AEROBIC NITROGEN FIXATION IN AZOTOBACTER VINELANDII
CENTRALE LANOBOUWCATALOGUS
0000 0086 9491 Ditproefschrif tme tstellinge nva n
HUBERTUSBONIFACIU SCHRISTOPHORU SMARI AHAAKE R doctorandus ind eChemie ,gebore no p2 4jul i 1946,t eBadhoevedorp ,i sgoedge - keurddoo rd epromoto rDr .C .Veeger ,hoogleraa r ind eBiochemie .
De Rector Magnifieus nan de Landbovwhogesahool, J.P.H.va nde rWan t
Wageningen, 17 deaembev 1976. : YVT) XO\ &7
H.B.C.M. Haaker Aerobic nitrogen fixation in Azotobacter vinelandii (with a summary in Dutch)
Proefschrift
terverkrijgin gva nd egraa d vandocto r ind eLandbouwwetenschappen , op gezagva nd eRecto rMagnificus , Dr. Ir.J.P.H .va nde rWant , hoogleraar ind eVirologie , inhe topenbaa r teverdedige n opwoensda g 16februar i197 7 desnamiddag st evie ruu ri nd eAul a vand eLandbouwhogeschoo l teWageninge n
l$n —/09///-& i These investigations were carried outunde r theauspice s of the Netherlands Foundation for Chemical Research (S.O.N.)wit h financial aid from the Netherlands Organization for theAdvancemen t ofPur eResearc h (Z.W.O.). /£////&>/ fa
STELLINGEN
i Zonderhe t tebeseffe n latenAppleb y et at. zien dat in Rhizobium bacteroiden het electronen transport naar denitrogenas e geremd wordt door eenontkoppelaar . Appleby,C.A. , Turner,G.L . and Macnicol,P.K . (1975) Biochim. Biophys.Acta ,387 ,461-474 .
2 Deuitspraa k dat denitrogenas e activiteit aanwezig ind ewortelknolletje sva n sojabonen gereguleerd wordt doorAT P alsmeded e energielading kan niet gedaan worden aan dehan d vand e gepresenteerde experimenten. Ching,T.M . (1976)Lif e Sciences 18,1071-1076 .
Deredoxpotentiaa l van Azotobaater vinelandii (4Fe-4S)„ ferredoxine Ii sori - danks de redoxpotentiaal metingen van Yoch enArno n en Sweeny et al.- nog steeds niet bepaald. Yoch,D.C . and Arnon,D.I . (1972)J . Biol. Chem. 247,'4514 - 4520. Sweeny,W.V. ,Rabinowitz ,J.C .an d Yoch,D.C . (1975)J . Biol. Chem. 250,7842-7847 .
4. Dedoo r Abramovitze n Massey aangedragen experimenten leveren onvoldoende bewijs voorhe tbestaa n van eencharge-transfe rcomple x tussen old yellow enzyme en fenolen. Abramovitz,A.S .an d Massey,V . (1976)J . Biol. Chem.251 , 5327-5336. 5 Deopvattin gva nEdmondso ne n Tollin,da td e ionisatie toestand van hetaa n fla- vodoxine gebonden flavine hydrochinon bepaalt ofvolledig e reductie van flavo- doxine kan worden verkregen ofniet ,i snie tbewezen . Edmonson,D .E .an d Tollin,G . (1971)Biochemistr y 10,133-145 .
6 Songheef t zich debeperkinge n van de gebruikte rekenmethodeo md e ligging van triplet niveaus te voorspellen niet gerealiseerd. Song,P.S . (1969)J . Phys. Chem. 72,536-542 .
7 Deverklarin g gegeven door Denairie et al. voorhe t feitda t adenine-behoeftige mutanten van Rhizobium zich niet tot stikstofbindende bacteroiden ontwikkelen, ishoogs t speculatief. Denarie,J. , Truchet,G . and Bergeron,B. : In:Symbioti c nitrogenfixation inplant s (P.S.Nutman ,ed. )47-6 1 Cambridge University Press,Cambridg e 1976.
Het invoeren van kabel-t.v. kan dedemocratiserin go pplaatselij knivea u sterk bevorderen enhe twar e gezicht van plaatselijke hoogwaardigheidsbekleders dui- delijk doen uitkomen.
9 Dehuidig e mogelijkheid rente onbeperkt aftrekbaar tedoe n zijn vanhe tbelast - baar inkomen isonrechtvaardi g enwerk t denivellerend.
Proefschriftva n H. Haaker
BIBLTOTHEEK nv'K LANDBOUW;-L ',) r ,ESCHOO L WAGEiNIMGEN Voor niets gaat de zon op.
