尿病并发症和按结构完成药物设计的研究工作中,纯品ＡＬＲ的应用可发挥重要作用     

Nitrate Reductase and Nitrite Reductase Activity in Dark-Grown Radish Seedlings: Relation to the Supply of NADPH

Functioning of nitrate reductase and nitrite reductase was measured in intact cotyledons from radish seedlings (Raphanus sativus L.) grown in the dark in a nitrate medium. Reduction of nitrate to nitrate did proceed during the whole period of 45 h, whereas the reduction of nitrite in the intact cotyledons dropped abruptly between 20 and 23 h after exposing the roots to nitrate. The activity of the enzymes glucose-6-P dehydrogenase (G6PDH) and 6-P-gluconate dehydrogenase (6PGDH), measured in cotyledon extracts, showed a sharp decline simultaneously with the drop in nitrite reductase activity of the intact cotyledons. It was concluded that the amount of NADPH generated by the enzymes G6PDH and 6PGDH is not sufficient to allow continuous functioning of nitrite reductase after 20 h in cotyledons of seedlings grown in the dark. Therefore, the results from our experiments point to the functioning of nitrite reductase as the rate limiting step in the reduction pathway of nitrate in the dark.     

Nitrite Reductase Activity of Nonsymbiotic Hemoglobins from Arabidopsis thaliana

Plant nonsymbiotic hemoglobins possess hexacoordinate heme geometry similar to that of the heme protein neuroglobin. We recently discovered that deoxygenated neuroglobin converts nitrite to nitric oxide (NO), an important signaling molecule involved in many processes in plants. We sought to determine whether Arabidopsis thaliana nonsymbiotic hemoglobins classes 1 and 2 (AHb1 and AHb2, respectively) might function as nitrite reductases. We found that the reaction of nitrite with deoxygenated AHb1 and AHb2 generates NO gas and iron-nitrosyl-hemoglobin species. The bimolecular rate constants for reduction of nitrite to NO are 19.8 ± 3.2 and 4.9 ± 0.2 M(-1) s(-1), respectively, at pH 7.4 and 25 °C. We determined the pH dependence of these bimolecular rate constants and found a linear correlation with the concentration of protons, indicating the requirement for one proton in the reaction. The release of free NO gas during the reaction under anoxic and hypoxic (2% oxygen) conditions was confirmed by chemiluminescence detection. These results demonstrate that deoxygenated AHb1 and AHb2 reduce nitrite to form NO via a mechanism analogous to that observed for hemoglobin, myoglobin, and neuroglobin. Our findings suggest that during severe hypoxia and in the anaerobic plant roots, especially in species submerged in water, nonsymbiotic hemoglobins provide a viable pathway for NO generation via nitrite reduction.     

Methyl Viologen-linked Nitrite Reductase from Bean Roots

Methyl viologen-linked nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, was isolated from bean roots. The isolated enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 62,000 by SDS-polyacrylamide gel electrophoresis. In the oxidized form, the enzyme had absorption maxima at 280, 397 (Soret band), 535, and 573 nm (α band), indicating that siroheme is directly involved in the catalysis of nitrite reduction. The absorbance ratios, A₃₉₇ : A₂₈₀ and A₅₇₃ : A₃₉₇, were 0.3 and 0.39, respectively. Antiserum to spinach leaf nitrite reductase failed to give a positive Ouchterlony result with bean root nitrite reductase, but this antiserum did inhibit the activity of the latter enzyme.     

C-terminal tail residue Arg1400 enables NADPH to regulate electron transfer in neuronal nitric-oxide synthase

The neuronal nitric-oxide synthase (nNOS) flavoprotein domain (nNOSr) contains regulatory elements that repress its electron flux in the absence of bound calmodulin (CaM). The repression also requires bound NADP(H), but the mechanism is unclear. The crystal structure of a CaM-free nNOSr revealed an ionic interaction between Arg(1400) in the C-terminal tail regulatory element and the 2'-phosphate group of bound NADP(H). We tested the role of this interaction by substituting Ser and Glu for Arg(1400) in nNOSr and in the full-length nNOS enzyme. The CaM-free nNOSr mutants had cytochrome c reductase activities that were less repressed than in wild-type, and this effect could be mimicked in wild-type by using NADH instead of NADPH. The nNOSr mutants also had faster flavin reduction rates, greater apparent K(m) for NADPH, and greater rates of flavin auto-oxidation. Single-turnover cytochrome c reduction data linked these properties to an inability of NADP(H) to cause shielding of the FMN module in the CaM-free nNOSr mutants. The full-length nNOS mutants had no NO synthesis in the CaM-free state and had lower steady-state NO synthesis activities in the CaM-bound state compared with wild-type. However, the mutants had faster rates of ferric heme reduction and ferrous heme-NO complex formation. Slowing down heme reduction in R1400E nNOS with CaM analogues brought its NO synthesis activity back up to normal level. Our studies indicate that the Arg(1400)-2'-phosphate interaction is a means by which bound NADP(H) represses electron transfer into and out of CaM-free nNOSr. This interaction enables the C-terminal tail to regulate a conformational equilibrium of the FMN module that controls its electron transfer reactions in both the CaM-free and CaM-bound forms of nNOS.     

