STUDIES ON THE OF ESCHERICHIA COLI IN THE CELL-FREE STATE

By W. JOKLIK'"

[Manuscript received August 29, 1949]

Summary A method of purifying the nitrate reductase obtained by crushing Escherichia coli cells in a ground glass mill is described, involving elution at pH 9.5 and precipitation into slightly acidified acetone, followed by treatment with phosphate buffer ( pH 8.0) and dialysis. Methods of estimating the activity of the without recourse to coupling with other are outlined, viz.: (a) the oxidation time method; (b) the use of photochemically reduced methylene blue as hydrogen donor. The action of various inhibitors on the enzyme was studied. The fol­ lowing significant results emerge: (a) The enzyme is strongly inhibited by cyanide and azide. (b) The enzyme is largely unaffected by reagents for iron and by carbon monoxide. (c) The enzyme is unaffected by the majority of the so-called -SH reagents, but is inhibited by heavy metal ions; however, this is most probably due to their action of precipitating the enzyme, as used here, in extremely small concentrations. ( d) N itrophenols inhibit the reduction of nitrate in the cell-free state. The reaction between hydrogenase and nitrate reductase depends on the presence of a certain carrier present in boiled bacterial suspensions and re­ placeable by Nile blue and . Evidence for the existence of a sep­ arate enzyme, nitrite reductase, is presented. The nature and physiological significance of nitrate reductase are discussed in the light of the above findings.

1. INTRODUCTION That nitrate can supplant the function of oxygen and act as a hydrogen acceptor has been known for a long time (e.g. Niklewski 1914; Ruhland 1924); nor is nitrate unique in this respect among inorganic ions, for nitrite, chlorate, sulphate, sulphite, and others, likewise accept hydrogen. Quastel and Wool­ dridge (1929) were the first to show that the reduction of nitrate was enzymic, for until then it had been thought that nitrate acted rather like methylene blue (MB ). It was noted quite early that even such closely related ions as nitrate and nitrite, or sulphate and thiosulphate, are not reduced by the same enzymes (Quastel, Stephenson, and Whatham 1925; Yamagata and Nakamura 1938) for there exist bacteria which reduce only one of each of these pairs of ions .

.. Department of Biochemistry, University of Sydney. This work was carried out during the tenure of a Commonwealth Junior Research Studentship. KITRATE REDUCTASE OF ESCHERICHIA COLI 29 Stickland (1931), studying the nitrate-reducing enzyme of Escherichia coli, found it to be uninhibited by carbon monoxide, not affected by toluene treat­ ment, but completely inhibited by cyanide. He found that the "nitrate reduc­ tase" could be coupled with many dehydrogenases which are thereby enabled to oxidize their substrates under anaerobic conditions. Aubel and Egami ( 1936) were able to isolate an organism which oxidatively de-aminated l-alanine under anaerobic conditions using nitrate, and nitrate only, as the hydrogen acceptor. Woods (1938) studied the reduction of nitrate to ammonia by means of molecular hydrogen in Clostridium welchii. Many other bacteria failed to do this, however, and only reduced nitrate to nitrite. The latter compound can easily be shown to be an intermediate in the reduction of nitrate to ammonia, but the further steps are obscure though Aubel (1938a, 1938b) has claimed that hydroxylamine and hyponitrous acid are intermediates. Lascelles and Still (1946) studied the system hydrogenase-nitrate reduc­ tase in E. coli. This organism reduced nitrate at a rapid rate, but nitrite was reduced only if a dye such as benzyl viologen was added; evidently hydrogenase and "nitrate reductase" needed a special carrier. They found that nitrate reduction was inhibited by cyanide and azide: Very little work has been done on nitrate reductase in the cell-free state. Lemoigne, Desveaux, and Gavard (1944) claimed that the nitrate reductase of E. coli consists of an enzyme complex, partly in the culture fluid and partly in the cell; either factor alone is inactive. Yamagata (1938a, 1938b), however, remains the most important contributor. He obtained a cell-free preparation of nitrate reductase from E. coli by autolysis at 30°C. The enzyme was ad­ sorbed on Berkefeld filters, failed to oxidize reduced coenzyme I and was strongly inhibited by O.OlM cyanide. The present work was undertaken with a view to preparing a cell-free (i preparation of nitrate reductase by crushing cells of E. coli in a ground glass . mill, purifying, and if possible, isolating the enzyme, and to study the effect of inhibitors on it in order to expose any prosthetic group.

