symposium on metabolism of inorganic compounds' … · symposium on metabolism of inorganic...

SYMPOSIUM ON METABOLISM OF INORGANIC COMPOUNDS'

II. ENZYMATIC PATHWAYS OF NITRATE, NITRITE, AND HYDROXYLAMINE METABOLISMS2

ALVIN NASON

McCollum-Pratt Institute, The Johns Hopkins University, Baltimore 18, Maryland

I. Introduction ......................................................... 16II. Reduction of Nitrate to Nitrite ........................................................ 17

A. Assimilatory Nitrate Reductase........................................................ 181. The molybdoflavoprotein system ..................................................... 182. Photoreduction of nitrate............................................................ 19

B. Respiratory Nitrate Reductase........................................................ 201. Studies of the enzyme system from E. coli............................................ 202. Studies of the enzyme system from other microorganisms ............................. 22

C. Factors Influencing the Pathways of Nitrate Reduction .. 24D. Adaptation of Nitrate Reductase ....................................................... 24E. Nitrate Reductase Activity in Animal Tissues .......................................... 25

III. Stepwise Reduction of Nitrite........................................................ 25A. Nitrite Reductase in Nondenitrifying Organisms ........................................ 26B. Nitrite Reductase in Denitrifying Organisms............................................ 28C. Hyponitrite as a Possible Intermediate in Nitrite Reduction .. 30D. Hydroxylamine Reductase........................................................ 31

IV. Reduction of Organo-Nitro Compounds .................................................... 33V. Cell-free Nitrification ........................................................ 33VI. Probable Evolution of Inorganic Nitrogen Pathways....................................... 35VII. Literature Cited ........................................................ 36

I. INTRODUCTIONThe last decade has witnessed important ad-

vances in our knowledge of the enzymaticpathways and mechanisms of inorganic nitrogenmetabolism as carried on by microorganisms,higher plants, and to a lesser extent, animals.Most of this new information has been concernedwith the reduction of nitrate to nitrite, and morerecently with the stepwise reduction of nitriteultimately to the most highly reduced state ofnitrogen as represented by ammonia and theamino group. In addition, it is only within the

1 This symposium was organized by Dr. HowardGest under the auspices of the Division of GeneralBacteriology and presented at the Annual Meetingof the American Society for Microbiology inChicago, Ill., on April 26, 1961. Dr. Jacques C.Senez served as honorary convener.

2 The survey of the literature pertaining to thisreview was concluded in April 1961. ContributionNo. 351 of the McCollum-Pratt Institute. A por-tion of the work described here was supported inpart by research grants (no. 2332) from the Na-tional Institutes of Health, U. S. Public HealthService, and The National Science Foundation.

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last 2 or 3 years that segments of the biologicalprocess of nitrification, namely, the successiveoxidation of ammonia to nitrite by Nitrosomonasand nitrite to nitrate by Nitrobacter have beenattained for the first time at the cell-free level.Finally, the heretofore seemingly impregnableproblem of preparing cell-free extracts capable ofcarrying out nitrogen fixation has at last beenpierced, largely through the efforts of Mortenson,Mower, and Carnahan (79) as described in thenext article of this Symposium.The biological significance of inorganic nitrogen

metabolism is evident from the realization thatthe ultimate source of nitrogen for all forms oflife is inorganic nitrogen. From one point of viewthe evolution of biological systems has resultedin a particular nutritional relationship amongorganisms with respect to nitrogen utilization.We can regard nearly all plants and manymicroorganisms which are capable of convertingthe nitrogen atom from its various inorganicoxidized states (other than molecular nitrogenin most cases) to the more reduced forms, namely,ammonia and amino groups, as representing

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METABOLISM OF INORGANIC NITROGEN

the base or foundation of a typical ecologicalpyramid. Dependent upon this base are allthe other forms of life (such as particular micro-organisms and virtually all animals includingman) who can fulfill their nitrogen require-ment only from an exogenous supply of organicnitrogen and ammonia since they are incapableof transforming the more oxidized states ofinorganic nitrogen to this level. Therefore, greenplants and numerous microorganisms, by virtueof possessing the necessary complement ofenzymes for carrying out nitrate assimilation,are ultimately responsible for providing nitrogento many heterotrophic forms of life on our planet.The essential features of inorganic nitrogen

metabolism center about oxidation-reductionreactions. The nitrogen atom occurs in nature ina variety of oxidation states ranging from thedisputed oxidation level of +6 as represented bythe presumed short half-lived N03 (Wells (154))through the oxidation states of +5 (N205 or itshydrated form, HN03); +4 (NO2); +3 (N203or its hydrated form, HNO2); +2 (NO); +1(N20), (HNO), H2N20, and N02*NH2 represent-ing nitrous oxide, nitroxyl, hyponitrous acid(the dimer of the hypothetical nitroxyl), andnitramide, respectively); 0 (N2); -1 (NH20H);-2 (NH2NH2); and -3 (NH3). With the excep-tion of the controversial +6 oxidation state,each has been implicated in the inorganic nitro-gen metabolic pathways of either intact organ-isms or cell-free preparations.The present review deals primarily with the

enzymatic properties and sequences of nitrate,nitrite, and hydroxylamine transformation. Inparticular, emphasis will be placed on thosepapers published during the last 3 years whichhave a direct bearing on the over-all pathwaysand on the mechanisms of action of the stepsconcerned. The important contributions madein this area up until 1958 have been compre-hensively and collectively treated in the Mc-Collum-Pratt Symposium on Inorganic NitrogenAletabolism (71), the symposium of the Societyfor Experimental Biology on "The Utilization ofNitrogen and Its Compounds by Plants" (111),and the review article by Nason and Takahashi(86). Portions of the earlier material have beenincluded in the present paper, however, whereverit was deemed necessary for a more integratedpresentation of the subject. Although it is evidentthat certain fundamental patterns are emerging

from the great welter of detail concerning thepathways of inorganic nitrogen metabolism, anumber of important aspects are still not clear.

II. REDUCTION OF NITRATE TO NITRITEAt first glance there appears to be a variety of

types of nitrate reduction in a wide range ofmicroorganisms. Further examination has re-vealed, however, that they fall tentatively intotwo general classes: (i) nitrate assimilation orassimilatory nitrate reduction, and (ii) nitraterespiration or dissimilatory nitrate reduction.The two types of nitrate reduction play impor-tant roles in metabolic function and have beencorrelated in the past with certain underlyingenzymatic constituents that are characteristicallyassociated with each of the two processes. Furtherstudies are revealing, however, that the enzy-matic similarities between these two types aregreater than their differences.

Nitrate assimilation represents the biologicalreduction of nitrate to ammonia or the aminolevel with the products being used for the bio-synthesis of nitrogen-containing cell constituents,for example, proteins and nucleic acids. Thetransformation of nitrate to nitrite in the courseof nitrate assimilation is the initial step in theenzymatic pathway of the 8-electron changerequired to attain the oxidation level of nitrogen(-3) as represented by the nitrogen of ammonia,amino acids, and proteins. In nitrate respiration,nitrate is used as the terminal electron acceptor inplace of oxygen by several microorganisms underanaerobic or partially anaerobic conditions. Thereduction products, which may include nitrite,nitric oxide (NO), nitrous oxide, molecular andother oxidation states of nitrogen, depending uponthe organism and the chemical and physicalconditions of its environment, are apparentlynot further utilized and are for the most partexcreted into the surrounding medium. If molec-ular nitrogen, nitrous oxide, or nitric oxide isthe product of nitrate respiration, the process iscalled denitrification. The latter is therefore aparticular aspect of nitrate respiration whichhistorically was designated by the term denitrifi-cation long before it was recognized to be essen-tially a form of nitrate respiration.

Because of its obvious physiological and enzy-mological similarity to oxygen respiration, itwould be expected that nitrate respiration in-volves energy-yielding reactions which under

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given conditions are necessary for the growth andwell-being of the organism. At best there is slightevidence for the coupling of phosphorylationto nitrate respiration (Takahashi, Taniguchi,and Egami (136); Ohnishi and Mori (99)). Ithas not yet been demonstrated in cell-free prep-arations. Perhaps one of the most distinguishingenzymological features of nitrate respiration isthe involvement of one or more cytochromes aselectron carriers in the process. In contrast, theassimilatory reduction of nitrate to nitrite doesnot include any of the heme proteins as com-ponents of the electron transport system. Molyb-denum, however, is a component of both systems.

A. Assimilatory Nitrate Reductase1. The molybdoflavoprotein system. The first

step in nitrate assimilation is catalyzed by thesoluble, sulfhydryl molybdo-FAD-protein,3 ni-trate reductase. The enzyme was first charac-terized from Neurospora (Nason and Evans (85))and soybean leaves (Evans and Nason (30)).Neurospora nitrate reductase is relatively specificfor TPNH as the electron donor, whereas TPNHand DPNH are equally effective with the soy-bean enzyme. The presence of a similar or closelyrelated nitrate reductase has since been reportedto occur in a variety of higher plants (14, 37, 39,98, 100, 117, 130, 142, 143) and indicated to beadaptive in nature (14, 37, 39, 117, 142, 143) asshown for Neurospora and other microorganisms(see review by Nason and Takahashi (86)).That molybdenum is the metal constituent of

the Neurospora enzyme (Nicholas and Nason(90)) was shown by the (i) increased nitratereductase activity in various protein fractionsaccompanied by increased molybdenum concen-tration, (ii) decrease in molybdenum concomi-tant with a decrease in enzyme activity duringdialysis against cyanide, (iii) specific reactivationof the cyanide-dialyzed enzyme by molybdenum,and (iv) specific effect of molybdenum deficiencyduring growth resulting in decreased nitratereductase activity.

3The following abbreviations are used: FAD,FADH2, flavin adenine dinucleotide and reducedform; FMN, FMNH2, flavin mononucleotide andreduced form; DPN, DPNH, diphosphopyridinenucleotide and reduced form; TPN, TPNH, tri-phosphopyridine nucleotide and reduced form;ATP, adenosine triphosphate; ADP, adenosine di-phosphate.

Nicholas and Nason (91) also demonstratedthat flavin and molybdenum function as electroncarriers in Neurospora nitrate reductase in thefollowing sequence:

TPNH -* FAD -* Mo - NO3-Flavin precedes molybdenum as shown by theinability of the molybdenum-free enzyme tocatalyze the reduction of nitrate to nitrite byTPNH or reduced flavin, although FAD (orFMN) was reduced by TPNH. Added molyb-denum specifically restored the ability of theenzyme to catalyze nitrite formation by reducedflavin, or TPNH plus flavin. That molybdenumfunctioned as an electron carrier was stronglyimplied by experiments in which reduced molyb-date (prepared by treatment with sodium hy-drosulfite) enzymatically reduced nitrate tonitrite, and by the observation that molybdateenzymatically oxidized FMNH2. The reportedisolation and separation of three oxidation statesof molybdenum (Mo+6, Mo+i and Mo+3) byNicholas and Stevens (95) and their claim thatMo+i was as effective as TPNH as an electrondonor for the enzymatic reduction of nitratesupport the suggestion by Nicholas and Nason(91) that molybdenum enzymatically undergoes areversible oxidation-reduction from the +5 tothe +6 oxidation states. Our present knowledgeof the mechanism of action of nitrate reductasefrom Neurospora can be summarized as follows:

TPNH + H+ -* FAD FADH2 * Mo6+Mo5+ N03-* N02- + H20

The observation by McElroy (70), Kinskyand McElroy (57), and Nicholas and Scawin(94) that inorganic phosphate stimulated themolybdenum-requiring step of the Neurosporanitrate reductase (namely, FADH2 -* 'Ilo -*NO -) led Kinsky and McElroy (57) to postulatethe existence of a phosphomolybdate complexin the enzyme. This is analogous to the situationin which specific reagents are known to reducephosphomolybdenum complexes but not freemolybdate. Final proof for the oxidation-reduc-tion role of molybdenum in nitrate reductase,however, must await direct evidence from experi-ments with the endogenous molybdenum of theintact enzyme.