Reduction of Nitrate and Nitrite by Subcellular Preparations of Anabaena cylindrica. I. Reduction of Nitrite to Ammonia

Reduction of nitrite by cell-free preparations of Anabaena cylindrica in the dark has been investigated. Nitrite-reducing activity was recovered in a supernatant fraction. The nitrite reductase system was partially purified by column chromatography on Sephadex G-75. NADPH could serve as an H-donor. NADH was completely inactive. The reduction required ferredoxin which mediated the transfer of electrons from NADPH to nitrite. Ferredoxin was successfully replaced with methyl viologen, benzyl viologen and diquat. The nitrite-reducing activity was inhibited by KCN, and by 2,4-dinitrophenol and arsenate at higher concentrations. The extent of nitrite reduction by NADPH was dependent on the oxidation-reduction states of NADP and ferredoxin.     

The FAD-shielding residue Phe1395 regulates neuronal nitric-oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain

Phe(1395) stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe(1395) was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP(+) binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP(+) with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP(+) dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP(+)-bound state as compared with wild type. We conclude that Phe(1395) regulates nNOSr catalysis in two ways. It facilitates NADP(+) release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer.     

Regulation of Nitrite Reductase Activity under CO2 Limitation in the Cyanobacterium Synechococcus sp. PCC7942

During photoautotrophic growth under CO2-limited conditions, cells of Synechococcus sp. PCC7942 excreted into the medium about 30% of the nitrite produced by reduction of nitrate. No nitrite was excreted under CO2-sufficient conditions. After transfer of high-CO2-grown cells to CO2-limited conditions, nitrite reductase activity started to decline within 0.5 h and decreased to 50% of the initial level in 3 h, whereas nitrate reductase activity was virtually unchanged. Nitrite started to accumulate in the medium about 3 h after the transfer of the cells to CO2-limited conditions and reached a concentration of >0.4 mM at 17 h. These findings suggested that the nitrite excretion was due to an imbalance of the activities of nitrite reductase and nitrate reductase. Since ammonium, the product of nitrite reduction, was not detected in the medium, it was concluded that the step of nitrite reduction limits the rate of nitrate assimilation under CO2-limited conditions. The extent of decrease in nitrite reductase activity under CO2-limited conditions was much larger than that caused by rifampicin (an inhibitor of RNA synthesis) treatment under high-CO2 conditions. Addition of CO2, in the form of sodium bicarbonate, to the CO2-limited culture increased the nitrite reductase activity, but rifampicin inhibited this increase. These findings suggested the presence of a mechanism that irreversibly inactivates nitrite reductase under CO2-limited conditions.     

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

In leaves of spinach plants (Spinacia oleracea L.) performing CO₂ and NO₃⁻ assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.     

Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities.     

Further Characterization of Ferredoxin Nitrite Reductase and the Relationship between the Enzyme and Methyl Viologen-dependent Nitrite Reductase

Ferredoxin-dependent nitrite reductase of spinach has been further characterized and the relationship between this enzyme and methyl viologen-dependent nitrite reductase studied.

Purified ferredoxin nitrite reductase, having a molecular weight of 86,000, showed 2.5 times higher ferredoxin-dependent activity than methyl viologen-linked activity. Besides 4 mol of labile sulfide the enzyme contained about 2 mol of siroheme per mol. When dithionite, methyl viologen and nitrite were added, ESR signals of a heme nitrosyl complex at g = 2.14, 2.07 and 2.02 were observed. Moreover, hyperfine splitting of the signal due to ¹⁴N nuclear spin was also observed at 2.033, 2.023 and 2.013. The sole addition of hydroxylamine to the ferric enzyme also caused the same but much less intense signals with the hyperfine splitting.