II. EXPERIMENTAL METHODS The strain of bacteria used in this investigation was strain B of E. coli, kindly supplied by Dr. M. Delbriick. The method of growing, collecting, and crushing the bacteria has been outlined in a previous communication (Joklik 1950). In order to increase the yield of nitrate reductase the addition of nitrate to the medium was tried, but no significant increases in nitrate re­ ductase content were observed. Among the various chemicals used in this study which were prepared in the laboratory were the following: (a) Ko;ic acid.-A strain of Aspergillus flavus oryzae, kindly supplied by Mr. J. M. Vincent of the School of Agriculture, University of Sydney, was grown at 37°C. under the most aerobic conditions possible, oxygen being con- 30 W. JOKLIK tinually passed through the medium. After three weeks the mycelium was filtered off and the mother liquor evaporated till crystals formed. In general, the procedure of Raistrick et al. (1931) was followed but not all the purifica­ tion steps were carried out. (b) Flavoprotein was prepared following the method of Straub (1939) to the a:dsorption on alumina Gy. (c) Coenzyme I was prepared according to the method of Umbreit, Burris, and Stauffer (1945). . (d) Carbon monoxide was prepared by the action of concentrated sul­ phuric acid on formic acid. The gas was passed through three traps contain­ ing respectively alkaline sodium hydrosulphite, alkaline pyrogallol, and con­ centrated sulphuric acid. (e) Janus red was prepared according to the method of Fischer and Eysenbach (1937) except that Janus green B was used as the starting com­ pound. Nitrite was estimated with the Griess-Ilosvay reagent. Since the concen­ tration of nitrite formed was usually very great, the practice adopted was to dilute the sample 1 in 150, whereby removal of any dye or deproteinization became unnecessary. Where, however, deproteinization was necessary, this was done by adding to the reaction mixture half a· volume of glacial acetic acid and one-sixth the volume of saturated ammonium sulphate, and placing in a boiling water-bath. Within one to two minutes the had usually flocculated completely, and were filtered off through a Whatman No. 41 filter paper. This procedure had to be worked out since all the other common precipitating reagents, even trichloracetic acid, interfered with the estimation of nitrite. Estimation of the colour developed was carried out by means of a photo­ electric absorptiometer, using a green filter. Standard curves were constructed frequently.

III. ESTIMATION OF THE ENZYME Many workers have estimated the rate of reduction of nitrate in tissues, but the method has always been to couple nitrate reductase with a dehydro­ genase, and to estimate the nitrite produced. Questel, Stephenson, and Whetham (1925), for instance, used enzymically reduced leucomethylene blue as a hydrogen donor, so that the rate of re-oxidation in the presence of nitrate and nitrate reductase represented the difference of the activities of nitrate re­ ductase and the dehydrogenase. Fischer and Eysenbach (1937), however, estimated fumaric hydrogenase by reducing a dye (Janus red) with a slight excess of sodium hydrosulphite, which was then oxidized away so that they were dealing with a known amount of non-enzymic hydrogen donor. In the present study, nitrate reductase was so active that the small volume of very dilute (O.OOOIM) hydrogen donor used by Fischer and Eysenbach would have been quite inadequate; hence larger quantities of sodium hydro- NITRATE REDUCTASE OF ESCHERICHIA COLI 31 sulphite had to be used. That substance acts as the donor, while the dye (Janus red), of which a very small amount suffices, acts as the carrier. It can be shown that the rate of the reaation is a function of dye concentration only when this is less than O.OOOO5M. The creation of anaerobic conditions pre­ sented a problem since sodium hydrosulphite decomposes in an unpredict­ able way in vacuo, depending on the sudace of the glass. Anaerobic condi­ tions were therefore maintained by placing a layer of paraffin oil about two centimetres thick over the reaction medium. By this means a given sample of reduced dye was easily kept in the reduced state for periods from two to six days; it was pedectly satisfactory for experiments lasting at the. most one hour. Aubel, Schwartzenkopf, and Glaser (1937) have suggested that nitrite· could re-oxidize leucodyes chemically. No evidence for this was found under the conditions used here; it is probable. that the amounts of nitrite formed were too small to cause such an effect. The general procedure was to place into a test-tube 0.1M Sorensen phos­ phate buffer (pH 7.6), enzYme, nitrate (equal to the amount of sodium hydro­ sulphite used), and sodium hydrosulphite (0.3 to 0.8 ml. of a O.lM solution, the amount being chosen so that the time the dye takes to change colour (the oxidation time) is between six and twenty minutes). The tube was then in­ cubated in a water-bath at 37°C. and left to attain the temperature. Any inhi­ bitors were now, added. Within four minutes after placing the tubes in the water-bath (for if left longer the sodium hydrosulphite decomposes) 1.0 ml. of a O.OOO4M solution of Janus red (JR) or other oxidation reduction indicator ( OR!) and 2.0 mi. paraffin oil were quickly added and the time noted on a stop watch. The tube was then gently agitated from time to time by rotating between the fingers, and the time when the OR! changed colour noted to the nearest five seconds. Duplicates done at the same time agreed to within 2 per cent.; in separate runs, however, oxidation times may differ by, 5 per cent. since the sodium hydrosulphite decomposes rapidly at 37°C. in aqueous solution, as shown in Table 1.

TABLE 1 DECOMPOSITION OF SODIUM HYDROSULPHITE AT 37°C. IN AQUEOUS SOLUTION·,

Time of Addition of JR Oxidation Time and Paraffin (min.) (min. & sec. ) o 15-00 10 12-00 20 10-20 30 8-15 40 6-1>0

• Each tube contained 2.0 ml. buffer, 0.2 ml. of enzyme solution,: 0.4 mi. 0.1M KNOg, and 0.8 m!. Na2S204' JR and paraffin were added to the first tube at time 0, and then 10, 20, 30, and 40 min: later to the other ones. The oxidation times were noted in each case ..