Studies have also been made with the nitratereductase from soybean leaves identifying molyb-denum (29, 92) as the metal component and

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demonstrating the sequence and mechanism (92)to be essentially the same as in Neurospora.The presence of a somewhat similar system inEscherichia coli, in addition to the usual respira-tory nitrate reductase, with FAD and molyb-denum as components, has been indicated (93),depending upon the conditions of growth of theorganism.

The findings of Kinsky and McElroy (57),that nitrate reductase preparations from Neuro-spora contained an inseparable TPN-cytochromec reductase with somewhat similar properties,suggested that the two activities are catalyzedby the same enzyme. Adaptation experimentsshowed that both nitrate reductase and cyto-chrome c reductase activities were induced in a

parallel manner when Neurospora was grownin a medium containing varying amounts ofnitrate. However, there was no nitrate reductasein mycelia grown on ammonia as the sole nitrogensource, although there was substantial TPN-cytochrome c reductase having approximately50% of the specific activity of that obtained fromnitrate-grown Neurospora. Their results pointto two kinds of TPN-cytochrome c reductases:(i) a constitutive enzyme with no associatednitrate reductase as indicated by the adaptationexperiments with ammonia-grown mycelia, and(ii) an enzyme associated with nitrate reductaseactivitv. The latter enzyme may well be a singlesystem which is responsible for nitrate reduction,and in part for cytochrome c reduction; or thetwo activities share a common enzymatic step,presumably the rate-limiting TPN-flavin reduc-tase reaction. Since several metal-binding agentswere potent inhibitors of nitrate reductase butnot of cytochrome c reductase, it seems plausiblethat the next step would involve the enzymatictransfer of electrons from flavin, either to nitratevia molybdenum, thus giving nitrate reductaseactivity, or to cytochrome c to give cytochromec reductase activity. The parallel inducement ofboth activities in nitrate-grown mycelia could beascribed primarily to the adaptive formationof nitrate reductase, the first step of which(TPNH -- flavin) could also be used by thecytochrome c reductase, thus reflecting itselfin an increased cytochrome c reductase activity.This is represented as follows:

Mo -* NO3-

TPNH -- FAD metal-binding agents

cytochrome c

The above hypothesis implies that at least twocomponent enzymes are involved in the nitratereductase system, although they have not as yetbeen separated.

Silver (127) observed a pyridine nucleotide-nitrate reductase in the extracts of the yeastHansenula anomala grown on a nitrate-containingmedium. The enzyme was similar to that foundin Neurospora and soybean leaves, having ametalloflavoprotein with FAD and molybdenumas probable prosthetic groups. Spectrophoto-metric observations eliminated a direct role of thecytochromes in nitrate reduction. Taniguchi andOhmachi (140) have recently studied an induci-ble pyridine-nucleotide nitrate reductase residingin the large particles of nitrate-grown Azotobactercells. Except for its particulate nature, the enzymestrongly resembles that observed in Neurosporaand higher plants, and is apparently of the assimi-latory type. It is a sulfhydryl, metalloenzyme inwhich no cytochromes participate. Added FADor FMN is significantly stimulatory with DPNHgiving twice as much maximal activity as TPNH.The same large particles which contain nitratereductase also possess DPNH oxidase activity.The latter activity in contrast to that of nitratereductase involved the participation of cyto-chrome components. The following proposedsequence (140) of electron transfer in Azoto-bacter from DPNH bears a strong similarity tothe suggested scheme indicated above for Neuro-spora:

cytochrome oxidase -* 02

cytochromesystem

FAD7DPNH or

FMN\, nitrate reductasemetal -- NO3-

If the above unidentified metal should prove tobe molybdenum, then the nitrate reductase ofAzotobacter would be essentially the same as thatalready described for Neurospora and higherplants.

2. Photoreduction of nitrate. Although the effectof illumination in accelerating the reduction of

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nitrate in green plants has been known for sometime, the explanation of this phenomenon hasbeen a highly controversial one (see reviews byNightingale (96, 97), Street (135), and Virtanenand Rautanen (148)). That photochemicalprocesses may furnish the hydrogen donors (i.e.,reduced pyridine nucleotides) for nitrate re-duction was shown by Evans and Nason (30)through the use of grana or broken chloroplastsand purified nitrate reductase from soybeanleaves. A similar concept based upon a com-petition between nitrate and carbon dioxidefor reducing power has been proposed by VanNiel, Allen, and Wright (146) as a result oftheir experiments with Chlorella. They ob-served that at low light intensity the uptakeof carbon dioxide, but not the production ofoxygen, was decreased in the presence of ni-trate. Kessler (55), in discussing the enhancedreduction of nitrate by light, concluded fromvarious lines of evidence that although the photo-chemical reduction of pyridine nucleotides islikely to be one of the important factors, itcannot explain all the available experimentalresults. He feels that the action of light is acomplicated phenomenon contributing to thesupply of hydrogen donors, energy-rich phos-phate bonds, and carbon compounds. In thisrespect it is of interest that Stoy (133), in testingthe possible role of riboflavin as a light-absorbingcatalyst in biological photoreduction, showedthat photochemically reduced riboflavin is amore efficient electron donor than DPNH withthe use of purified nitrate reductase of wheatleaves. He had previously observed (132) asignificant increase in nitrate reduction by de-tached wheat leaves in the blue and violet partsof the spectrum, suggesting involvement of ayellow pigment.

B. Respiratory Nitrate Reductase1. Studies of the enzyme system from E. coli.

The first demonstration of nitrate respiration innondenitrifying bacteria was made by Quastel,Stephenson, and Wetham (114) in 1925. Theyobserved that their strain of E. coli failed to growanaerobically on lactate unless a suitable electronacceptor such as nitrate was furnished in themedium. Subsequent experiments by Yamagata(156), with cell-free preparations from aerobicallygrown E. coli and reduced methylene blue as theelectron donor for the reduction of nitrate, pro-

vided direct proof for the existence of the enzymenitrate reductase. Detailed investigations someyears later by Egami and his collaborators indi-cated that the nitrate-reducing system of E. coliwas bound to a subeellular particle which wasalso intimately involved in aerobic respiration.On the basis of their experiments they formu-lated (141) in 1956 the following sequence ofelectron transport involving the reduction ofnitrate and oxvgen:

2-n-heptyl-4-hydroxyquinoline-N-oxideDPNH 02

oxidase tFAD -- cytochrome b1

Ax nitrate reductaseFormate 7 NO3-

reduced methylene blue

In support of this scheme were the observationsthat the rate of oxidation of DPNH by nitratewas considerably increased by catalytic quantitiesof FAD. Cytochrome bi was implicated by (i)spectral studies showing oxidation of the reducedheme complex upon addition of nitrate in theabsence of oxygen, and (ii) the large inhibitioncaused by 2-n-heptyl-4-hydroxyquinoline-N-ox-ide. The latter is considered to be a specificinhibitor of cytochromes b and bi (66). In thissystem, nitrate reductase is defined as the termi-nal member of the electron transport chain react-ing directly with the nitrate. It was concludedthat nitrate served as a cellular oxidant throughthe action of the above terminal nitrate reductaseduring anaerobiosis, in place of oxygen and theterminal respiratory oxidase. Except for theterminal steps, the same electron transportchain appeared to be involved in both the aerobicand anaerobic states. As seen in the above se-quence, the use of reduced methylene blue orother suitable reduced dye (e.g., reduced methylviologen) offers a distinct advantage for thestudy of the terminal nitrate reductase itselfapart from the other members of the organizedelectron transport chain.

Iida and Taniguchi (41), in further studies ofthe complex E. coli particulate electron transportsystem extending from DPNH or formate tonitrate, confirmed the sequence proposed above.They found in addition that an unidentifiedacid-labile, heat-stable, soluble factor (not re-placeable by FAD, menadione, or ferrous ion,singly or in combination) was necessary for full

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activity of the DPNH- or formate-nitrite reduc-tase system and the DPNH oxidase. Menadionewas observed to reverse partially the inhibitioncaused by Dicumarol. Solubilization by meansof isobutanol and deoxycholate treatment of theorganized electron transport system resulted inits inactivation. The marked inhibition of theterminal nitrate reductase (using reduced methyl-ene blue as the electron donor) by several metal-binding agents suggested the presence of a metalcomponent.When the same E. coli strain was grown aerobi-

cally in a medium similar to that used by Nicho-las and Nason (93) for E. coli strain B, withthe exception that yeast extract was also added,it was observed by Itagaki and Taniguchi (48)that a considerably increased amount of solublenitrate reductase could be easily extracted fromthe cells. Although the soluble crude extract incontrast to the particulate fraction was incapableof using formate as an electron donor, it pos-sessed DPNH-nitrate reductase activity (TPNHwas less effective) which was also sensitive to2-n-heptyl-4-hydroxyquinoline-N-oxide. It appar-ently contained a small quantity of activelyparticipating cytochrome bi ; and, after dialysis,was markedly stimulated by the addition ofFAD, menadione, and Fe++ ions. The lattercould also serve as an electron donor; and Dicu-marol inhibition was shown to be reversed bymenadione. On the basis of their results, Itagakiand Taniguchi (48) proposed that theDPNH-nitrate reductase system of theYanagutchi strain, grown as describedconsists of the following sequence:

