Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium.

Cellular ATP level, ATP/ADP ratio and nitrogenase activity rise when oxyleghaemoglobin is added to respiring suspensions of Rhizobium japonicum bacteroids from soybean root nodules. Increased gaseous O2 tension is much less efficient than oxyleghaemoglobin in stimulation of bacteroid ATP production. Studies with the inhibitor carbonyl cyanide m-chlorophenylhydrazone show this ATP to be generated as a consequence of oxidative phosphorylation. N-Phenylimidazole, a specific cytochrome P-450 inhibitor, also lowers the efficiency of bacteroid oxidative phosphorylation. An approximately linear relationship is observed between ATP/ADP ratio and nitrogenase activity as N-phenylimidazole concentration is lowered. It is suggested that cytochrome P-450 is a component of the leghaemoglobin-facilitated respiration pathway and that it may act as intracellular O2 carrier rather than terminal oxidase. A less efficient oxidase appears to function when cytochrome P-450 is inhibited.     

Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)

The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea ( Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O₂ evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO₂ (1.0 m M NaHCO₃) than that at limiting CO₂ (0.1 m M NaHCO₃). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO₂. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO₂ than at optimal CO₂. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO₂ (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.     

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450_arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450_arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (20 cigarettes a day) than in normal and moderate smokers (<20 cigarettes a day). At term, the mean aromatase activity and P-450_arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450_arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450_arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450_arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.     

Effects of phenobarbital and sodium salicylate on cytochrome P-450 mixed function oxygenase and glutathione S-transferase activities in rat brain

The effects of acute and therapeutic doses of phenobarbital and sodium salicylate on cytochrome P-450 mixed function oxygenase (EC 1.14.14.1) and glutathione S-transferase (EC 2.5.1.18) activities have been studied in rat brain and compared with those of rat liver. P-450 enzymic activity was assayed by N-demethylation of p-chloro-N-methylaniline and 1-chloro-2,4-dinitrobenzene was used as substrate for glutathione S-transferase activity. The acute effects of a single daily dose of phenobarbital (75 mg/kg/day;i.p.) and sodium salicylate (500 mg/kg/day;i.p.) for 3 days increased cytochrome P-450 as well as glutathione S-transferase in rat liver. But the same doses of both drugs decreased glutathione S-transferase levels in rat brain and increased cytochrome P-450 dependent N-demethylation of p-chloro-N-methylaniline. The therapeutic doses of sodium salicylate (50 mg/kg/day;i.p.) and phenobarbital (10 mg/kg/day;i.p.) daily for 21 days increased cytochrome P-450 in rat liver as well as in brain. The increase in brain glutathione S-transferase by prolonged treatment of phenobarbital was significant compared to the control values.     

Mitochondria from human term placenta. II. Characterization of respiratory pathways and coupling mechanisms

Pathways of electron transport utilized for respiration in human term placental mitochondrial preparations were differentiated and characterized through the use of classical respiratory chain inhibitors and multiple sources of reducing equivalents. Mechanisms of associated energy conservation and utilization were examined in these preparations with uncouplers and inhibitors of phosphorylation.

Inhibition by rotenone, antimycin A and cyanide established the classical electron transport chain as the major pathway of respiration with glutamate and succinate as substrates. Approximately 20% of glutamate-supported respiration was insensitive to inhibitors and may proceed by the cytochrome P-450 linked pathway of electron transport. Approximately 50% of ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine supported respiration was insensitive to 10⁻³ M cyanide and must utilize an undefined by-pass of cytochrome oxidase. A rotenone- and antimycin-insensitive, exterior pathway for NADH oxidation was demonstrated which could be artificially linked by exogenous cytochrome c to the cytochrome oxidase region of the classical electron transport system. Glycerol 3-phosphate also supported oxidative phosphorylation yielding ADP/O ratios of 2.

Respiration of placental mitochondria was stimulated by 2,4- dinitrophenol and gramicidin. With succinate, dinitrophenol-stimulated respiration exceeded that obtain-red in the presence of ADP. Oligomycin and atractyloside prevented the stimulation of respiration by ADP. Thus, respiration appeared coupled through normal mechanisms to ATP formation and ion transport. A preferential coupling of respiration to the energy-utilizing processes of steroid hormone biosynthesis may exist.     