When no enzyme was added to the tubes, the OR! stayed in the reduced condition for over 24 hours, since, even though all sodium hydrosulphite might 32 W. JOKLIK have long been decomposed, the ORI will not become re-oxidized because no oxidizing agent is present. Similarly, if small amounts of enzyme are used, the ORI will remain reduced for a long time, since only very little sodium hydro sulphite will be used up by it. Readings were rarely continued for more than 60 minutes. When the nature or colour of a reagent used rendered clear observation of the end-point by eye impossible, the nitrite formed was estimated. It was found that, with the amount of sodium hydrosulphite generally used, 0.1 ml. of the reaction mixture mixed with 15.0 ml. of distilled water gave, after addi­ tion of the Griess-Ilosvay reagent, a colour which could be read accurately on the instrument used. This large dilution made it unnecessary to either deproteinize or to remove the ORI; it also stopped the reaction immediately. It is necessary, when estimating nitrite after a certain time, to have excess reducing agent still present; otherwise nitrite production has stopped and the amount of nitrite formed under these conditions is no measure of the activity of the enzyme. However, the amount of sodium hydrosulphite left must not be large, otherwise the pink diazo compound is not formed. In practice the dilution of 1 : 150 depresses the concentration of hydrosulphite sufficiently to permit the colour to develop normally. After 20 minutes the colours were read on the instrument, and referred to a standard curve. The results were usually expressed in micrograms nitrite/ml. of the solution, the colour of which was measured; to express this figure as mg. of nitrite formed in the reaction mixture, this would have to be multiplied by 0.17 x, where x is the volume of the reaction mixture in ml. Experiments were carried out to test whether the amount of nitrite formed was actually related to the amount of sodium hydrosulphite used and whether the reaction stopped completely after the ORI had been re-oxidized. The relative efficiencies of JR, benzyl viologen (BV), and methylene blue (MB) were also estimated by comparing the time which elapsed before they changed' colour (Table 2). Table 2 indicates that the nitrite concentration is apparently a linear function of the time of incubation, that the reaction stops when the ORI changes colour, and that the amount of nitrite produced is proportional to the amount of sodium hydro sulphite used. Similar experiments confirmed the fact that the reaction with BV as the carrier is quicker than that with MB. One other method was used in the estimation of the activity of nitrate reductase. Yamagata (1938a) had suggested that the photochemical reduction of MB by kojic acid in strong light could be used for this purpose: a mixture of the correct amounts of the two compounds in buffered aqueous solution and illuminated with two 200-watt globes results in the reduction of the MB within 15 minutes. In order to carry out these experiments, enzyme, buffer, nitrate, and in­ hibitor were pipetted into the lower part of a Thunberg tube, while into the stopper were placed the MB and kojic acid solutions. The tube was then evacuated and filled with nitrogen, the stopper illuminated till the MB was NITRATE REDUCTASE OF ESCHERICHIA COLI 33 reduced, and the tube placed in a water-bath and tipped. The rate of re­ colouration was observed. Owing to the high activity of the enzyme used here, a strong solution of MB had to be used. This results in the precipitation of the reduced MB; and the intense colours involved made impracticable their removal for the colori­ metric estimation of nitrite formed. Yamagata used the method only to de­ monstrate the re-oxidation of reduced ORI; in the work described in this paper the method was used to observe the action of several inhibitors which were reduced by sodium hydrosulphite and to confirm results obtained with other methods. The results, however, were not quantitative.

TABLE 2 EFFECT bF SODIUM HYDROSULPHITE AND NITRATE ON THE EXTENT OF NITRITE FORMATION BY NITRATE REDUCTASEO

Time of Oxidation

Na2S20 4 KNO:1 Incubation Time Nitrate Formed (m!. ) (m!. ) OR! (min. ) (min. & sec. ) (/-tg.!m!. ) 0.6 0.2 MB 10 0.27 0.6 0.2 MB 20 0.56 0.6 0.2 MB 30 0.95 0.6 0.2 MB 40 38-25 1.33 0.6 0.2 MB 50 37-50 1.44 0.6 0.2 MB 60 37-40 1.46 0.6 0.2 BV 30 24-10 1.20 0.2 0.1 MB 30 12-45 0.39 0.2 0.3 MB 30 11-55 0.33 0.4 0.2 MB 30 23-45 0.79 0.6 0.1 MB 30 1.16 0.6 0.3 MB 30 1.08 " Each tube contained 2.0 m!. buffer (pH 7.6), 0.5 m!. enzyme solution, 1.0 m!. of the ORI indicated, the specified amount of O.lM sodium hydrosulphite, and O.lM nitrate.