FAD Fe++DPNH - or, menadione, It -+

FMN Fe+++nitrate reductasecytochrome bil (unknown heavy metal)

quinoline oxide

solubleE. coliabove,

NO3-

The marked inhibition of the terminal nitratereductase by cyanide and azide implied thepresence of a heavy metal component as indicatedabove. The proposed scheme is essentially inagreement with the results obtained some 5 yearsearlier by Wainright (149), who observed that thepyridine nucleotide-nitrate reductase, in a cell-free preparation of aerobically grown E. colistrain 1433, probably included a cytochromecomponent and was stimulated by menadioneand ferrous ions in the presence of added FAD orFMN. Studies by other workers with intactcells and extracts of E. coli had also implicatedthe participation of cytochrome (9) and iron(28) in the nitrate reduction process.Heredia and Medina (38, 75), with extracts

of aerobically grown E. coli strain 86, obtainedevidence for two nitrate reductase pathways ofa particulate nature. They concluded thatthrough the action of menadione reductase (155),vitamin K3 (menadione) or one of its analoguesserves as an electron carrier in one of the path-ways for the ultimate enzymatic reduction ofnitrate by DPNH under aerobic as well as anaer-obic conditions. The strikingly inhibitory effectsof several metal-binding agents indicated theinvolvement of a metal constituent. The addi-tion of menadione to their enzyme system provedto be absolutely essential for the reduction ofnitrate aerobically and caused as much as a 30-fold stimulation in the reduction of nitrateanaerobically by the same preparations. The fact,however, that in the absence of added menadionethere was still some anaerobic nitrate reductionwhich was stimulated by FAD, suggested that asecond mechanism of nitrate reduction is alsopresent, probably of the oxygen-sensitive respira-tory type already indicated by Egami and hiscollaborators (141). They proposed the followingrelationship between the two pathways:

vitamin K3analogue

menadionereductase

/' - nitrate reductase -- N03-DPNH

\,DPNH /dehydrogenase -+ FAD -* cytochrome b, -* oxidase -- 02

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The postulated scheme of MIedina and Herediafor the involvement of menadione in the enzy-matic reduction of nitrate by E. coli differs fromthat previously indicated by Itagaki and Tanigu-chi (48). Further experimentation is necessary toresolve the differences between the electrontransport sequences proposed by these two groups.Using E. coli grown anaerobically in a medium

containing 0.1 % KNO3, Taniguchi and Itagaki(137, 138) obtained a particulate formate-nitratereductase system essentially similar to that al-ready indicated earlier for the aerobically growncells (141) but possessing a 20- to 30 fold greaterspecific activity. In contrast to the particulatesystem of aerobically grown cells, the particleswere devoid of DPNH and TPNH oxidases aswell as various dehydrogenases with the notableexception of formic dehydrogenase. Solubilizationof the enzyme by alkaline cold incubation afterheat treatment (or by steapsin or chymotrypsindigestion) followed by purification to a homoge-nous state yielded only the terminal nitrate re-ductase (reduced methyl viologen as electrondonor) of the original particulate system. Thepurified enzyme contained neither formic dehy-drogenase, flavin, nor cytochrome b1, and pos-sessed a molecular weight of 1 million. The dif-ference spectrum (oxidized minus reduced) ofthe homogeneous enzyme preparation displayeda broad peak at 445 to 450 mu, which instantlydisappeared under anaerobic conditions uponaddition of nitrate, accompanied by the simul-taneous production of nitrite. Spectrographicanalvsis showed the presence of 1 atom of boundmolybdenum and about 40 molecules of boundiron per protein molecule (138). Jida and Yama-saki (42) at the same time also reported thepresence of molybdenum and iron in the nitritereductase of E. coli.A comparison of the properties of the assimi-

latory nitrate reductase of Neurospora with thoseof the respiratory nitrate reductase of E. coli isgiven in Table 1. Both systems share at least onefundamental characteristic, namely, the possessionof molybdenum as an active enzymatic compo-nent. For the Neurospora enzyme, the evidenceindicates that molybdenum serves as an electroncarrier (apparently undergoing a reversiblechange in oxidation state from +6 to +5) tonitrate. A similar mechanism of action of molvb-denum has now been implicated for the E. colienzyme (49). The recent finding of molybdenum

in the respiratory nitrate reductase of E. colilends further support to the generally emergingpattern that molybdenum is a necessary com-ponent of those enzymes capable of catalyzingthe reduction of nitrate to nitrite (the nitratereductase of Neurospora (90), higher plants (92),and E. coli (42, 138); xanthine oxidase from milkand liver (26, 116); and aldehyde oxidase (68)).

2. Studies of the enzyme system from other micro-organisms. Sadana and _McElroy (123) purifiedand characterized a nitrate-reducing system fromthe salt-water luminous bacterium, Achromobacterfischeri, and proposed the following pathway ofelectron transport:

DPNH (TPNH) -* FMN (FAD)1

Fe+++ -* bacterial -+ 02cytochrome

reduced benzyl viologen -- nitratereductase

N03The electron transport chain was separated intotwo soluble portions, (i) the electron-donor sys-tem, namely, DPNH-cytochrome c reductasewith a requirement for FMN or FAD, and (ii)the terminal nitrate reductase system, by whichelectrons were transported from the reducedcytochrome to nitrate. The cytochrome compo-nent which was auto-oxidizable had absorptionbands similar to mammalian cytochrome c. Whenreduced benzyl viologen supplied electrons, thebacterial cytochrome was not involved as indi-cated by spectral studies as well as the lack ofinhibition by CO in contrast to the photorevers-ible inhibition by this reagent with DPNH as theelectron donor. The proposed electron transportchain is similar in some respects to that alreadyindicated for E. coli. In view of the cytochromeinvolvement and the apparent competition by 02for electrons in the conversion of nitrate to nitrite,the nitrate-reducing system of A4. fischeri isclassified under nitrate respiration.A somewhat similar system has been reported

by Fewson and Nicholas (32) in the denitrifyingbacterium Pseudomonas aeruginosa. The purifiedDPNH-specific, sulhydryl flavoenzyme containedcytochrome c and apparently involved molyb-denum as a component of the system. The fol-lowing sequence of electron transport was sug-gested:

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TABLE 1. Comparison of Neurospora nitrate reductase (nitrate assimilation type) with Escherichia colinitrate reductase (nitrate respiration type)

Properties Neurospora nitrate reductase E. coli nitrate reductase

1. Original physical state

2. Sensitivity to 02

3. Electron donors

4. Components of electrontransport chain

FlavinCytochromesMoFeVitamin K derivativeOther factors

5. Sequence of electron trans-port chain

6. Separation of nitrate re-ductase activity fromremainder of electrontransport chain

7. Other activities associatedwith electron trans-port chain

8. Km for nitrate9. Metabolic role

Soluble, molecular weightnot reported

Unaffected

TPNH, FADH2, FMNH2,Mo5+, reduced indophenoldyes, other artificial dyesnot reported

FADNonePresentNot reportedNot reportedNot reportedTPNH -- FAD > MoNO3-

Not reported

TPNH-cytochromeductase activity

c re-

1.4 X 10- MNitrate assimilation

Particulate, mol wt ca. 1,000,000

Decreases enzyme activity by compet-ing for electrons

DPNH, FADH2, reduced cytochromeb6, various reduced dyes (including

reduced methylene blue, methyl vio-logen, benzyl viologen, phenosafra-nine, etc.)

FADCytochrome bi1 atom/enzyme moleculeCa. 40 atoms/enzyme molecule possiblyPossiblyUnidentified soluble factorDPNH- FAD, menadione(?),Fe++tred () cytochrome bl -e Mo-eh NO3-Fe... ~ ~ F~ ?

TIFeFA

Cytochrome bi -* Mo -* NO3-Fe++IT (?)Fe+++

a) With intact chain: formate dehy-drogenase, and in some preparationsother dehydrogenases and reducedpyridine nucleotide oxidases

b) Separated nitrate reductase: none ofthe above activities other than ni-trate reductase

5.1 X 10-4 MNitrate respiration

DPNH -- FAD -> cytochrome c -- Mo -- NO3-

cytochrome oxidase

02

In extending their earlier reports (16, 17) of aparticulate DPNH-specific nitrate reductase fromthe Rhizobium japonicum of soybean nodules,Cheniae and Evans (18) concluded that thesystem compared most favorably with the ni-trate-respiratory type from E. coli. The enzymewas a sulfhydryl protein which was inhibited by

various metal-binding agents, antimycin A, andDicumarol. It exhibited a menadione requirementand possibly involved the cytochrome b com-plex. Thus far the participation of flavin has notbeen shown. Succinate or several reduced dyesserved as an electron source in place of DPNH.Cheniae and Evans (19) are still of the opinionthat the nodule nitrate reductase system is re-lated in some manner to the nitrogen fixationprocess. They found that the activity of the en-zyme in Rhizobium from nodules of soybean

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plants grown without combined nitrogen was atleast 8- to 10-fold greater than that of pure cul-tures of Rhizobium grown in media containingnitrate.A unique nitrate reductase, apparently of the

respiratory type, from Aerobacter aerogenes wasinvestigated by Pichinoty and Senez (104, 108).The enzyme which apparently catalyzed the firststep in the ultimate conversion of nitrate to am-monia neither utilized pyridine nucleotides norcontained cytochromes. Molecular hydrogenwhich served as the electron source for nitratereduction to nitrite (as well as to ammonia)stoichiometrically reduced nitrate in the presenceof benzyl viologen:

H2 -hydrogenase benzyl viologen nitrate ) NO3-reductase

The system was inhibited by a number of metal-binding agents.

C. Factors Influencing the Pathways ofNitrate Reduction

Walker and Nicholas (153) recently reportedthat Neurospora grown at low oxygen tensionsexhibited the enzymatic and other metabolicproperties characteristic of nitrate respiration, incontrast to that of nitrate assimilation whengrown under a more aerobic environment. Underthe former conditions nitrate reductase was de-pendent on iron as well as molybdenum in thenutrient medium; nitrite accumulated and cyto-chromes b and c increased in the mycelia. Whenoxygen was no longer limiting, the nitrite whichhad accumulated earlier was now reutilized andnitrate reductase activity became independent ofthe iron status of the medium.The data of Walker and Nicholas have essen-

tially confirmed the earlier report of Lenhoff,Nicholas, and Kaplan (65) and that of Higashi(40), who indicated that the oxygen tension of themedium during the growth of Pseudomonasfluorescens determined the alternative routes ofterminal electron transfer formed by the or-ganism. Lenhoff et al. had observed that cellsgrown at low oxygen tension in a nitrate-contain-ing medium had large quantities of cytochromepigment, a higher iron requirement, and a lowermolybdenum requirement than those grownunder more aerobic conditions. Their data sug-gested that in aerobically grown cells the molyb-doflavoprotein functioned primarily in the nitrateassimilatory process, whereas in anaerobically

grown cells the cytochrome-nitrate reductasechain served in terminal respiration (5).

Earlier findings by Sacks (121), Allen and VanNiel (5), Baalsrud and Baalsrud (10), and Rosen-berger and Kogut (118) had already attributedthe typically pink color of denitrifying bacteria,when grown under anaerobic conditions in thepresence of nitrate, to an increase in bacterialcytochrome. These results, in addition to pro-viding further support for our current conceptregarding the physiological roles of nitrate res-piration and assimilation, also illustrated theadaptability of the two processes within thesame organism, with one pathway often domi-nating the other, depending, among other factors,upon the availability of molecular oxygen.

D. Adaptation of Nitrate Reductase

The literature dealing with the adaptive na-ture of nitrate-reducing enzyme systems hasbeen collectively reviewed through 1957 byKluyver (59), Delwiche (24), and Nason andTakahashi (86). Early studies, particularly byPollock (109), with nitrate-respiring organismsincluding the denitrifiers demonstrated theadaptive nature of the nitrate-reducing enzymesystem. This was indicated by a lag period inthe reduction of nitrate by cells not originatingin a nitrate-containing medium. Wainwright andNevill (150), using nitrogen-depleted E. coli, ob-served a marked increase in nitrate reductaseactivity with a maximal value being attainedwithin 2 hours after adding nitrate. The strikingstimulation brought about by the addition ofcasein hydrolyzate suggested that the limitingfactor in adaptation was the slow rate of syn-thesis de novo of amino acids. They also showedthat auxotrophic mutants of E. coli requiringspecific amino acids did not grow or form nitratereductase in the absence of the appropriate aminoacid. Their results with mutants requiring uracilor thymine suggested that ribonueleic acid syn-thesis is necessary for the synthesis of the nitrate-reducing system. Egami, Hayase, and Taniguchi(27) were unable to induce the formation offormic dehydrogenase and nitrate reductase(cytochrome b - N03-) in an E. coli mutantauxotrophic for hemin in aerobic or anaerobiccultures in the presence or absence of hemin.

