STUDIES ON THE NITRATE REDUCTASE 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 enzyme without recourse to coupling with other enzymes 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 flavoprotein. 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 redox 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 proteins 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 protein 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.