IV. PURIFICATION The source of the enzyme was the organism E. coli, which had been ground in a ground glass mill according to the method outlined previously (Joklik 1950). When the bacteria had been crushed two or three times in distilled water and the extract centrifuged at 4500 r.p.m. for 20 to 25 minutes, most of the nitrate reductase was found to be in the insoluble residue (either still on major particles of the cell wall or on smaller submicroscopic particles) while the rest was either in solution or in such a finely divided state that centri­ fuging at the above-mentioned speed failed to deposit it. This latter fraction was largely precipitated by one-twelfth saturated ammonium sulphate; concen­ trations higher than half saturated were not tolerated by the enzyme. Both the enzyme precipitated by ammonium sulphate and that adhering to the larger cell fragments could be brought into solution by dialysis against 34 W. JOKLIK borate buffer (pH 9.6) at 5°C. for 24 hours. The supernatant, which was slightly opalescent, was then added to six volume~ of dioxane at WC., just above its freezing point. The precipitate should not flocculate and was cen­ trifuged quickly; the dioxane was then drained off the precipitate, which was dissolved in 0.2M sodium phosphate (dibasic). After standing overnight, the precipitate was centrifuged down, and the nitrate reductase remained in solu­ tion. Acetone could be substituted for dioxane, and better results were some­ times obtained if the organic solvent was acidified slightly (about lO-llM with regard to hydrochloric acid); it must be emphasized that, though the method has worked well and given active, soluble nitrate reductase, sometimes the precipitate after acetone or dioxane precipitation has been inactive. Probably all the factors involved in tha.t precipitation are not yet known. A further step in the purification would be to concentrate the enzyme by freeze-drying, for, although, in the preparation described above, the enzyme was present in a perfectly soluble state and free from other enzymes (especially hydrogenase and nitrite reductase)· except catalase, the actual amount present was relatively small. Freeze-drying did not affect the enzyme when unpurified in the bac­ terial extract; however, this process was not carried out, since the ordinary preparations were quite satisfactory for the work at hand.

V. PROPERTIES ( a) Behaviour on Heating The enzyme is sensitive to heat. When two m!. portions of enzyme solu­ tion were plunged into a boiling water-bath and heated to different tempera­ tures for 50 to 60 seconds, a temperature of 70°C. caused a loss in activity of almost 50 per cent., while at 80°C. the loss was over 75 per cent. (Table 3).

TABLE 3 EFFECT OF HEAT ON NITRATE REDUCTASE ACTIVITY"

Temperature 20 50 60 70 80 90 95 attained (oC.)

Oxidation time 9--30 9-55 13-15 16--30 40--00 >60 >60 (min. & sec. )

" Each tube contained 1.0 ml. enzyme solution, 1.0 ml. buffer (pH 7.6), 0.3 ml. nitrate (O.IM), 0.3 ml. Na2S:P4 (O.IM), 1.0 ml. JR (0.0004M), and 2.0 ml. paraffin oil.

The heat treatment adopted for hydrogenase (J oklik 1950) would thus destroy most of the nitrate reductase. ( b) Dilution No dilution effect could be demonstrated. That and the fact that nitrate reductase is not inactivated by dialysis make it probable that leuco-Janus red acts directly as the hydrogen donor without an intennediate carrier. NITRATE REDUCTASE OF ESCHERICHIA COLI 35 (c) Substances Shortening the Oxidation Time It was noticed frequently that acetone seemed to have an accelerating effect on the enzyme activity; that is, the oxidation time was greatly reduced

TABLE 4 EFFECT OF ACETONE ON THE OXIDATION TIMEo Amount of Nil 0.1 0.2 0.3 {).4 Acetone (m!.) Oxidation time 11-30 5-00 1-05 str. t str. t (min. & sec. )

.. Each tube contained 0.5 m!. enzyme solution, 2.0 ml. buffer, 0.2 ml. KN03 (0.2M), 0.4 ml. sodium hydrosulphite (O.lM), 1.0 ml. JR, and the specified amount of acetone. t str. denotes that the JR remained in the oxidized state.

(Table 4). These results could be repeated even when partially heat-denatured enzyme was used. If, however, no nitrate was present, the ORI stayed in the reduced form in the presence of acetone as well as in its absence. It was clear, the;efore, that the effect depended either on the amount of enzyme or on the amount of sodium hydro sulphite present; for these are the two factors controlling the oxidation time. The latter was found to be responsible, since evidence was obtained that, in the presence of a large excess of acetone (and even 0.1 ml. of acetone in 4.0 ml. would constitute a large excess), sodium hydrosulphite is destroyed, probably in the reduction of acetone. Sodium selenite and very small amounts of ferrous sulphate acted similarly; that is, the oxidation time was again greatly decreased (Table 5).

TABLE 5 EFFECT OF FERROUS SULPHATE AND SODIUM SELENITE ON THE OXIDATION TIMEo

Concentration of Concentration of Ferrous Sulphate Sodium Selenite Oxidation Time (molarity) (molarity) (min. & sec. ) Nil 15-45 0.001 12-00 0.0005 9-00 0.00033 8-15 0.0002 9-15 0.0001 10-00 Nil 0.002 4-30 .. Each tube contained 0.5 m!. enzyme solution, 2.0 ml. buffer, 0.2 ml. nitrate (0.2M), 0.4 ml. sodium hydrosulphite (O.lM), 1.0 ml. JR, and the specified amount of ferrous sulphate or sodium selenite.