Aan Ursula mijn ouders Maruscha Misoha Voorwoord
Hetwer kbeschreve n indi tproefschrif t isuitgevoer d inhe tLaboratoriu m voor Biochemieva nd eLandbouwhogeschoo l teWageninge ne nmogelij kgemaak tdoo r financielesteu nva nd eNederlands eOrganisati evoo r ZuiverWetenschappelij k Onderzoek (Z.W.O.). Iedereen zalbegrijpe nda t zonderd edirect ehul pva nvele n iknie tin - staat zouzij ngewees tdi tproefschrif tvoor t tebrengen . Omhe trisic o tevermijden ,da t ik iemand zouvergete n tenoemen ,wi l ik volstaanme tbi jdez e iedereenhartelij k tebedanken ,di eheef tbijgedrage n tot deverwezenlijkin gva ndi tproefschrift . Voorwoord
Hetwer kbeschreve n indi tproefschrif t isuitgevoer d inhe tLaboratoriu m voor Biochemieva nd eLandbouwhogeschoo l teWageninge ne nmogelij k gemaaktdoo r financiele steunva nd eNederlands eOrganisati e voor ZuiverWetenschappelij k Onderzoek (Z.W.O.). Iedereenza lbegrijpe nda t zonderd edirect ehul pva nvele n iknie tin - staat zou zijngewees tdi tproefschrif tvoor t tebrengen . Omhe trisic o tevermijden ,da t ikieman d zouvergete nt enoemen ,wi l ik volstaanme tbij dez e iedereenhartelij k tebedanken ,di eheef tbijgedrage n tot deverwezenlijkin gva ndi tproefschrift . Contents
Listo fabbreviation s 10 Listo fenzyme s 11 Introduction 13
I Regulationo fdinitroge nfixatio ni nintac t Azotobaater vinelandv 21
II Regulationo frespiratio nan dnitroge nfixatio ni ndifferen ttyp e 35 of Azotobaater vinelandii
III Aerobicnitroge nfixatio ni n Azotobaater vinelandii 45
IV Involvement ofth ecytoplasmi cmembran ei nnitroge nfixatio nb y 61 Azotobaater vinelandii
Discussion 81 Summary 91 Samenvatting 93 List of abbreviations
ADP adenosine 5'-diphosphate AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate CoA coenzymeA DEAE diethylaminoethyl EDTA ethylene-diaminetetra-acetate NAD+ oxidized nicotinamide-adenine dinucleotide NADH reduced nicotinamide-adenine dinulceotide NADP+ oxidized nicotiamide-adenine dinucleotide phosphate NADPH reduced nicotiamide-adenine dinucleotide phosphate P phosphate residues Tes N-tris[hydroxymethyl jmethyl-2-amin o ethane TPP thiamine pyrophosphate tricine N-[tris(hydroxymethyl)-methyT)-glyci n tris trisfhydroxymeth yll-aminom ethan e
10 List of enzymes
In this thesis thenon-systemati c names of enzymes areused . This list includes the trivial and the systematic names of the enzymes;als o is included the enzyme number according to theRepor t of the Commission for Enzymes of the International Union of Biochemistry.
EC number Systematic name Trivial name
3.6.1.3 ATP phosphohydrolase ATPase 4.1.2.14 3-deoxy-2-keto-6-phosphogluconate glyceraldehyde-3-phosphate-lyase 1.1.3.4 S-D-glucose: oxygen 1-oxido- glucose oxidase reductase 1.1.1.49 D-glucose-6-phosphate:NAD P glucose-6-phosphatedehydrogenas e 1-oxidoreductase 1.11.1.6 hydrogen-peroxide: hydrogen catalase peroxide oxidoreductase 1.1.1.44 6-phospho-D-gluconate:NAD P 6-phosphogluconate 2-oxidoreductase dehydrogenase (decarboxylating) 4.2.1.12 6-phosphogluconatehydro-lyas e 1.2.4.1 pyruvate: lipoateoxido pyruvate dehydrogenase reductase complex (acceptor acylating) 1.6.1.1 Reduced-NADP+: NAD+ oxido NAD(P) transhydrogenase reductase nitrogenase
11 Introduction
GENERAL INFORMATIONABOU TNITROGE N FIXATION
Thereductio no fmolecula rnitroge nt oammoni aha slon gbee nknow nt ob ea propertyo fmicroorganism spresen ti nth eroo tnodule so flegumenes . Hellriegel& Withfart h (1) wereth efirs tone st odemonstrat ebiologica l nitrogen fixationbac ki n1880 ,bu ti nth efollowin g6 0year s onlyfou r organismswer e shownt ocarr you tthi sreaction :th efree-livin gbacteri a Azotobaatev sppan d Clostridium spp; theobligator ysymbioti c Rhizobium spp- legumeassociation san dth eblue-gree nalga e Nostoa. Inth epas t2 0year s thereha sbee na grea tupsurg eo fresearc h intoman y aspectso fnitroge n fixation,a chang etha tha sbee ndu elargel yt oth edevelopmen to fne wmethod s formeasurin gth eprocess .I nth eearl ystudies ,nitroge nreductio nwa sdetect edb yusin gth einsensitiv eKjehldah lmetho dt omeasur eth etota lnitroge n contento fmicroorganism so rplant sbefor ean dafte rgrowt hi nmedi ai nwhic h thesol esourc eo fnitroge nwa smolecula rnitrogen .Th eintroductio nb yBurri s andMille ri n194 2o fisotopi cmethod sprovide da mor esensitiv ean dconvenien t wayo ffollowin gth ereaction .Wor ko nth ebiochemistr yo fth eproces sstart ed,followin gth edemonstratio ni n196 0tha tcell-fre eextract so f Clostridium pasteurianum produceammoniu m ions fromnitroge n (2).