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M_r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg⁻¹ (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min⁻¹·mmg⁻¹ and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min⁻¹·mg⁻¹. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.     

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.     

The membrane ATPase of Escherichia coli I. Ion dependence and ATP-ADP exchange reaction

The properties of the membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli have been reexamined using membranes obtained by mechanical disruption of exponentially growing cells.

The activity exhibited an absolute requirement for Mg²⁺ in the neutral pH range, while Ca²⁺ was found able to activate ATPase at more alkaline pH. Optimal activity was observed at pH 7.5, with a Mg/ATP ratio of 0.5.

ADP was found to inhibit ATP hydrolysis and to transform the Michaelian ATP concentration dependence with a K_m of 0.5 mM into a sigmoid curve with increasing K_m and decreasing V.

In contrast ADP activated an ATP-ADP exchange process and this shift from hydrolysis to exchange was stimulated by high Mg²⁺ and by orthophosphate.

All nucleoside triphosphates tested interfered with ATP hydrolysis, all could be hydrolyzed and could donate their terminal phosphate group to ADP. The relative efficiencies of nucleoside triphosphates in these three processes varied in parallel with minor discrepancies.

ATP hydrolysis was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD) Dio 9, NaN₃ and pyrophosphate, the first two being ineffective against ATP-ADP exchange, the third being stimulatory and the last inhibitory.

ATP hydrolysis and ATP-ADP exchange are tentatively attributed to the terminal enzyme of oxidative phosphorylation.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Cytochrome P-450 inducer/inhibitor effects on cell cultures of Catharanthus roseus

Cytochrome P-450 inducers phenobarbitone and β-naphthoflavone and cytochrome P-450 inhibitor ketoconazole were examined for their effect on Catharanthus roseus. Treatment was during growth on medium M3 which supports alkaloid synthesis. The inhibitor ketoconazole was found to inhibit serpentine accumulation prior to an effect on growth, while the inducers inhibited growth and in the case of phenobarbitone increased serpentine accumulation. No direct evidence of induction or inhibition of plant cytochrome P-450 is shown and the results are discussed in relation to possible effects on geraniol hydroxylase and other plant cytochromes P-450.     

Regulation of dinitrogen fixation in intact Azotobacter vinelandii

1. In intact Azotobacter vinelandii the influence of oxygen on the levels of oxidized nicotinamide adenine dinucleotides and adenine nucleotides in relation to nitrogenase activity was investigated.

2. The hypothesis that a high (NADH + NADPH)/(NAD⁺ + NADP⁺) is the driving force for the transport of reducing equivalents to nitrogenase in intact A. vinelandii was found to be invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of the whole cells increases.

3. By measuring oxidative phosphorylation and using 9-amino acridine as a fluorescent probe, it could be demonstrated that respiration-coupled transport of reducing equivalents to the nitrogenase requires a high energy level of the plasma membrane or possibly coupled to it, a high pH gradient over the cytoplasmic membrane. Furthermore nitrogen fixation is controlled by the presence of oxygen and the ATP/ADP ratio.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

Mechanism of O2 generation in reduction and oxidation cycle of ubiquinones in a model of mitochondrial electron transport systems

O⁻₂ generation in mitochondrial electron transport systems, especially the NADPH-coenzyme Q₁₀ oxidoreductase system, was examined using a model system, NADPH-coenzyme Q₁-NADPH-dependent cytochrome P-450 reductase. One electron reduction of coenzyme Q₁ produces coenzyme Q₁ and O⁻₂ during enzyme-catalyzed reduction and O₂ + coenzyme Q₁ are in equilibrium with O⁻₂ + coenzyme Q₁ in the presence of enough O₂. The coenzyme Q₁ produced can be completely eliminated by superoxide dismutase, identical to bound coenzyme Q₁₀ radical produced in a succinate/fumarate couple-KCN-submitochondrial system in the presence of O₂. Superoxide dismutase promotes electron transfer from reduced enzyme to coenzyme Q₁ by the rapid dismutation of O₂⁻ generated, thereby preventing the reduction of coenzyme Q₁ by O⁻₂. The enzymatic reduction of coenzyme Q₁ to coenzyme Q₁H₂ via coenzyme Q₁ is smoothly achieved under anaerobic conditions. The rate of coenzyme Q₁H₂ autoxidation is extremely slow, i.e., second-order constant for [O₂][coenzyme Q₁H₂] = 1.5 M⁻¹ · s⁻¹ at 258 μM O₂, pH 7.5 and 25°C.     