Pichinoty and D'Ornano (105, 106) concludedthat the adaptive formation of nitrate reductaseactivity in cell suspensions of Aerobacter aerog-enes under anaerobic conditions is due to syn-

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thesis de novo of the enzyme from free aminoacids. The induced formation of nitrate reductasein this organism (H2 -- benzyl viologen -- NOO)by nitrate or nitrite ions was markedly stimulatedby the addition of casein hydrolyzate to themedium and was prevented by chloramphenicol.Molecular oxygen was reported to inhibit re-versibly enzyme activity as well as adaptive for-mation of the enzyme. The latter phenomenon,namely that the adaptation of cells is reversiblyinhibited by oxygen, had already been demon-strated by Pollock (110) for E. coli. The classicobservation of little or no denitrification in deni-trifying organisms subjected to aerobic conditionsmay well reside in the inhibition of both thebiosynthesis and activity of the nitrate reductase.

Although most of the evidence illustrated theadaptive nature of the respiratory nitrate reduc-tase in microorganisms, there are a few reportsin the literature which fail to support this view-point. Straughn (134) was unable to obtain anenhanced nitrate reductase activity by the addi-tion of nitrate to cells grown in a peptone broth.Farkas-Himsley and Artman (31) found that al-though the kinetics of nitrate reduction by non-proliferating suspensions of old E. coli cells withand without nitrate was suggestive of an adaptivesystem, it could be ascribed instead to a permea-bility barrier in old cells toward nitrate. Althoughold E. coli cells grown on a medium supplementedwith nitrate reduced nitrate at a faster rate andwithout a lag period as compared to similar cellsfrom a nitrateless medium, the cell-free extractsfrom both groups reduced nitrate at the samerapid rate. In further support of their viewpointwas the observation that nonproliferating youngcells reduced nitrate at the same fast rate re-gardless of the presence or absence of nitrate inthe growth medium. In general their results aresuggestive of a substrate-inducible permease.The adaptive nature of the assimilatory nitrate

reductase (flavomolybdoprotein) appears to beclearly established in Nuerospora (85) and higherplants (14, 17, 37, 39, 142, 143). Most of theevidence is based on the demonstration that cell-free preparations of tissues exposed to ammoniumsalts or amino acids showed little or no nitratereductase activity in comparison to the highenzymatic activity in nitrate-grown material.The markedly inhibiting effect of ammonia on thenitrate reductase of certain fungi (not includingNeurospora) has been ascribed by Morton (80) to

its adverse effect on the formation and stabilityof the enzyme.

E. Nitrate Reductase Activity in AnimalTissues

The ability of animal tissues to reduce nitratewas first demonstrated in liver preparations ofvarious species by Bernheim and Dixon (11).They showed that nitrate was enzymatically re-duced by simply functioning as a hydrogen ac-ceptor of aldehyde oxidase in place of methyleneblue or oxygen. The recent studies of Omura andTakahashi (101, 102), however, have led to thesuggestion that the nitrate reductase activity ofanimal cell preparations may not necessarily bedue to the aldehyde oxidase and xanthine oxidaseactivities. Further evidence on this point and onthe possible metabolic significance of nitrate re-duction in animal tissues is necessary, however,before any conclusions can be drawn.

III. STEPWISE REDUCTION OF NITRITE

With the continued elucidation of the enzy-matic mechanisms for the conversion of nitrateto nitrite, attention has been turning increasinglyto the subsequent reduction of nitrite. Althoughthere has been some degree of success in separat-ing and isolating one or two of the enzymaticsteps presumed to be involved in the ultimatereduction of nitrate via nitrite to ammonia, thepathway, intermediates, and mechanisms be-yond the nitrite stage are still very much in needof clarification. Perhaps most indicative of ourlimited knowledge in this area is the fact that ithas not yet been conclusively established whetherthe subsequent reduction of nitrite by microor-ganisms and higher plants proceeds via organicintermediates or by way of an inorganic pathway.The present status of the problem can best beevaluated by indicating the progress that hasbeen made to date in various aspects of the field.

If we assume that the biological reduction ofnitrite proceeds via the inorganic pathway andthat two electron changes are involved for eachenzymatic step, then the following sequence ofintermediates with the indicated oxidation statesfor the nitrogen atom can be postulated.N02- (HNO),N02 -NH2,+3 +1

or H2N202 -- NH20H -> NH3-1 -3

The enzyme concerned with the reduction ofnitrite to the +1 oxidation state would be called

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nitrite reductase, whereas that which mediatesthe reduction of hydroxvlamine to ammonia ishydroxylamine reductase. Wte are still very un-certain as to the +1 oxidation state nitrogenintermediate in the inorganic pathway. As seenfrom the above sequence at least three possibili-ties exist: the hypothetical nitroxyl (HNO),nitramide (NO2 NH2), and hyponitrous acid(H2N202). The evidence that hyponitrous acid(and nitramide) may or may not be involved inbiological systems is discussed below.

A4. Nitrite Reductase in NondenitrifyingOrganisms

Yamagata (157) was the first to demonstratethe presence of nitrite reductase by using cell-freepreparations of Bacillus pyocyaneus (Pseudomonasaeruginosa). Subsequently Taniguchi et al. (139)observed the enzyme in Bacillus pumilus withthe use of reduced methvlene blue as the electrondonor. The detection of l)yridine-nucleotide-ni-trite reductase in extracts of Neurospora andsoybean leaves (30, 85) subsequently led to partialpurification and characterization of nitrite andhydroxylamine reductases from these sources(84, 119, 159). Both enzymes in Neurospora,which catalyzed the reduction of their respectivesubstrates to ammonia, were shown to be metallo-flavoproteins with unidentified metal compo-nents, whereas the corresponding systems fromsoybean leaves appear at this time to be some-what different, exhibiting a specific requirementfor Mn+ and an as yet unidentified cofactor(119).The nitrite and hydroxylamine enzymes have

been recently purified from soybean leaves byRoussos and Nason (119) and shown to requiresubstrate quantities of nitrite and hydroxylamine,respectively, in order to catalyze the oxidation ofreduced pyridine nucleotides. Both enzyme ac-tivities are inhibited by several metal-bindingagents and are stimulated 3- to 6-fold specificallyby Mun+ . They also show an absolute require-ment for an unidentified, dissociable, heat-stableorganic factor obtained from soybean leaf ex-tracts. DPN was demonstrated to be one of theproducts of both the nitrite and hydroxylamineenzyme reactions but the fate of nitrite andhydroxylamine is still unknown, although thelatter disappeared in quantities stoichiometricwith the oxidation of DPNH. The failure todemonstrate either that flavin was required orthat ammonia was an end product of the enzv-

matic reactions suggested the possibility that afragmentation of the nitrite reductase and hy-droxylamine reductase chains had been achieved.

Recent studies of a 50-fold purified Neurosporanitrite reductase by Nicholas, Medina, and Jones(89) failed to yield substantially more conclusiveinformation about its properties. No mentionwas made of the nitrite reduction product of theenzymatic reaction; whether it was ammonia or asubstance of an intermediate oxidation state wasnot indicated. The tentative identification of thenative flavin as FAD (no data were presented)and the conclusion based on the following evidencethat iron and copper were also active constituentsof the enzyme system must await further experi-mental verification. The nitrite reductase activityof extracts of mycelia deficient in Mg, Fe, Cu, orZn was significantly depressed, whereas a de-ficiency in Mo or Mn was without effect on theenzyme. Although the addition of iron compounds(Fe++) and copper compounds (Cu+ ) to ex-tracts of Fe-deficient and Cu-deficient mycelia,respectively, markedly stimulated enzyme ac-tivity, there was no indication of the specificityof these metal effects. Nor were any attemptsreported to remove the metal component fromthe purified enzyme by dialysis against metal-binding agents followed by restoration studies, aprocedure which proved to be of great value inthe identification of molybdenum as the metalconstituent of nitrate reductase (90). The copperand iron contents of various enzyme fractions,however, were shown to be related to their nitritereductase activities. Cuprous ion was implicatedas an electron donor in the enzymatic reductionof nitrate, whereas ferrous ion and reducedcytochrome c were inactive in this respect. Theabove Mg requirement was attributed to its in-direct effect in enzyme formation, since it did notaccumulate in the purified fractions of the enzymenor did its addition in vitro activate the nitritereductase. The zinc requirement, however, wasignored. The observation that the spectra ofpurified enzyme fractions reduced with dithioniteor DPNH showed no definite cytochrome bandscontradicted an earlier report from the samelaboratory (87) in which cytochromes b and clwere identified in the purified Neurospora nitritereductase. The latter report also claimed that (i)the enzyme was particulate, (ii) hyponitrite wasthe product of the reaction, and (iii) there was a"phosphorylation during nitrite reductase actionbecause uncoupling reagents inhibited the enzyme

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and inorganic phosphate was incorporated intothe ATP." These points were notably absent inthe recent and more detailed presentation of theproperties of the enzyme by the same group (89)and one can only assume that they no longerapply. With regard to the last point, Kessler andBucker (56), on the basis of experiments withgreen algae, concluded that the reduction ofnitrite required (rather than generated) highenergy phosphate in contrast to the first step ofnitrate reduction where it is not necessary.The pyridine nucleotide-nitrite reductase of

Azotobacter was shown by Spencer, Takahashi,and Nason (131) to be an adaptive FAD-metallo-protein which catalyzed the reduction of nitriteto ammonia. Unlike hydroxylamine reductasefrom the same organism, it exhibited no require-ment for manganous ions. Lazzarini and Atkinson(64) characterized a cyanide-sensitive TPNH-specific nitrite reductase from E. coli which wasnot stimulated by FMN or FAD or a variety ofmetal ions. Although the reduction product of thereaction was ammonia, the enzyme was inactivetoward nitrous oxide and hyponitrite (and ni-trate), but rapidly reduced hydroxylamine. Infact, TPNH-specific hydroxylamine reductaseactivity closely paralleled the nitrite reductaseactivity throughout the 100- to 200-fold purifica-tion. All attempts to alter the proportions of thetwo activities were unsuccessful, indicating thatthe same enzyme probably accounted for bothactivities. Interestingly enough the evidence ob-tained with isotopically labeled nitrite indicatedthat free hydroxylamine was not an obligate in-termediate in the reduction of nitrite to ammonia.It seems possible that one enzyme catalyzes thecomplete reduction of nitrite and hydroxylamineto ammonia without the intervention of freeintermediates. The purified enzyme also hadTPNH-specific cytochrome c and sulfite reductaseactivities. The latter activity is especially note-worthy in view of the recent results of Mager(67) implicating the TPNH-hydroxylamine re-ductase activity of E. coli as secondary to theTPNH-sulfite reductase which it always accom-panied (see Section III, D, below, on hydroxyl-amine reductase). The cell-free extract of E. coligrown in deep standing cultures also displayedtwo other nitrite reductases. One was an enzymein which DPNH served as the electron donor, andthe other was a particulate system utilizing re-duced benzyl viologen or FMNH2 as a donor.