On treatment of the ferredoxin nitrite reductase (native enzyme) with DEAE-Sephadex A-50 chromatography, a modified nitrite reductase having a molecular weight of 61,000 and a protein fraction having an apparent molecular weight of 24,000 were separated. The modified enzyme contained about one mol of siroheme and 4 mol of labile sulfide per mol and showed essentially the same heme ESR signals as the native enzyme. Contrary to the native enzyme, this modified enzyme accepted electrons more efficiently from methyl viologen than ferredoxin and the reduction of nitrite to ammonia catalyzed by the modified enzyme was not stoichiometric. The observed nitrite to ammonia ratio was 1 to less than 0.6. Cyanide at concentrations between 0.02 to 0.2 mm inhibited the activity of the native enzyme almost completely but the modified enzyme was inhibited only partially.

From the results obtained, it is suggested that the native ferredoxin-linked nitrite reductase consists of two components (or subunits) and removal of the light component results in formation of a modified enzyme with increased relative affinity to methyl viologen.     

15N Studies on the In Vivo Assay of Nitrate Reductase in Leaves: Occurrence of Underestimation of the Activity Due to Dark Assimilation of Nitrate and Nitrite

The reduction of nitrate and nitrite in leaf disks from sevendi- and two monocotyledonous species under in vivo nitrate reductaseassay conditions was studied using 15N-labeled substrates. Significantreduction of both nitrate and nitrite into ammonia and aminoacids was detected under aerobic conditions (in an atmosphereof air): in some cases, the amount of nitrate-N reduced to ammoniaand amino acids was more than that remaining as nitrite. Anaerobicincubation (under an atmosphere of N₂ gas) enhanced the accumulationof nitrite, but the subsequent reduction to the basic nitrogencompounds was 40 to 180% of the aerobic rates. The present examinationindicates that in vivo assays of nitrate reductase under aerobicconditions may give greatly underestimated results due to nitritereduction and that exclusion of oxygen from the in vivo assaymixture is desirable in terms of the quantity of nitrite formed.n-Propanal treatment increased nitrite accumulation under aerobicbut not under anaerobic conditions, and depressed the incorporationof nitrate-N into basic fractions under both conditions. Therefore,addition of n-propanol may be desirable for assays under aerobicconditions. No significant difference in the reduction of nitratesupplied as sodium and potassium salts was observed on the nitriteformation and on the incorporation of nitrate-N into basic fractions. ¹⁵N experiments on dark assimilation of nitrate, nitrite andammonia into amino acids in wheat leaves showed that these threenitrogen sources were assimilated through the same route andthat the glutamine synthetase/glutamate synthase pathway wasthe major route. With anaerobic treatment, the incorporationof nitrogen into alanine and serine remained at relatively high,but the incorporation into aspartate and asparagine was muchlower than in the cases of aerobic treatment. (Received July 11, 1981; Accepted October 3, 1981)     

Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2

Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b₅, and NADH-dependent cytochrome b₅ reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar K_cat and V_max values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.     

Purification and Properties of Nitrite Reductase from Spinach Leaves

Nitrite reductase (EC 1.6.6.4) has been purified 730-fold from spinach leaves. The enzyme catalyzes the reduction of nitrite to ammonia, with the use of reduced form of methyl viologen and ferredoxin. A stoichiometry of one molecule of nitrite reduced per molecule of ammonia formed has been found. KCN at 2.5×10^-4 m inhibited nitrite reductase activity almost completely. Purified enzyme was almost homogeneous by disk electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 61,000 from gel filtration. Nitrite reductase, in the oxidized form, has absorption maxima at 276, 388 and 573 mμ. Both methyl viologen and ferredoxin linked nitrite reductase activities of the enzyme were inactivated on exposure to low ionic strength.     

Elucidation of a complete kinetic mechanism for a mammalian hydroxysteroid dehydrogenase (HSD) and identification of all enzyme forms on the reaction coordinate: the example of rat liver 3alpha-HSD (AKR1C9)

Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3alpha-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP(+) (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP(+) controlled k(cat), whereas the chemical event also contributed to this term. k(cat) was insensitive to [(2)H]NADPH, whereas (D)k(cat)/K(m) and the (D)k(lim) (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated k(cat), whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall K(eq) favored reduction. The kinetic Haldane and free energy diagram confirmed that K(eq) was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.     

Cytochrome cd1 Nitrite Reductase NirS Is Involved in Anaerobic Magnetite Biomineralization in Magnetospirillum gryphiswaldense and Requires NirN for Proper d1 Heme Assembly