Thyroid hormone-induced changes in the hepatic monooxygenase system, heme oxygenase activity and epoxide hydrolase activity in adult male, female and immature rats

In 8-day-old rat pups, pretreatment with a single injection of L-triiodothyronine or L-thyroxine decreased hepatic cytochrome P-450 content, aminopyrine N-demethylase activity and epoxide hydrolase activity but increased hepatic microsomal cytochrome c reductase, 7-ethoxyresorufin O-deethylase and heme oxygenase activities without significantly altering UDP-glucuronosyltransferase activity (towards o-aminophenol) or the microsomal yield.

In adult rats of either sex such single injections of L-triiodothyronine failed to significantly alter these enzyme activities. However, multiple injections evoked changes similar to those observed in the pups, in all these enzyme activities, except that 7-ethoxyresorufin O-deethylase activity was slightly decreased rather than increased.

These findings demonstrate that: (1) The hepatic monooxygenase system in the rat pup is more responsive to thyroid hormones than that in adult. (2) Thyroid hormones can decrease rat liver cytochrome P-450 content and its dependent monooxygenase activity independently of sexual maturity. (3) Thyroid hormones also decrease hepatic epoxide hydrolase activity in both pups and adults. Thus, hyperthyroidism could render the rat pup more susceptible to hepatotoxicity from electrophilic epoxides which utilize microsomal epoxide hydrolase as the major detoxication pathway.     

Molecular orbital studies of oxygen activation and mechanisms of cytochromes P-450-mediated oxidative metabolism of xenobiotics

Molecular dimensions and molecular orbital calculations of the electronic structures of 56 substrates, inhibitors and inducers of the cytochromes P-448 and other families of the cytochromes P-450 are reported. Substrates of the cytochromes P-448 are shown to be planar molecules with relatively large values of area/depth², and to have electronic structures with relatively low values for ΔE, the difference in energy between the frontier orbitals (E(LEMO) − E(HOMO)). Substrates of other families of the cytochromes P-450 are globular molecules, with relatively low values of area/depth² and relatively high values of ΔE. Molecular orbital calculations of the active oxygen species, singlet oxygen and superoxy anion, have also been made. Singlet oxygen is a poor electron donor (low values of E(HOMO)) but a good electron acceptor (low values of E(LEMO)), whereas superoxy anion is a good electron donor and a poor electron acceptor. Cytochrome P-448 substrates, which are good electron donors, would preferentially accept singlet oxygen, a good electron acceptor; substrates of the other families of cytochrome P-450, which are less effective electron donors, would preferentially accept superoxy anion, a good electron donor, although substrates of both cytochromes P-448 and other P-450s may accept both species of active oxygen. Together with recent published evidence, these data provide a greater understanding of the mode of activation of oxygen by the various families of the cytochromes P-450, and to the insertion of active oxygen into the substrates. Mechanisms are proposed for the oxygenation of substrates, namely, epoxidation involving singlet oxygen and hydroxylation by superoxy anion. Finally, a detailed explanation of the cytochrome P-450 cycle is discussed, and mechanisms of the different types of oxidative metabolism are presented.     

ORG 33201: A new highly selective orally active aromatase inhibitor

Org 33201 has been selected as a very potent aromatase inhibitor. The compound is an enantiomer of a SC₂H₅ substituted imidazoylethylphenalene. Org 33201 inhibited human aromatase activity for 50% at a concentration of 2.2 × 10⁹ mol/l. More than 200-fold higher concentrations were needed for the inhibition of other cytochrome P-450 enzymes. In vivo the compound was active in rats (ED₅₀ = 0.035 mg/kg) and dogs (1 mg/kg gave 70% inhibition) after oral administration. It can be concluded that Org 33201 is a potent and highly selective orally active aromatase inhibitor.     