Pichinoty and Senez (107, 124) found that cell

suspensions or extracts of Desulfovibrio desulfuri-cans reduced nitrite and hydroxylamine to am-monia with the uptake of molecular hydrogencorresponding to stoichiometric expectations (3moles of H2 per mole of nitrite and 1 mole of H2per mole of hydroxylamine). Cytochrome C3 wasimplicated as a component of these enzymes.Krasna and Rittenberg (63) also observed thereduction of nitrite and hydroxylamine to am-monia by the same species with the uptake ofhydrogen gas being consistent with reduction toammonia. McNall and Atkinson (73) isolated anE. coli strain (Bn) which reduced nitrate ornitrite completely to ammonia at the expense ofmolecular hydrogen, and showed (74) that itcould utilize hyponitrite, hydroxylamine, ornitrous oxide as its sole nitrogen source. Theability of certain green algae after adaptation inthe dark to a hydrogen-containing atmosphere tocarry out the stoichiometric reduction of nitriteto ammonia by molecular hydrogen was reportedby Kessler (54).Vanecko and Varner (145), with the use of

nitrite-infiltrated wheat leaves, identified oxygenas the gas evolved upon exposure to light andconcluded that photolysis of water was the pri-mary and immediate source of reducing substancein the light reduction of nitrite, especially since1 /' moles of oxygen were evolved per mole ofnitrite reduced.

HNO2 + H20 -* HN3 + 1/f 02

They concluded that nitrite was apparently re-duced to the amino level and probably incorpo-rated into proteins since there was no significantincrease in the levels of ammonia and amide.The interesting observation by Kessler (55), withthe use of intact cells of the algae Ankistrodesmusbravnii, that the reduction of nitrite was markedlyaccelerated by exposure to light under anaerobicconditions has recently been extended by Huzisigeand Satoh (40a). The latter workers found thatintact cells of Euglena gracilis experienced adoubling in the rate of nitrite reduction (aerobi-cally or anaerobically) upon exposure to light.The addition of phosphate as well as suitablehydrogen donors such as malate or pyruvatefurther enhanced the reaction. Huzisige andSatoh (40b) subsequently reported the isolationfrom spinach leaves of a soluble enzyme (desig-nated as photosynthetic nitrite reductase) whichwas required in addition to grana for the photo-chemical reduction of nitrite. The similarity in

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the methods of preparation and the properties ofphotosynthetic nitrite reductase to those ofphotosynthetic pyridine nucleotide reductase(San Pietro and Lang (123a)) suggests that thelatter enzyme is probably involved in the photo-synthetic nitrite-reducing system.

B. Nitrite Reductase in Denitrifying OrganismsDenitrification is the process by which particu-

lar microorganisms convert nitrate and certainintermediate reduction products, such as nitrite,to molecular nitrogen, nitrous oxide, or nitricoxide. This process represents in part a form ofnitrate respiration since the first step in theprocess utilizes nitrate (in place of oxygen) underanaerobic conditions for the ultimate oxidationof organic substrates. Denitrifying bacteria are

facultative organisms occurring almost univer-sally in soil and water and utilizing nitrate as a

hydrogen acceptor for energy-yielding oxidativereactions. The more recent developments in thisarea through 1957 (including references to re-

views covering details of earlier findings) were

indicated by Nason and Takahashi (86). Theyevaluated and summarized the evidence concern-

ing the metabolic characteristics of most denitri-fyers, namely (i) that organic compounds ingeneral serve as an energy source with eitheroxygen or nitrate as the ultimate electron ac-

ceptor (aromatic acids and aliphatic straightchain compounds, however, cannot be oxidizedanaerobically in the presence of nitrate); (ii) thatoxygen is a potent inhibitor of the denitrificationprocess by virtue of its effective competition withnitrate as an acceptor of electrons in the oxida-tive functions of the cell. Pichinoty and D'Ornano(106), as stated in Section II, D on adaptation)recently pointed out, however, that the inhibitoryeffect of oxygen may well be due to its action inpreventing both the biosynthesis and activity ofthe respiratory nitrate reductase; (iii) that cyto-chromes are involved as components in the elec-tron transport chain of the denitrification process;

and (iv) that the biochemical pathway for denitri-fication is believed to be essentially as follows:

NO3-

NO2- NO

(HNO) --* H2N2O2 0 N2

or INO2 *NH2 > N2

The above scheme postulates 2-electron steps.The first involves the conversion of nitrate to

nitrite, and is apparently mediated by a respira-tory nitrate reductase pathway as discussed in a

previous section. Fewson and Nicholas (32), on

the basis of recent experiments with cell-freepreparations of the denitrifying bacteriumPseudomonas aeruginosa, suggested a sequence ofelectron transport as shown previously in SectionII, B2. The next step in denitrification, namely,the reduction of nitrite on the basis of a presumed2-electron change would result in formation ofthe hypothetical nitroxyl intermediate, perhapsas a nitroxyl-enzyme complex. The presumedspontaneous dimerization of nitroxyl to yieldpossibly hyponitrite (or nitramide) might befollowed by a spontaneous or enzymatic decom-position of hyponitrite involving the removal ofwater thereby yielding nitrous oxide (N20). Thereduction in vitro of the latter to molecularnitrogen was demonstrated for the first time byNajjar and Chung (82) with cell-free preparationsof Pseudomonas stutzeri. The alternate possi-bility that the enzymatic hydrogenation ofhyponitrite (or nitramide) might lead directlyto the formation of nitrogen gas seems to bemore or less eliminated. The original claim byAllen and Van Niel (5) that P. stutzeri hydro-genated nitramide to gaseous nitrogen was sub-sequently ruled out by Kluyver and Verhoeven(60), who found that nitramide was decomposedtoo rapidly upon addition of phosphate bufferto be used as substrate. The latter workers alsoshowed that neither Micrococcus denitrificans nor

Pseudomonas aeruginosa evolved nitrogen gas

from sodium hyponitrite, confirming the resultsof Allen and Van Niel (5) with P. stutzeri.With regard to the other steps in the over-all

denitrification pathway, Sacks and Barker (122)concluded that nitrous oxide was not an obliga-tory intermediate in the formation of molecularnitrogen since (i) nitrous oxide was not detectablein their denitrification experiments with Pseudo-monas denitrificans; (ii) nitrous oxide utilizationwas selectively blocked by azide or dinitrophenolunder conditions which permitted the formationof nitrogen from nitrite; and (iii) a lag frequentlypreceded the utilization of nitrous oxide byresting cells, a phenomenon not observed in theconversion of nitrite to N2. Allen and Van Niel(5) also observed that the conversion in P.stutzeri of nitrous oxide to N2 was inhibited bycyanide, whereas the reduction of nitrite to N2was unaffected.The first report of cell-free denitrification,

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namely, the enzymatic reduction of nitrate ornitrite to molecular nitrogen and nitrous oxide(as well as nitric oxide), was made by Najjarand Allen (81) with preparations from P. stutzeriand Bacillus subtilis. Chung and Najjar (20, 21,82), with crude ammonium sulfate fractions ofP. stutzeri extracts, subsequently showed thatin the reduction of nitrite to nitric oxide TPNHand DPNH served as electron donors, and FADand FMN gave a 2-fold stimulation. The crudeenzyme fractions also contained substantialamounts of cytochrome c and were inhibited byseveral metal-bindings agents. The partial loss inactivity after successive dialysis against solutionsof metal-binding agents and water was restoredto a small extent by the addition of cupric andiron salts. Nitric oxide reductase activity (NO2 --

N2) in similar fractions exhibited almost identicalproperties. Najjar and Chung therefore sug-gested the following electron transport sequenceto yield N2 and NO in the reduction of NO2-, andN2 in the reduction of NO.

NO2-TPNH -- FAD -- Cu++ -- cytochrome

or or orDPNH FMN Fe+++ NO

Walker and Nicholas (151), with the use of a600-fold purified nitrite reductase from Pseudo-monas aeruginosa, also reported nitric oxide to bethe product of the reaction. Except for the factthat DPNH and TPNH could not be used aselectron donors, the enzyme was quite similarto the corresponding P. stutzeri system reportedpreviously by Najjar and Chung (82). As anFAD enzyme possessing a cytochrome c-typespectrum, it also contained iron and coppercomponents. Fewson and Nicholas (34), how-ever, questioned the claim by Chung and Najjar(21) that the nitrite oxide reductase of P. stutzerirequired both iron and copper for activity. Theybelieved that the action of iron, copper, and, toa smaller extent, zinc, in reactivating the enzymeafter prolonged dialysis against salicylaldoxime,resulted from the removal of residual chelatebound to the enzyme, especially since the reversalof inhibition was in the order of affinities of themetals for salicylaldoxime. Fewson and Nicholas(33) also purified a nitric oxide reductase fromPseudomonas aeruginosa and characterized it as aflavoprotein which did not use reduced pyridinenucleotides. They claimed it contained iron butnot copper, and that nitric oxide is an inter-

mediate not only in the nitrate respiratory path-way (e.g., denitrification), but also in the nitrateassimilatory pathway and possibly in nitrogenfixation and nitrification.Yamanaka and Okunuki (158) isolated a highly

purified cytochrome oxidase particle from P.aeruginosa which also catalyzed the reductionof nitrite by the reduced cytochrome c-likecomponent of the bacterium. According to theseresults, the purified cytochrome oxidase particleof Pseudomonas functioned as a nitrite reductase.Iwasaki and Mori (51), working with extracts of adenitrifying bacterium tentatively identified asPseudononas denitrificans, reported that in thepresence of lactate, nitrite was reduced only tomolecular nitrogen, whereas, in the absence ofadded lactate, the evolved gas was almost ex-clusively nitrous oxide. Hyponitrite could not beutilized but hydroxylamine had a stimulatoryeffect on N2 gas evolution from nitrite. On thebasis of these results they suggested that, indenitrification, nitrite is converted to N2 byelectrons which are enzymatically transferredfrom a substrate such as lactate. According totheir hypothesis, a portion of the nitrite is reducedto a substance such as hydroxylamine whichin turn reacts with the remaining nitrite in eitherof two presumed ways: (i) hydroxylamine under-goes an enzymatic reaction with nitrite to producenitrous oxide; or (ii) in the presence of lactateand lactic dehydrogenase, nitrite is first reducedto NO and then reacts enzymatically with hy-droxylamine to produce N2.