These results are difficult to explain as sodium hydro sulphite could not have been used up in the reduction of the reagent added. Estimations done after experiments in which acetone, ferrous ions, and sodium selenite had been 36 W. JOKLIK used, revealed that only very small amounts of nitrite were formed; the re­ action appeared to have stopped even before the ORI had changed colour. This would be caused, as has been discussed above, by the destruction of sodium hydrosulphite; by its oxidation in the presence of acetone, and, possibly, sodium selenite; and probably by a catalytic effect of very small concentrations of iron salts. However, references to none of these effects could be found in the common text-books of chemistry, nor does Beilstein mention sodium hydro sulphite as a possible reducing agent for acetone.

( d) Effect of Acidified Acetone on the Enzyme In an attempt to split a prosthetic group from nitrate reductase, partially purified preparations were added to acetone containing from 1O-6M to 10-1 M hydrochloric acid. The precipitate was in each case centrifuged down, drained well, and re-suspended in phosphate buffer (pH 8.0). It was shown that this treatment had little effect on the enzyme provided conditions were kept as cool as possible till the concentration of hydrochloric acid reached lO-2M, when complete inactivation resulted. Attempts to resynthesize the enzyme using inactive apoenzyme and haem failed.

60 rl------r------r------r------~------~------_,

~ 40 r z 01 0 '"M :;- o o

20

o I I 6·0 6·5 7·0 7·5 8·0 8·5 9·0

pH

(e) Relation of Nitrate Reductase Activity to pH A pH curve over a wide range was constructed; however, it was impossible to proceed far into the acid region, since sodium hydro sulphite decomposes very readily at pH values below 5.5. Three different series of buffers were NITRATE REDUCTASE OF ESCHERICHIA COLI 37 used: citric acid-phosphate between pH 5.2 and 7.2; phosphate between pH 5.5 and 8.0; and glycine-sodium hydroxide between pH 8.0 and 10.0 The enzyme has two pH maxima, at pH 6.9 and 8.0 (Fig. 1).

(f) Effect of Inhibitors (i) Reagents for Iron- and Iron Porphyrin-containing Enzymes.-The only reagents in this group which inhibited nitrate reductase were cyanide (inhibition 100 per cent. in O.01M and 80 per cent. in O.OOlM KeN); azide (100 per cent. inhibition in O.OlM solution); and potassium sulphocyanide (40 per cent. in 0.003M solution), the effect of which, however, is not due to its reaction with iron, since all iron was present in the reduced state. Among other substances tried and found to be without effect were hydrazine and hydroxylamine, thioglycollic acid, 8-hydroxyquinoline, a a'-dipyrridyl, o-phenan­ throline, and, as a general heavy metal reagent, diethyldithiocarbamate; none of these reagents caused any change in the oxidation time in concentrations ranging from M/lOO to M/2000.

TABLE 6 EFFECT OF -SH REAGENTS AND HEAVY METALS ON NITRATE REDUCTASE

Inhibitor Concentration Precipitation Inhibition (molarity) (%) o-Mercuribenzoate 0.0025 90 o-Iodosobenzoate 0.0025 20 Benzamide 0.0025 20 Iodine 0.001 66 Copper sulphate 0.001 50 (?) Mercuric sulphate 0.001 Cobalt sulphate .0.001 40 Chromic sulphate 0.002 + 40 Chromic sulphate 0.001 Ceric sulphate 0.002 + 50 Ceric sulphate 0.001 ± 20 Ceric sulphate 0.0005 Stannous chloride 0.001 ± 40 Stannous chloride 0.0005 10 Cadmium chloride 0.0002 + 30

(ii) Reagents Specific for -SH G1'OUpS and Heavy Metal Ions.-While in­ vestigating the effects of these two groups of inhibitors two difficulties were encountered. The first was that certain of the compounds tried were reduced by sodium hydro sulphite (e.g. o-mercuribenzoate, mercuric sulphate). The second was that most metal ions (cerium, cadmium, copper, iron, etc.) preci­ pitated the enzyme in concentrations above M/500 and thereby caused inhi­ bition. Whether highly purified enzyme would also be precipitated is not certain, as the enzyme preparation used in these experiments was not one . of the purest obtainable by the method outlined above. However, the con­ centrations of metal ions used were low as it was assumed that if the metal 38 W. JOKLIK combined with -SH groups, or otherwise inhibited the enzyme to a significant degree, the effect would be noticeable at 0.001M concentration. Table 6 shows the results obtained. Iodine and o-mercuribenzoate are seen to be the most important of the -SH inhibitors for nitrite reductase, but all inhibit to a certain extent. Several of the results set out in Table 6 were checked by estimating the nitrite formed (Table 7). TABLE 7 EFFECT OF CERTAIN INHIBITORS ON THE FORMATION OF NITRITE