The alphaproteobacterium Magnetospirillum gryphiswaldense synthesizes magnetosomes, which are membrane-enveloped crystals of magnetite. Here we show that nitrite reduction is involved in redox control during anaerobic biomineralization of the mixed-valence iron oxide magnetite. The cytochrome cd₁-type nitrite reductase NirS shares conspicuous sequence similarity with NirN, which is also encoded within a larger nir cluster. Deletion of any one of these two nir genes resulted in impaired growth and smaller, fewer, and aberrantly shaped magnetite crystals during nitrate reduction. However, whereas nitrite reduction was completely abolished in the ΔnirS mutant, attenuated but significant nitrite reduction occurred in the ΔnirN mutant, indicating that only NirS is a nitrite reductase in M. gryphiswaldense. However, the ΔnirN mutant produced a different form of periplasmic d₁ heme that was not noncovalently bound to NirS, indicating that NirN is required for full reductase activity by maintaining a proper form of d₁ heme for holo-cytochrome cd₁ assembly. In conclusion, we assign for the first time a physiological function to NirN and demonstrate that effective nitrite reduction is required for biomineralization of wild-type crystals, probably by contributing to oxidation of ferrous iron under oxygen-limited conditions.     

Participation of ferredoxin in oxygen reduction by the photosynthetic electron transport chain

Oxygen reduction by isolated pea thylakoids was studied in the presence of ferredoxin (Fd), Fd + NADP, and cytochrome c. At Fd concentrations optimal for NADP reduction, it contributed 30–50% of the reducing equivalents (as deduced by comparing the rates of oxygen reduction and light oxidation of reduced Fd). The oxygen reduction rate in the presence of Fd + NADP was 3–4 times lower than with Fd alone, and comparable to that with cyt c. It is supposed that the process involves a photosystem I component whose reaction with oxygen depends on the rate of electron efflux from the PS I terminal acceptors, and that this component is phylloquinone.     

The redox state of the NADP system in illuminated chloroplasts

Pyridine nucleotide levels were measured in intact spinach chloroplasts. The NADPH/NADP ratio was close to unity in darkened chloroplasts. On illumination, chloroplast NADP levels decreased rapidly. The decrease was more prominent at low than at high light intensities. In the presence of bicarbonate, NADP subsequently increased to reach a steady-state level. The kinetics of the increase were related in general, but not in detail, to the lag phase of photosynthesis. In the steady state, chloroplast NADP was sometimes, particularly during photosynthesis at high light intensities, less reduced in the light than in the dark. In the dark-light transition, phosphoglycerate reduction is driven by increases in the ratios NADPH/NADP and ATP/ADP. When photosynthesis accelerates after the initial lag phase, the NADPH/NADP ratio decreases and a high ratio of phosphoglycerate to triose phosphate becomes an important factor in driving carbon reduction. Under photosynthetic flux conditions, the redox state of the chloroplast NADP system appeared to be governed largely by the chloroplast ratio of phosphoglycerate to dihydroxyacetone phosphate and by the phosphorylation potential [ATP]/[ADP] [P_i]. The inhibitor of cyclic electron transport, antimycin A, increased reduction of the chloroplast NADP system. Even when reduction was almost complete in the presence of 5 μM antimycin A, photosynthesis was still significant at low light intensities. Electrons appeared to be effectively distributed between the cyclic electron-transport pathway and the noncyclic route to NADP at NADPH/NADP ratios as low as about 1. When bicarbonate was absent, the NADP system remained largely reduced in the light. The energy-transfer inhibitor, Dio-9, and uncouplers and agents which interfered with pH regulation of the Calvin cycle increased reduction of the NADP system while decreasing photosynthesis.     

Kinetics of hydrogen-dependent denitrification under varying pH and temperature conditions

It is important to determine the effect of changing environmental conditions on the microbial kinetics for design and modeling of biological treatment processes. In this research, the kinetics of nitrate and nitrite reduction by autotrophic hydrogen-dependent denitrifying bacteria and the possible role of acetogens were studied in two sequencing batch reactors (SBR) under varying pH and temperature conditions. A zero order kinetic model was proposed for nitrate and nitrite reduction and kinetic coefficients were obtained at two temperatures (25 +/- 1 and 12 +/- 1 degrees C), and pH ranging from 7 to 9.5. Nitrate and nitrite reduction was inhibited at pH of 7 at both temperatures of 12 +/- 1 and 25 +/- 1 degrees C. The optimum pH conditions for nitrate and nitrite reduction were 9.5 at 25 +/- 1 degrees C and 8.5 at 12 +/- 1 degrees C. Nitrate and nitrite reduction rates were compared, when they were used separately as the sole electron acceptor. It was shown that nitrite reduction rates consistently exceeded nitrate reduction rates, regardless of temperature and pH. The observed transitional accumulation of nitrite, when nitrate was used as an electron acceptor, indicated that nitrite reduction was slowed down by the presence of nitrate. No activity of acetogenic bacteria was observed in the hydrogenotrophic biomass and no residual acetate was detected, verifying that the kinetic parameters obtained were not influenced by heterotrophic denitrification and accurately represented autotrophic activity.