4H +HN02HN02\ INH20H- + N20

\HjH

NO

N2+2H20

Iwasaki (50) subsequently purified two inactivefractions from the extract which upon recombina-tion served as a denitrification system. One of thefractions had the typical absorption spectrumof a c-type cytochrome, and the other, which heconsidered to be the denitrifying enzyme itself,was red due to a so-called "cryptocytochrome c."The above enzymatic reaction of nitrite withhydroxylamine to produce nitrous oxide wasapparently mediated by the latter fraction. Thecytochrome c-like fraction was considered to bean electron carrier to the denitrifying or "crypto-

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cytochrome c" system with the latter also mediat- and a soluble component, both of which wereing the conversion of nitrite and hydroxylamine necessary for denitrification. The activity of theto N2: soluble component during purification paralleled

HNO2 + NH20H

H donor cytochrome c > cryptocytochrome c(e. g., lactate) X

N2

Another denitrifying bacterium, a halotolerant hydroxylarnine reductase activity. Using partiallyM1icrococcus (strain 203), has also been the subject purified dialyzed preparations, he found (8)of cell-free denitrification studies. Detailed in- that the soluble and particulate componentsvestigations by Asano of the nitrite-reducing were activated by copper and iron, respectively.system from this organism revealed certain Asano's conclusion that neither component wasdifferences in properties as compared to the cor- of a cytochrome nature, unlike that reported forresponding Pseudomonas enzyme. Using a par- the Pseudomonas nitrite reductase system bytially purified enzyme preparation from Mllicro- Iwasaki and Mori (51), leaves unexplained hiscoccus, Asano (6) reported that the electron trans- earlier proposal that cytochrome b1 was an elec-port system functioning in the reduction of tron carrier as indicated in the above sequence.nitrite was similar to the electron transport chainto oxygen. He observed that virtually all the C. Iyponitrite as a Possible Intermediate innitrite reduced was converted to nitrogen gas with Nitrite Reduction1 mole of N2 appearing for every 2 moles of The original reports by Medina and Nicholasnitrite disappearing. The preparation possessed (76-78) in 1957 constitute the only claim for aDPNH-nitrite reductase activity in the presence cell-free system which catalyzes the reduction ofof high NaCl concentrations (0.6 M), was stimu- hyponitrite. Taniguchi et al. (141) had pointedlated by added FAD and menadione, and was out earlier that hyponitrous acid was not reducedinhibited by amytal, quinine, Dicumarol, anti- by a crude extract of halotolerant bacteria con-mycin A, and carbon monoxide. The system taining all the enzyme systems for reduction ofalso appeared to include a cytochrome b4 as an nitrate to ammonia with reduced methyleneelectron carrier. In view of his results, Asano blue as the hydrogen donor. Medina and Nicholassuggested the following electron transport se- indicated the presence of a DPNH-hyponitritequence for Mlicrococcus nitrite reductase. reductase in crude extracts of Neurospora and

02

D)PNH -* flavoprotein -- vitamin K - (?) antimycin A-sensitive factor -e cytochrome b4T\I

succinate dehydrogenase nitriteT reductase\\

succinate N02-

The Micrococcus nitrite reductase preparationalso possessed a powerful hydroxylamine reduc-tase activity (in contrast to the absence of suchactivity in the nitrite reductase preparations ofPseudomonas), but nitrite was not reduced tohydroxylamine and ammonia. The observedinhibitory effect of hydroxylamine indicatedthat the mechanism of denitrification was dif-ferent from that reported for Pseudornonas byIwasaki and -Mori (51) as discussed above.Asano (7) subsequently resolved the Micro-

coccus nitrite reductase denitrifying system intotwo protein fractions, a particulate component

designated it as a metalloflavoprotein catalyzingthe conversion of hyponitrite to ammonia. Itsunusual similarity to the accompanying Neuro-spora nitrite reductase including its reportedsensitivity to a number of inhibitors (e.g., naph-thoquinone and 2-n-heptyl-4-hydroxyquinoline-N-oxide) as well as to a nutritional deficiency ofcopper or iron raises the question as to whetheror not the two systems are identical.More important, however, is the question of

the stability of hyponitrite in buffered solutionsnear neutrality. Frear and Burrel (35) observedthat although aqueous solutions of sodium hypo-


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nitrite were stable for 2 hours at room tempera-ture at pH 11.3, they spontaneously decomposed(apparently to nitrite and nitrate) at pH 7.3 ata much faster rate displaying half-lives of about11 minutes. Chaudhary, Wilson, and Roberts(15) had already emphasized that hyponitritewas quite easily and spontaneously oxidizedunder neutral or slightly acidic conditions. Intheir own experiments on the possible reductionof added hyponitrite by infiltrated soybeanleaves, Frear and Burrell (35) concluded thathyponitrite nitrogen was first partially or com-pletely oxidized to the level of nitrite (or nitrate)prior to its biological reduction to ammonia.Their results, therefore, neither established thathyponitrite per se was assimilated nor that it wasan intermediate in the reduction of nitrate ornitrite to ammonia. The previous report byVanecko and Frear (144) that hyponitrite was anintermediate in nitrate reduction, based on theconversion of N'5-labeled hyponitrite into re-duced nitrogen fractions by green leaves, musttherefore be re-evaluated in view of the estab-lished instability of hyponitrite. The possiblerole, if any, of this compound in inorganic nitro-gen metabolism is therefore still in a highlyunsettled state.

D. Hydroxylamine Reductase

Yamagata (157) in 1939 was among the firstto note hydroxylamine reductase in bacteriawith the aid of an artificial dye as an electrondonor. Some 15 years later, further studies ofthe enzyme from bacteria by Egami's group (141)and Klausmeier and Bard (58) and from Neuro-spora and soybean leaves (84, 159) by Nasonand his colleagues were undertaken. The Neuro-spora hydroxylamine reductase was demonstratedto be a pyridine nucleotide-specific metalloflavo-protein which catalyzed the stoichiometric reduc-tion of hydroxylamine to ammonia (159), whereasthe corresponding pyridine nucleotide-specificmetalloprotein from soybean leaves showed aspecific requirement for Mn+ ions (84, 119).The properties of the latter system have alreadybeen discussed in Section III, A. Mn+ wasalso observed to be a highly specific activator forthe flavin-stimulated adaptive pyridine nucleo-tide-hydroxylamine reductase of Azotobacteralthough the product of hydroxylamine reduc-tion was not identified (131). Bulen (12) alsoindependently observed a particulate pyridine

nucleotide-hydroxylamine reductase in the sameorganism.

Reinvestigation of "ammonium dehydro-genase" in Bacillus subtilis extracts which werereported to catalyze the reversible reduction ofhydroxylamine by DPNH to ammonia, DPN,and water (Klausmeier and Bard (58)), showedthat the system had only hydroxylamine re-ductase activity similar to that of Neurospora(Zucker and Nason, (159)). Roussos, Takahashi,and Nason (120), although confirming the ob-servations of Klausmeier and Bard, found thatthe apparent enzymatic reduction of DPNH byNH40H resulted instead from an indirect pHeffect. The results were in keeping with thecalculated equilibrium constant of 1035 for hy-droxylamine reduction by DPNH, making itunlikely that the reverse reaction could be demon-strated.

Kono, Taniguchi, and Egami (62) purified asoluble, autoxidizable, and carbon monoxide-binding cytochrome from a halotolerant bac-terium. The pigment catalyzed the reductionof hydroxylamine by reduced methylene blue andwas stimulated by Mn+ . Ishimoto,. Yagi, andShiraki (46), using cell-free extracts of sulfate-reducing bacteria, showed a cytochrome require-ment for the reduction of hydroxylamine toammonia by hydrogen. Chemically reduced cvto-chrome also evolved hydrogen gas in the presenceof hydrogenase.

In continuing their earlier investigations(107, 126) on the reduction of hydroxylamine toammonia by molecular hydrogen in the presenceof cell-free preparations of Desulfovibrio desulfuri-cans, Senez and Pichinoty (124, 125) concludedthat hydrogenase was the only enzymatic factorin the process. They proposed that the hydroxyl-amine-reducing mechanism included reductionby hydrogenase of a natural electron carrier,cytochrome C3 (originally noted in this organismby Postgate (112, 113) as being involved in thereduction of sulfate and sulfite), which was non-enzymatically reoxidized by hydroxylamine with-out the intervention of a specific reductase.Although benzyl viologen could be substituted forthe cytochrome, it was a less efficient electroncarrier. Purified ferrocytochrome c from beefheart, like cytochrome C3, was nonenzymaticallyreoxidized by hydroxylamine but its rate ofoxidation was lower. On the basis of experimentswith other anaerobic and aerobic bacteria, theyobtained supporting evidence that the reduction

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of hydroxylamine by hydrogenase and a non-specific natural or artificial electron carrier wasnot confined to the sulfate-reducing bacteriabut could be effected by any organism possessinghydrogenase. DPN was apparently not involvedsince it was neither reduced enzymatically by H2in the presence of bacterial extracts, nor wasDPNH oxidized by hydroxylamine or cyto-chrome C3.The above property of certain heme proteins

to serve as electron donors or carriers in the"nonenzymatic" reduction of hydroxylamine

Electron donor -+(cytochrome b4 orreduced methyleneblue)

had already been observed by Raw (115) formammalian cytochrome and even earlier byColter and Quastel (22) for hemoglobin. Thelatter workers reported that hemoglobin underanaerobic conditions acted as an enzyme bring-ing about the reduction of hydroxylamine toammonia with the concomitant oxidation ofhemoglobin to methemoglobin. It also catalyzedthe reduction of hydroxylamine to ammoniaby cysteine or ascorbic acid. The reverse reactionwas also demonstrated, namely, the oxidation ofhydroxylamine to N2 by methemoglobin toyield hemoglobin. Heat treatment of hemoglobin,or the presence of cyanide (10-2 M) greatlydiminished its power to catalyze the breakdownof hydroxylamine. Hemin itself was far lesseffective than an equivalent quantity of hemo-globin in catalyzing the breakdown of hydroxyl-amine. The question as to whether the reactionbetween heme proteins and hydroxylamine isenzymatic or nonenzymatic is philosophical andnot readily answered.Kono and Taniguchi (61) purified and charac-

terized the hydroxylamine reductase of the de-nitrifying halotolerant Micrococcus strain 203using reduced methylene blue as the electrondonor. It is of some interest that this organismand other typical denitrifiers (e.g., Micrococcusdenitrificans and Pseudomonas denitrificans) pos-sess an active hydroxylamine reductase evenwhen strongly denitrifying.

NO3- -* NO2- -* N2 NH20H -+ NH3Denitrification Hydroxylamine

reductase

The 200-fold purified enzyme showed a typicalcytochrome c-type spectrum, was inhibited byseveral metal-binding agents, and contained bothiron and manganese. The inactivated, dialyzedenzyme was specifically restored by added Mn++which the authors believed experienced a possiblereversible change to manganic ions (Mn+§).The reduced enzyme also was reported to reactwith 02. On the basis of their experimentalresults they postulated the following electrontransport sequence for the hydroxylamine re-ductase of Micrococcus:

cytochrome C554 NH20HFe.++ Fe++ -lk (Mn+++ * Mn++)-A

,

sensitive to CN- 02or CO (in dark)

The hydroxylamine reductase of anotherdenitrifier, Pseudomonas denitrificans, was puri-fied about 50-fold by Walker and Nicholas (152)and shown to catalyze the reduction of hydroxyl-amine to ammonia using reduced dyes or flavinsas the electron source. DPNH, TPNH, or reducedcytochrome c did not serve as electron donors.The system appeared to be an FAD, manganeseenzyme possessing a cytochrome of the c type,although there was no evidence that the latterfunctioned in electron transfer during enzymeaction. The major difference between this hy-droxylamine reductase and that of Micrococcusappeared to be the absence of an FAD require-ment by the latter.Mager (67) has made the highly interesting

observation that the TPNH-specific hydroxyl-amine reductase and TPNH-specific sulfitereductase activities (both are FAD dependent)of E. coli may well be catalyzed by the sameenzyme. The facts that they followed a strictlyparallel pattern of quantitative response in termsof inhibition, denaturation, feed back repressionby cysteine, cystine or methionine, and stimula-tion of formation by serine, as well as an apparentcompetitive inhibition between the substrateshydroxylamine and sulfite, led him to this con-clusion. The Michaelis constant for sulfite wasmore than 100-fold lower than the correspondingKm for hydroxylamine, reflecting a large dif-ference in enzyme affinity for the respectivesubstrates. Mager is of the opinion that sulfitereduction represents the "true" physiologicalfunction, whereas reduction of hydroxylamine

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"constitutes an incidental capacity of the sameenzyme molecule." At most this can only be atentative conclusion. The curious substratespecificity of the enzyme for sulfite and hydroxyl-amine, as well as the inhibition of catalyticfunction by cyanide, led to the suggestion that afree carbonyl group is the "active site" of theenzyme.