Inhibitor Concentration Nitrite Found Inhibition (molarity) (!tg'/ml. ) (%) None 0.90 Mercuric sulphate 0.002 0.45 50 Ceric sulphate 0.002 100 . COpper sulphate 0.002 0.10 90 Stannous chloride 0.002 0.94 Silver nitrate 0.002 0.73 30 Potassium ferrocyanide 0.0045 0.40 55 Potassium sulphocyanide 0.002 100 Sodium pyrophosphate 0.003 0.86 o-Mercuribenzoate 0.002 0.10 90 Phenyl mercuric acetate 0.002 0.30 66 0-Iodoso benzoate 0.002 0.86 '" c/-DipyrridyJ 0.002 100 Saturated toluene 0.87

Discrepancies were found for mercuric sulphate which caused a 50 per cent. inhibition of nitrite accumulation, and copper sulphate, which seemed to form a complex with JR and almost completely inhibited nitrite formation; likewise '" "" -dipyrridyl and potassium sulphocyanide here caused total inhi­ bition (d. Section (f) (i)). The effect of carbon monoxide and oxygen was tested also, but neither gas had any effect on the enzyme. Alcohol and toluene did not affect the activity, while capryl alcohol and dioxane had an effect similar to that of ace­ tone, though to a much smaller degree.

(g) Donor and Carrier Specificity A series of experiments was carried out to determine whether nitrate re­ ductase can use carriers other than reversibly reducible dyes, such as JR, MB, or BV. Among likely substances were those capable of being oxidized and reduced easily; but ferrocyanide, iron salts, cysteine, and ceric ions were all inactive. Coenzyme I, flavoprotein, and cytochrome C were also used, but the amount of nitrite formed in their presence was no larger than in the absence of any carrier whatsoever; nor did these substances increase the rate of re­ action when they were used in the presence of MB. NITRATE REDUCTASE OF ESCHERICHIA COLI 39 (h) Estimation of Nitrate Reductase Activity with Photochemically Reduced MB The limitations of this method 4ave already been outlined, as also the ~; method of carrying out the experiments. It was used mainly for three reasons: I to study the effect of substances reduced by sodium hydrosulphite, to deter- 'i mine whether nitrophenols are themselves reduced by nitrate reductase, and to \ detect any nitrite reductase present in the bacterial extracts. The last point ' will be commented on later. No evidence was obtained that nitrophenols themselves are reduced. The results with a variety of inhibitors are given in Table 8. TABLE 8 EFFECT OF INHIBITORS ON NITRATE REDUCTASE, AS DETERMINED BY THE RE-OXIDATION OF PHOTOCHEMICALLY REDUCED MBo

Concentration Inhibitor (molarity) Comment None The colour changes from light green to dark blue Cyanide 0.002 } The colour stayed light green; full inhibition Azide 0.002

Mercuric sulphate 0.002 } Phenyl mercuric acetate 0.002 No inhibition 0-Iodosobenzoate 0.002 o-Mercuribenzoate 0.002 Greatly retarded; 90% inhibition Sodium selenite 0.002 No inhibition Silver nitrate 0.002 Colour changed to and stayed dark green; inhibition Stannous chloride 0.002 } Slight inhibition Urethane 1.0 2,4-Dinitrophenol 0.002 } Strong inhibition 2,4-Dinitrophenylhydrazine 0.002 m-Nitrophenol 0.002 } Inhibition 70% (?) p-Nitrophenol 0.002 .. Each Thunberg tube contained from between 0.2 to 0.5 ml. enzyme solution, 2.0 ml. buHer (pH 7.6), and 0.3 ml. KNOa (O.IM), while the stoppers contained 0.5 mI. MB ( 0.2% ), and 0.5 ml. kOjic acid solution. The gas used was nitrogen; illumination was provided· by three ISO-watt globes for 20 minutes.

It appears from Table 8 that sometimes this method of estimating nitrate reductase activity gave results different from those obtained with other methods; especially was this so for mercuric sulphate, which showed no inhibi­ tion by this method. Possibly mercury ions, even though present only to the extent of about 4 p.p.m. in the final solution, had interfered with the estima­ tion of nitrite. On the other hand, silver nitrate showed much more inhibition by this method than by nitrite estimation with JR as the carrier. 40 W. JOKLIK The nitrophenols, none of which were reduced by the enzyme, were strongly inhibitory. Lascelles (1946), however, had found that, measuring the rate of hydrogen uptake in the presence of nitrate and intact E. coli cells, nitrate reduction was not inhibited by nitrophenols. Finally, it may be said that molar urethan causes slight inhibition. This has been confirmed with the oxidation time technique, when an inhibition of about 20 per cent. was found. However, this large concentration of urethan precipitated the enzyme (at least in the state in which it was used), so that in all probability urethan itself has only a very slight effect on the enzyme. This is not in agreement with Yamagata (1938a).

VI. OBSERVATIONS ON NITRITE REDUCTASE Frequently, throughout this work, preparations were tested for nitrite re­ ductase. The results obtained, however, were made doubtful by the experi­ ments of Aubel, Schwartzenkopf, and Glaser ( 1937), who suggested that nitrite could re-oxidize leuco-dyes chemically.