The nitrogen fixation system of photosynthetic bacteria II. Chromatium nitrogenase activity linked to photochemically generated assimilatory power

The nitrogenase activity (measured by N₂ or acetylene reduction) of cell-free extracts of the photosynthetic bacterium Chromatium was coupled to photochemically generated ATP and reductant. The ATP was formed through cyclic photophosphorylation by bacterial chromatophores. The reductant (reduced ferredoxin) was generated by a heated preparation (incapable of O₂ and ATP production) of spinach chloroplasts. The nitrogenase activity of Chromatium extracts was supported by reduced Chromatium or Clostridium pasteurianum ferredoxin but not by that of spinach chloroplasts.     

Cytochrome P-450 distribution in rat liver and the effect of sodium phenobarbitone administration

A microspectrophotometric method for assaying cytochrome P-450 in fresh 24 μm unfixed cryostat sections of rat liver has been developed. When used to assay this cytochrome in sections of microsomal preparations it has yielded results equivalent to those obtained by the conventional spectrophotometric assay of the same preparations. Random measurements made throughout sections of liver have given mean values for cytochrome P-450 concentrations which are twice those measured in microsomes prepared from the livers of the same animals (not corrected for the yield in the homogenate).

Measurements of the cytochrome P-450 content of liver cells by the microspectrophotometric method show that in liver from male Wistar rats, cells nearer to the central veins contain up to twice as much cytochrome P-450 as those nearer to the portal tract (mean cell concentrations of 26.4 (±4.4) μmol/l and 17.5 (±3.0) μmol/l respectively). In the livers from similar rats, killed at the same time, but which had received 1 mg/ml sodium phenobarbitone in their drinking water for one week, the cells near the central vein contained up to five times as much cytochrome P-450 as those near the portal tract (mean cell concentrations of 77.3 (±25.0) μmol/l and 28.3 (±9.6) μmol/l respectively).

The results show a selective increase in cytochrome P-450 content by the cells in the centrilobular region after treatment with sodium phenobarbitone and a smaller increase by some of the cells in the periportal region.     

Characterization of the particulate nitrite oxidase and its component activities from the chemoautotroph Nitrobacter agilis

1. Difference spectra, at room and liquid N₂ temperatures, of S₂O₄²⁻-, and NO₂⁻-reduced intact cells and cell-free preparations of Nitrobacter agilis demonstrated the presence of cytochromes of the c- and a-types. Reduction of cytochromes by succinate, and to a limited extent, by NADPH also occurred, provided KCN (0.1 mM) was also present.

2. A particulate, heat-labile nitrite oxidase having an absolute requirement for O₂ was prepared from N. agilis cells using sonic oscillation and differential centrifugation. The particles also possessed NADH oxidase, succinoxidase, formate oxidase and traces of NADPH oxidase activity. The stoichiometry of the nitrite oxidase reaction approached the theoretical value of 2 moles of NO₂⁻ consumed per mole of O₂ consumed. The pH optimum of the nitrite oxidase system shifted to progressively more alkaline values as the NO₂⁻ concentration was increased, changing from a pH value of 6.8 at 0.6 mM KNO₂ to pH 8.0 at 0.01 M KNO₂ with apparent K_m's of 0.2 and 1.2 mM NO₂⁻, respectively. Computations of the HNO₂ concentrations present under the above conditions showed an approx. 500-fold greater affinity for HNO₂ which was independent of pH, suggesting the involvement of HNO₂ as both a substrate and an inhibitor (at higher concentrations) of the nitrite oxidase system. The marked inhibition by NaN₃, NaCN and Na₂S, as well the light-reversible inhibition by CO, indicated the presence of cytochrome oxidase which was subsequently characterized. NO₂⁻ proved to be a competitive inhibitor of the nitrite oxidase system.

3. The particulate preparation also possessed a heat-labile nitrite-cytochrome c reductase activity which was energy independent and routinely measured under anaerobic conditions. As in the case of nitrite oxidase, the affinity of the enzyme for NO₃⁻ increased as the pH was lowered, but the pH optimum remained unaffected. In terms of calculated HNO₂ concentration an approximately constant K_m of about 0.2 μM was estimated at the several pH's examined. The inhibition by NO₃⁻ was shown to be competitive. The marked sensitivity of the reductase to several metal-binding agents implicated a metal component in the electron transport chain at the site prior to cytochrome c.

4. The membrane-like composition of the nitrite oxidase system is indicated.