IV. REDUCTION OF ORGANO-NITRO COMPOUNDS

The metabolic significance of the reductionof organo-nitro compounds by intact cells andpurified enzyme preparations is still obscure. Thepossibility that nitrate assimilation may proceedby way of an organic pathway, whereby aninorganic reduction product of nitrate other thanammonia is introduced into organic compoundsand further reduced, was postulated by McEl-roy and Spencer (72). The experiments of Silverand McElroy (128), using Neurospora mutantsblocked at different genetically controlled stepsin nitrite utilization, suggested a pathway leadingfrom hydroxylamine through pyridoxaloximephosphate and pyridoxamine phosphate to aminonitrogen. The mutants required pyridoxine inthe media for growth on nitrite but not on am-monia. They proposed that an alternative inor-ganic assimilative pathway via hydroxylamineand ammonia supplemented the organic route.

Nicholas et al. (89) subsequently reported thatthe pyridine nucleotide-nitrite reductase wasactivated in vitro in crude extracts of the pyri-doxine-requiring mutant of Neurospora by add-ing either pyridoxine, pyridoxal, or pyridoxalphosphate. Although they reported that thiseffect in vitro was not obtained with the puri-fied enzyme, they did not indicate whetherthe purified enzyme was prepared from the wild-type strain or the mutant. In contrast to themutant which required pyridoxine only whengrown on nitrite or nitrate (but not on ammonia)used by Silver and McElroy, the mutant em-ployed by Nicholas et al. also displayed a vitaminB6 requirement when grown on ammonia. Thelatter workers, in addition, indicated that theyfound no evidence for the enzymatic reduction ofoximes to amino acids by Neurospora.McElroy and Spencer (72) also considered that

the pyridine nucleotide-nitroaryl-reducing sys-tem might be utilized in the organic pathway.The characteristics of a number of enzymessuch as xanthine oxidase, diaphorase,, L-aminooxidase, hydrogenase, cytochrome c reductase,

several unidentified pyridine nucleotide-linkedflavoproteins and a number of molybdenum-dependent enzymes in catalyzing the reductionof various nitroaryl compounds (including picricacid, nitrofurazone, and chloramphenicol), nitro-prusside, and methemoglobin were discussed inearlier reviews (83, 86). The possibility was pre-sented that these compounds, like methyleneblue and indophenol, were simply nonspecificelectron acceptors for numerous enzymes, espe-cially for flavoprotein systems. Virtually no newwork has been reported in this area since 1957.Cain (13) recently isolated species of Nocardiaand Pseudomonas from soil and polluted streamswhich were capable of metabolizing nitrobenzoicacids. o- and p-Nitrobenzoate under aerobicconditions could provide the sole source of nitro-gen as well as carbon for two Nocardia species,resulting in a rapid initial ammonia production.The process of oxidation of both o- and p-nitro-benzoic acids was an adaptive one and wascompetitively inhibited by the m-isomer. Gun-derson and Jensen (36) had previously obtaineda strain of Corynebacterium simplex from the soilwhich could utilize the herbicide 4,6-dinitro-o-cresol as its sole source of carbon and nitrogen.More recently, Villanueva (147) prepared a 200-fold purified enzyme from a Nocardia specieswhich catalyzed the reduction of p-dinitrobenzeneby DPNH.

V. CELL-FREE NITRIFICATION

Important breakthroughs have taken placein our heretofore meager knowledge of the bio-chemical pathways and mechanisms of auto-trophic nitrification since this area was lastsummarized and evaluated 3 years ago by Nasonand Takahashi (86). The primary reason forthis unusual progress originated from the successof Aleem and Alexander (1) in growing adequatequantities of nitrifying organisms in pure culture.Thus it made available sufficient experimentalmaterial for cell-free studies.Aleem and Alexander (1) demonstrated for the

first time a nitrite-oxidizing system in cell-freeNitrobacter extracts and showed that it wasstimulated by the addition of iron and inhibitedby low concentrations of cyanide. Aleem andNason (2) subsequently found that the nitrite-oxidizing activity in cell-free preparations ofNitrobacter resided solely in a cytochrome-con-taining particle designated as nitrite oxidase.Their data implicated the action of the nitrite

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oxidase system to involve the enzymatic transferof electrons from nitrite to molecular oxygen viacvtochrome c- and cytochrome oxidase-like com-ponents according to the following sequence:

NO2- -_ cytochrome c -* cytochrome a, -*02

Although a specific requirement for iron could beconsistently demonstrated by means of thecyanide-dialysis procedure of Nicholas and Nason(90) the role of iron and its site of action in theabove electron transport chain has not yet beenestablished. Thus far it has not been possible toobserve a flavin component spectrophotometri-cally or to show a flavin requirement for thenitrite oxidase system. Aleem and Nason (3)also showed that partially purified nitrite oxidaseparticles catalyzed the formation of high energyphosphate bonds as ATP (when ADP was usedas the phosphate acceptor) concomitant with thespecific enzymatic oxidation of nitrite by molecu-lar oxygen. Inosine diphosphate and guanosinediphosphate served as phosphate acceptors inplace of ADP, whereas uridine diphosphate andcytidine diphosphate were ineffective. The highestP:0 ratios attained thus far with nitrite as theoxidizable substrate were about 0.2. It is quitepossible that this value may be substantiallyincreased as the system is further characterizedand more favorable conditions are found. Theelectron transport system mediating nitrite oxi-dation is sensitive to relatively high concentra-tions of such respiratory-chain inhibitors asantimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide. Dinitrophenol, thyroxine, and Dicu-marol, however, failed to uncouple the nitrite-specific oxidative phosphorylation. Malovolta,Delwiche, and Burge (69) also observed phos-phate esterification accompanying nitrite oxida-tion with crude cell-free extracts of Nitrobacterand indicated that the incorporation of C14-labeled CO2 into organic components resembledthat of photosynthesis. -More recently Aleemand Nason (unpublished data) partially purifiedand characterized a nitrite-cytochrome c re-ductase from Nitrobacter which was inhibitedby metal-binding agents and was apparentlydifferent from pyridine nucleotide-cytochrome creductase. The further transport of electronsfrom cytochrome c to 02 was catalyzed by thecytochrome oxidase present in the extracts.

Imshenetskii, Ruban, and their colleagues

reported in a series of papers some 5 years ago(43-45) their observations of cell-free oxidation ofammonia and hydroxylamine to nitrite by autoly-sates and filtrates of N\itrosomonas europea.Unfortunately, no precautions were indicated toeliminate unbroken cells or to avoid contamina-tion by other microorganisms, which was espe-cially likely in view of incubation periods rangingas long as 5 days at 37 C.

Nicholas and Jones (88) recently found thatcell-free extracts of the same species catalyzedthe oxidation of hydroxylamine but not of am-monia to nitrite, provided a suitable acceptorsuch as cytochrome c or phenazine methosulfatewas added. When cytochrome c was the acceptor,phosphate was required, but at higher concentra-tions it proved to be a competitive inhibitor withcytochrome c. Hydrazine when used in place ofhydroxylamine enzymatically reduced cyto-chrome c but no nitrite was produced. The enzymewas purified about 40-fold. Delwiche, Burge, andMalovolta (25) recently indicated that phosphateesterification accompanied hydroxylamine oxi-dation by cell-free extracts of Nitrosomonas.The recent observations by Creswell and He-

witt (23), with a partially purified enzyme fromextracts of marrow plants which catalyzed theoxidation of hydroxylamine, can probably bestbe ascribed to the \In-dependent peroxidasesystems elucidated by Kenten and Mann (52, 53).02 or H202 was required and horseradish peroxi-dase was found to replace the enzymatic com-ponent of the marrow leaf preparation, whenboiled leaf extract and manganese were alsopresent. The system is analogous to that pre-viously demonstrated by Heppel and Porterfield(37a) in which nitrite was oxidized to nitrate bythe peroxidase-like action of liver catalase.The area of nitrification has recently been

reviewed by Aleem and Nason (4). The impor-tant question as to how Nitrosomonas and Nitro-bacter cells obtain their reducing power, which ispresumably derived from ammonia and nitriteoxidation, respectively, is still untouched. If weassume that electrons provided by ammonia andnitrite ultimately reach the level of pyridinenucleotides or succinate, then energy must beprovided to make possible the transfer of theirelectrons "uphill" in view of the high positivepotential of these substrates (viz., ammonia andnitrite).

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VI. PROBABLE EVOLUTION OF INORGANICNITROGEN PATHWAYS

Several proposals have been recently madewith regard to the evolutionary development ofthe various pathways of inorganic nitrogenmetabolism and their significance in the economyof the organism (47, 129). At our present stageof knowledge they represent at best rationalspeculations based for the most part on currentbiochemical information. A number of importantquestions, however, remain unanswered.

According to the reasonable and well con-structed hypothesis of the origin and evolutionof life presented by Oparin (103), anaerobicheterotrophic organisms were the forerunners ofthe aerobic forms of life because the latter weredependent upon the accumulation of free oxygenin the atmosphere at the expense of photosyn-thesis. If one further assumes that the appearanceand functioning of porphyrin-conjugated pro-teins (e.g., cytochromes and chlorophylls) oc-curred not in the earlier periods of heterotrophdevelopment but just prior, relatively, to thedevelopment of the first photosynthetic organ-isms, then the following sequence in the evolu-tion of nitrate reduction seems reasonable.

oxidizing DPNH. The subsequent evolutionof light-sensitive pigmented organisms capable ofimparting a high reduction potential to the hy-drogen of water (and possibly to other substances)to produce reduced pyridine nucleotides presuma-bly accounts for the phenomenon of photo-chemical nitrate assimilation. The evolution ofcytochromes, presumably starting with a lowpotential cytochrome as exemplified by thecytochrome c3 of Desulfovibrio desulfuricans(112, 113), was probably succeeded by the de-velopment of cytochromes of higher and higherredox potential to yield nitrate respiratory elec-tron chains. This in turn could have easily evolvedinto an aerobic respiratory pathway in whichmolecular oxygen replaced nitrate. The chemo-autotrophs by virtue of their absolute require-ment for free oxygen (with the exception ofThiobacillus. denitrificans) and their allegedlyhighly advanced morphological characteristicsare presumed to have arisen later than thephotoautotrophs or photosynthetic forms (129).One of the major discrepancies of the above

evolutionary scheme has to do with the originof nitrate. If it is assumed that prior to the originand evolution of photosynthesis, nitrogen, like

assimilatoryFermentation -* nitrate reduction

appearance of porphyrin-protein conjugatescytochromes and chlorophylls

sulfate respiration photosynthesis and photochemicalnitrate assimilation

Vnitrate respiration -

aerobic respirationnitrification (and other chemoauto-

trophic processes)

In view of its lack of a cytochrome componentas well as its independence of molecular oxygen,the nitrate reduction pathway of the assimilatorytype seems to be quite primitive. In addition toproviding a means of reducing nitrate to theammonia level for the ultimate synthesis of pro-teins, it can be speculated that by virtue ofbeing a pyridine nucleotide-linked system, it maywell have served in facilitating fermentation by

many of the elements constituting the earth'senvironment, was in a reduced state as repre-sented by ammonia, we are unable to accountsatisfactorily for the presence of large quantitiesof nitrate without invoking molecular oxygen.How did nitrate arise in a reducing atmosphereunless of course it was derived after the evolutionof photosynthesis? Did nitrate respiration there-fore precede aerobic respiration? How do the

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actual biological energetics in terms of ATPformation compare between these two processes?These are some of the questions that are inneed of an answer.