TABLE 9 EFFECT OF DIFFERENT SUBSTRATES ON THE OXIDATION TIMEo

Substrate Oxidation Time with Enzyme Preparation (min. & sec.) ~ "--- ES Ia ES Ib ES II ES III Nitrate 1-40 5-50 14-20 10-00 Nitrite 0-40 1-00 >60 >60 Hydroxylamine 24-30 34-55 >60 >60 .. Each tube contained enzyme solution (0.1 ml. of ES Ia and b, 1.0 ml. of ES II and ES III; 0.5 ml. O.lM substrate; 0.5 ml. O.lM Na2 S20 4 ; 1.0 ml. 0.0004M JR; phosphate buffer (pH 7.6) to 4.0 ml.; and a layer of paraffin oil.

On the other hand it was shown repeatedly that, under the conditions used here, tubes containing buffer, nitrite, sodium hydro sulphite, and ORI, but no enzyme, did not re-oxidize the dyestuff. Hence Table 9 provides some evidence that under certain conditions nitrite reductase can be very active and is an entity different from nitrate reductase. The experiment was carried out with enzyme preparations which had just been obtained from crushed bacteria. Fractions ES la, ES 1b, ES II, and ES III have been described pre­ viously (Joklik 1950): they refer to ammonium sulphate fractions of the extract of crushed bacteria. E (nzyme) S ( olutions) I were those before ammonium sulphate treatment, and ES III were suspensions in phosphate buffer (pH 7.6) of the precipitate obtained by between one-third and one-half saturation with ammonium sulphate; ES II fractions were intermediate. Suspensions of this type were by no means pure preparations of nitrate reductase, but they were convenient to prepare and other enzymes did not interfere. NITRATE REDUCTASE OF ESCHERICHIA COLI 41

It appears from Table 9 that, since the amounts of sodium hydrosulphite and substrate were the same in all tubes, an explanation based on simple chemical reduction of the substrate, is not adequate. However, in view of the extraordinarily short oxidation times in the presence of nitrite, it seems that the factors involved in its reduction are unknown. Experiments using photochemically reduced MB as hydrogen donor provided evidence of the presence of a weak nitrite-reducing enzyme in certain preparations. The best method of studying the enzyme at the present time would be with a purified preparation of hydrogenase, measuring the hydrogen uptake. Nevertheless, even then studies of the enzyme, such as pH optima, effect of inhibitors as well as the possible need for a carrier between the two enzymes, would be greatly complicated.

VII. RECONSTllUCTION OF THE HYDROGENASE-NITRATE REDUCTASE SYSTEM With the separation of the two enzymes, it is now possible to reconstruct the system hydrogenase-nitrate reductase. A number of workers have noticed dilution effects in studying reactions involving hydrogenase and nitrate reductase, but the nature of the carriers involved is entirely unknown. Hoberman and Rittenberg (1943) suggested that an enzyme system, and not only a carrier, is involved in the reduction of MB by hydrogenase. So far as nitrate reductase is concerned, the position is still more uncertain. Yamagata (1938b) states that reduced coenzyme I is not a donor and this fact has been confirmed here, while Green, Stickland, and Tarr (1934) found that except for oxidation-reduction indicators, pyocyanine was the only substance capable of transferring hydrogen atoms from dehydro­ genases to nitrate reductase. Accordingly a short series of experiments was carried out using both enzymes in dialysed preparations. Before carrying out experiments with the two purified enzymes, a dialysed fraction of ES I was tested for the effect of certain chemical reagents, from which, as Hoberman and Rittenberg suggest, "the hydrogen ion and electron pair, necessary for the reduction of MB" could be contributed. The dialysis of the ES I (which contained both hydrogenase and nitrate reductase) was done very carefully, first against distilled water containing a few drops of thioglycollic acid, then against running water for a short time, and finally for a few short periods against glass-distilled water; this was necessary to prevent excessive denaturation of hydrogenase. Table 10 shows the results. Table 10 shows that none of these reagents except boiled bacteria had any effect on the reaction; the carrier responsible was thus not replaceable by any of the above reagents. The relatively large activity left after dialysis showed that this process was not the one which removed so much of the carriers that the purified preparations of the two enzymes, as is discussed below, only reacted together at a very low rate. A boiled bacterial suspension, however, gave a significant result, showing that some carrier involved in the transport of hydrogen was heat-stable and present in deficient amounts in the fraction ES I. 42 W. ]OKLIK Experiments were then carried out with the two purified enzyme pre­ parations. The hydrogenase used was of the type ES III (Joklik 1950), i.e. heat-treated, dialysed, and precipitated with ammonium sulphate; the nitrate reductase used was in the purest form available (after acetone precipitation).

TABLE lO. EFFECT OF VARIOUS CARRIERS ON THE HYDROGENASE-NITRATE REDUCTASE REACTION IN DIALYSED SOLUTIONS CONTAINING BOTH ENZYMES"

Reagent Added Hydrogen Uptake

------~------None 84 0.3 ml. coenzyme I solution (15 mg./ml.) 86 0.1 ml. Nile blue (0.2%) 79 0.3 ml. coenzyme I solution + 0.1 ml. Nile blue 94 0.1 ml. fumarate (O.lM) 100 0.1 ml. fumarate + 0.3 ml. coenzyme I solution 90 0.3 ml. boiled suspension of bacteria 139 .. Warburg manometers were used. Each cup contained the following reagents: dialysed enzyme preparation, 1.0 ml.; 0.1 ml. nitrate (0.1 M); 0.4 ml. MB (2%); where indicated, the reagents mentioned; and phosphate buffer (pH 7.6) to 3.0 ml. Incubation time was half an hour, and the readings are in 1Ll.!30 min.