VII. LITERATURE CITED

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2. ALEEM, M. I. H., AND A. NASON. 1959. Nitriteoxidase, a particulate cytochrome electrontransport system from 1itrobacter. Bio-chem. Biophys. Research Communs. 1:323-327.

3. ALEEM, M. I. H., AND A. NASON. 1960. Phos-phorylation coupled to nitrite oxidation byparticles from the chemoautotroph, Nitro-bacter agilis. Proc. Natl. Acad. Sci. U. S.46:763-769.

4. ALEEM, M. I. H., AND A. NASON. 1962. Meta-bolic pathways of bacterial nitrification.In Symposium on marine microbiology (inpress).

5. ALLEN, M. B., AND C. B. VAN NIEL. 1952.Experiments on bacterial denitrification. J.Bacteriol. 64:397-412.

6. ASANO, A. 1959. Studies on enzymic nitritereduction. I. Properties of the enzyme sys-tem involved in the process of nitrite re-duction. J. Biochem. (Tokyo) 46:781-790.

7. ASANO, A. 1959. Studies on enzymic nitritereduction. II. Separation of nitrite reduc-tase to particulate and soluble components.J. Biochem. (Tokyo) 46:1235-1242.

8. ASANO, A. 1960. Studies on enzymic nitritereduction. III. Effects of metal ions onsoluble and particulate components ofnitrite reductase. J. Biochem. (Tokyo)47:678-684.

9. ASNIS, R. E., V. G. VELY, AND M. C. GLICK.1956. Some enzymatic activities of a partic-ulate fraction from sonic lysates of Esch-erichia coli. J. Bacteriol. 72:314-319.

10. BAALSRUD, K., AND K. S. BAALSRUD. 1954.Thiobacillus denitrifipans. Arch. Mikrobiol.20:34-62.

11. BERNHEIM, F., AND M. DIXON. 1928. The re-duction of nitrates in animal tissues. Bio-chem. J. 22:125-134.

12. BULEN, W. A. 1956. Hydroxylamine reductasefrom Azotobacter vinelandii. Plant Physiol.31 :xxix.

13. CAIN-, R. B. 1958. Microbial metabolism ofnitro-aromatic compounds. J. Gen. Micro-biol. 19:1-141.

14. CANDELA, M. I., E. G. FISCHER, AND D. J.HEWITT. 1957. Molybdenum as a plant

nutrient. X. Some factors affecting theactivity of nitrate reductase in cauliflowersources grown with different nitrogensources and molybdenum levels in sand cul-ture. Plant Physiol. 32:280-288.

15. CHAUDHARY, M. T., T. G. G. WIISON, ANDE. R. ROBERTS. 1954. Studies in biologicalfixation of nitrogen. II. Inhibition in Azo-tobacter vinelandii by hyponitrous acid.Biochim. et Biophys. Acta 14:507-513.

16. CHENIAE, G. M., AND H. J. EVANS. 1956.Studies on "nodule nitrate reductase."Plant Physiol. 31 (suppl.), x.

17. CHENIAE, G. M., AND H. J. EVANS. 1957. Onthe relation between nitrogen fixation andnodule nitrate reductase of soybean rootnodules. Biochim. et Biophys. Acta 26:654-655.

18. CHENIAE, G. M., AND H. J. EVANS. 1959.Properties of a particulate nitrate reduc-tase from the nodules of the soybean plant.Biochim. et Biophys. Acta 35:140-153.

19. CHENIAE, G. M., AND H. J. EVANS. 1960.Physiological studies on nodule-nitrate re-ductase. Plant Physiol. 35:454-462.

20. CHUNG, C. W., AND V. A. NAJJAR. 1956. Co-factor requirements for enzymic denitrifi-cation I. Nitrite reductase. J. Biol. Chem.218:617-625.

21. CHUNG, C. W., AND V. A. NAJJAR. 1956. Co-factor requirements for enzymic denitrifi-cation. II. Nitric oxide reductase. J. Biol.Chem. 218:627-632.

22. COLTER, J. S., AND J. H. QUASTEL. 1950.Catalytic decomposition of hydroxyla-mine by hemoglobin. Arch. Biochem.27:368-389.

23. CRESWELL, C. F., AND E. J. HEWITT. 1960.Oxidation of hydroxylamine by plant en-zyme systems. Biochem. Biophys. ResearchCommuns. 3:544-548.

24. DELWICHE, C. C. 1956. Denitrification, ).233-259. In W. D. McElroy and B. H. Glass,[ed.], Symposium on inorganic nitrogenmetabolism. Johns Hopkins Press, Balti-more.

25. DELWICHE, C. C., W. D. BURGE, AND E.MALOVOLTA. 1961. Fractions of cell-freepreparations of Nitrosornonas europaea.Federation Proc. 20:350.

26. RENZO, E. C., E. KALEITA, P. G. HEYTLER,J. J. OLESON, B. L. HUTCHINGS, AND J. H.WILLIAMS. 1953. Identification of the xan-thine oxidase factor as molybdenum. Arch.Biochem. Biophys. 45:247-253.

27. EGAMI, F., Y. HAYASE, AND S. TANIGUCHI.1960. Tentative d'induction de la nitrate-reductase chez un mutant d 'Escherichia coli

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auxotrophe pour l'h6mine. Ann inst. Pas-teur 98:429-438.

28. ERKAMA, J., J. HXGGMAN, AND 0. WAHLROOS.1955. The role of iron in the nitrate-reduc-ing system of Escherichia coli. Ann. acad.Sci. Fennicae, (Ser. A) II, No. 60:460-470.

29. EVANS, H. J., AND N. S. HALL. 1955. Associa-tion of molybdenum with nitrate reductasefrom soybean leaves. Science 122:922-923.

30. EVANS, H. J., AND A. NASON. 1953. Pyridinenucleotide-nitrate reductase from extractsof higher plants. Plant Physiol. 28:233-254.

31. FARKAS-HIMSLEY, H., AND M. ARTMAN. 1957.Studies on nitrate reduction by Escherichiacoli. J. Bacteriol. 74:690-692.

32. FEWSON, C. A., AND D. J. D. NICHOLAS. 1961.Nitrate reductase from Pseudomonas aeru-

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33. FEWSON, C. A., AND D. J. D. NICHOLAS. 1960.Nitric oxide reductase from Pseudomonasaeruginosa. Biochem. J. 78:9P.

34. FEWSON, C. A., AND D. J. D. NICHOLAS. 1960.Utilization of nitrite oxide by microorgan-isms and higher plants. Nature 188:794-796.

35. FREAR, D. S., AND R. C. BURRELL. 1958. Theassimilation of N"5 from labelled hypo-nitrite by soybean leaves. Plant Physiol.33:105-109.

36. GUNDERSEN, K., AND H. L. JENSEN. 1956. Asoil bacterium decomposing organic nitro-compounds. Acta Agr. Scand. 6:100-114.

37. HAGEMAN, R. H., AND D. FLESHER. 1960.Nitrate reductase activity in corn seedlingsas affected by light and nitrate content ofnutrient media. Plant Physiol. 35:700-708.

37a. HEPPEL, L. A., AND V. T. PORTERFIELD.1949. Metabolism of inorganic nitrite andnitrate esters. I. The coupled oxidation ofnitrite by peroxide-forming systems andcatalase. J. Biol. Chem. 178:549-556.

38. HEREDIA, C. F., AND A. MEDINA. 1960. Ni-trate reductase and related enzymes inEscherichia coli. Biochem. J. 77:24-30.

39. HEWITT, D. J., AND M. M. R. K. AFRIDI.1959. Adaptive synthesis of nitrate reduc-tase in higher plants. Nature 183:57-58.

40. HIGASHI, T. 1960. Physiological study on theoxygen- and nitrate-respiration of Pseudo-monas aeruginosa. J. Biochem. (Tokyo)47:326-334.

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40b. HuzISIGE, H., AND K. SATOH. 1961. Photo-

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41. IIDA, K., AND S. TANIGUCHI. 1959. Studies onnitrate reductase system of Escherichiacoli. I. Particulate electron transport sys-tem to nitrate and its solubilization. J.Biochem. (Tokyo) 46:1041-1055.

42. IIDA, K., AND K. YAMASAKI. 1960. Spectro-graphic determination of molybdenum inthe nitrate reductase from Escherichia coli.Biochim. et Biophys. Acta 44:352-353.

43. IMSHENETSKII, A. A., E. L. RUBAN, AND 0. D.BUZINA. 1955. Noncellular nitrification.III. Kinetics of nitrite accumulation.Mikrobiologiya 24:539-544 (Chem. Abstr.50:8803e, 1956).

44. IMSHENETSKII, A. A., E. L. RUBAN, AND L. I.ARTEMOVA. 1956. Noncellular nitrificationIV. Inactivating thermophilic Nitrosomo-nas europaea enzymes which oxidize am-monia. Mikrobiologiya 25:12-15 (Chem.Abstr. 50:12146b, 1956).

45. IMSHENETSKII, A. A., AND E. L. RUBAN.1957. Ammonia oxidation by Nitrosomonasenzymes. Folia Biol. (Prague) 3:141-148(Chem. Abstr. 51:16270g, 1957).

46. ISHIMOTO, M., T. YAGI, AND M. SHIRAKI.1957. Sulfate-reducing bacteria. VIII. Thefunction of cytochrome of sulfate-reducingbacteria in decomposition of formate andreduction of sulfur and hydroxylamine. J.Biochem. (Tokyo) 44:707-714.

47. ISHIMOTO, M., AND F. EGAMI. 1959. Meaningof nitrate and sulphate reduction in theprocess of metabolic evolution, p. 555-561.In F. Clark and R. L. M. Synge, [ed.],International Union of Biochemistry Sym-posium Series. I. The origin of life on theearth. Pergamon Press, New York.

48. ITAGAKI, E., AND S. TANIGUCHI. 1959. Studieson nitrate reductase system of Escherichiacoli. II. Soluble nitrate reductase system ofaerobically grown cells in a synthetic me-dium. J. Biochem. (Tokyo) 45:1419-1436.

49. ITAGAKI, E., AND S. TANIGUCHI. (in prepa-ration).

50. IWASAKI, H. 1960. Studies on denitrification.IV. Participation of cytochromes in thedenitrification. J. Biochem. (Tokyo) 47:174-184.

51. IWASAKI, H., AND T. MORI. 1958. Studies ondenitrification. III. Enzymatic gas produc-tion by the reaction of nitrite with hy-droxylamine. J. Biochem. (Tokyo) 45:133-140.

52. KENTEN, R. H., AND P. J. G. MANN. 1952.

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The oxidation of manganese by enzymesystems. Biochem. J. 52:125-130.

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54. KESSLER, E. 1956. Reduction of nitrate withmolecular hydrogen in algae containinghydrogenase. Arch. Biochem. Biophys.62:241-242.

55. KESSLER, E. 1959. Reduction of nitrate bygreen algae. Symposia Soc. Exptl. Biol.13:87-105.

56. KESSLER, E., AND W. BUCKER. 1960. Uber dieWirkung von Arsenat auf NitratreduktionAtmung und Photosynthese von Grunal-gen. Planta 55:512-524.

57. KINSKY, S. C., AND W. D. McEL ROY. 1958.Neurospora nitrate reductase: The role ofphosphate, flavin and cytochrome c reduc-tase. Arch. Biochem. Biophys. 73:466-483.

58. KLAUSMEIER, R., AND R. BARD. 1954. Ammo-nium dehydrogenase. J. Bacteriol. 68:129-130.

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