Table 11 shows the results obtained. They suggest that flavoprotein· is in­ volved somewhere in the transport of hydrogen from the gaseous phase to nitrate; but it would be too early to assign to it a definite role either as carrier or as a prosthetic group of the enzyme.

TABLE 11 REACTION BETWEEN HYDROGENASE AND NITRATE REDUCTASE IN PURIFIED PREPARATIONS" Nitrate reductase + + + + + Hydrogenase + + + + + Reagent None BB FP FP NB Co I Co I Activity 13 33 38 43 35 .. Warburg manometers were used. Each cup contained 0.5 ml. of nitrate reductase and hydrogenase solutions; 0.4 ml. MB (2%); where indicated the following: 0.3 ml. boiled suspension of bacteria (BB), 0.1 ml. flavoprotein solution (FP), 0.3 ml. coenzyme I solu­ tion (15 mg./ml.) (Co I), and Nile blue (0.3%) (NB); and phosphate buffer (pH 7.6) to 3.0 ml.

It is remarkable how slow the hydrogen uptake was under these condi­ tions since the particular hydrogenase preparation used took up 250 ILL in 30 minutes in the presence of MB, and the activity of the nitrate reductase was such that with 0.2 ml. of O.IM sodium hydro sulphite the oxidation time was only 15 minutes; if these systems had reacted at something like the optimum speed for each, the hydrogen uptake would have been over ten times the value found. However, even in the presence of whole bacteria, the uptake of NITRATE REDUCTASE OF ESCHERICHIA COLI 43 gas is much greater in the presence of ME than in the presence of nitrate, so that even under conditions where the structural relationships between the two enzymes are preserved; the absence of a sufficient amollnt of carrier limits the rate of the reaction. Regarded in that way, activation with boiled sus­ pensions of bacteria is not surprising.

VIII. DISCUSSION The evidence presented here does not make it possible to speculate on the nature of nitrate reductase. The only observation which could strengthen the evidence for the presence of a prosthetic group in the enzyme would be that by precipitating the enzyme into acidified acetone relatively little activity is lost until a molarity of hydrochloric acid of 10-3 is reached (Section V (d»; in the step from there to 1O-2M, however, all activity disappears. This might indicate the splitting off of a prosthetic group rather than sudden enzymic denaturation. The claim of Lemoigne, Desveaux, and Gavard (1942), however, that nitrate reductase consists of two parts, one of which is in the cell-free culture fI~id, could not be confirmed. When the question of the nature of the prosthetic group is considered, two alternatives are open. On the one hand, the strong inhibition with cyanide and azide suggests that an iron porphyrin prosthetic group might be impli­ cated; the enzyme is not, however, affected by carbon monoxide, nor by re­ agents for free iron. On the other hand, there is also evidence for a flavine prosthetic group, as indicated by the fact that flavoprotein serves as a hydro­ gen carrier between hydrogenase and nitrate reductase. It is also of particular significance that the relation between nitrogen metabolism and molybdenum, which has been known to exist Jor some time in (Bortels 1930; Burk and Horner 1935), Rhizobium (Jensen and Betty 1943), and Clostridium (Jensen and Spencer 1947), and in particular that between nitrate metabolism and molybdenum (Bortels 1936; Jensen and Spencer 1947) has been recently observed in E. coli (Pinsent 1949). The only definite statement which can be made about the structure of the enzyme at the present time, is that free -SH groups do not seem to be necessary for enzymic activity, at least not to the extent of enabling the enzyme to be classed with the -SH enzymes. It is also interesting that nitrophenols inhibit the reduction of nitrate; this was rather unexpected, since they do not markedly affect the reduction of nitrate in intact cells (Lascelles and Still· 1946). Toluene likewise did not cause any inhibition; Lascelles and Still (1946) had found slight inhibition, while Yamagata (1938a) claimed that it inhibited appreciably. As far as carriers between hydrogenase and nitrate reductase are con­ cerned, only dyes and flavoprotein seem to be active. An unidentified carrier occurs in boiled bacteria, which also increases the rate of interaction between nitrate reductase and other enzymes. . The physiological significance of nitrate reductase is fairly clear, as was shown by Quastel, Stephenson, and Whetham (1925); it may be assumed 44 W. JOKLIK that the reaction between hydrogen and nitrate rarely, if ever, proceeds under physiological conditions. The function of the enzyme might be summed up by the statement that since its E' 0 is quite high (about 0.4 volts), which places it above most dehydrogenases in the scale of oxidation-reduction potentials, it can act as a terminal oxidizing agent under anaerobic conditions.

IX. ACKNOWLEDGMENTS The author gratefully acknowledges the continued interest shown in this work by Professor H. Priestley and Mr. G. Humphrey.

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