The enzymeresponsibl efo rth ecatalysi so fnitroge nreductio nt oammoni aha s beenterme dnitrogenase .Th ediscover y thatth eenzym ewil l alsoreduc e acetylenet oethylene ,a compoun dwhic hca nrapidl yan dsensitivel y determined byga schromatographi c techniques,ushere di nth eexplosio no fresearc ho n nitrogenfixatio ntha texist sa tth epresen t time.A historica lrevie wo fth e developmentsi nth efiel dbetwee n192 0an d197 0ha sbee nwritte nb yBurris(3) .
Nitrogenfixatio nresearc hca nroughl yb edevide d intoth efollowin gdirec tions.Recen treviews ,book so rarticle sar egiven . a.biolog y (6,7) b.molecula rbiolog y (8-10) c.enzymolog y (11-15) d.physiolog y (14-17) e.chemica lnitroge nfixatio n (14,15,18,19)
13 INTRODUCTIONT OAEROBI CNITROGE NFIXATIO N
Themai nai mo fthi sthesi si st odescrib eho wobligat eaerobe ssuc ha s Azotobacter vinelandii, areabl et ocarr you tth ever yreductiv eproces so f nitrogenfixatio ni na naerobi cenvironment .Th eproble mi sdivide dint o twoparts :1 )Generatio no freducin gequivalent sfo rnitrogenase ; 2)protectio no fnitrogenas eagains tinactivatio nb yoxygen .
1.GENERATIO NO FREDUCIN GEQUIVALENT S
Inanaerobi cnitrogen-fixin gorganism sreduce dferredoxi ni sth ephysiologica l electrondono rfo rnitrogenase .Ferredoxi nca nb ereduce di ndifferen tways : bypyruvat ei nth eso-calle dphosphoroclasti creaction ,a nactivit ywhic h hasbee ndetecte di n Clostridium pasteurianum (20), Bacillus polymyxaffl), Klebsiella pneumoniae(22); andb yothe rstron greducin gagent ssuc ha sF L orformat e (22,23).
Ferredoxini sals oth eelectro ndono rfo rnitrogenas ei nphotosyntheti c bacteriaan dblue-gree nalgae ,th eferredoxi ni ntur nbein greduce di na ligh t reactionwit hphotosyste mI o rb ypyruvat ean dpyruvat edehydrogenase .I twa s showntha tNADP Hvi aferredoxin:NAD P oxidoreductase,whic henzym ewa sdemon stratedt ob epresen ti nthes eorganism san dferredoxi nca nac ta sa nelectro n donorfo rnitrogenas e (24-28).
Althoughlo wpotentia lelectro ncarrier swhic hwil lreac twit hnitrogenas e havebee nisolate dfro maerobi corganism s (20,29),th ephysiologica lelectro n donoran dth eultimat esourc eo freducin gequivalent sfo rN ,reductio ni nsuc h organismsa s Azotobacter spp, Mycobacterium flavum and Rhizobium sppi sstil l unknown.Ferredoxin shav ebee nisolate dfro m Azotobacter (31), Rhizobium bacteriods (32)an d Mycobacterium flavum. Thelo wpotentia lflavoprotei n electroncarrier ,flavodoxin ,ha sbee nisolate dfro m Azotobacter (34,35)an d Rhizobium bacteriods (36,37).Bot htype so fcarrier swer efoun dt omediat eth e transfero felectron sfro milluminate dspinac hchloroplast st oa nitrogenas e preparation.Muc hlowe ractivitie sar efoun dwhe nth ereducin gequivalent s areobtaine dfro moxidizabl eorgani ccompounds ,suc ha sglucose-6-phosphat e orisocitrat ean dcouple dt onitrogenas ethroug hNAD P, spinac hferredoxin - NADP oxidoreductasean dferredoxin .
Enzymeswhic hcoupl eth eoxidatio no fpyruvat eo rNAD(P) Ht oth ereductio n offerredoxi no rflavodoxi nhav eno tye tbee ndetecte di nobligat eaerobi c nitrogen-fixingorganisms .Nevertheles sa nelectro ntranspor tchai nt o
14 nitrogenase in Azotobaoter vinelandii or Rhizobium bacteriodsha sbee n postulated (29,30). Itwa spropose d thata hig hNADPH/NAD P ratioprovide s thereducin gpowe ran d thatreducin gequivalent s fromNADP Har etransfere d tonitrogenas evi aa heat-labil efactor ,ferredoxi nan dflavodoxi n (29).I n paper Ihoweve r itwil lb eshow ntha t inintac t Azotobaoter vinelandii electrontranspor tt onitrogenas e isregulate dno tb y theNAD(P)H/NAD(P ) ratiobu tb y theenergize d stateo fth ecytoplasmi cmembrane .Thi s conclusion suggests thatth eelectro ntranspor tchai nt oth enitrogenas ema yb elocaliz edwithi nth ecytoplasmi cmembrane . Inpape r IIIi twil lb e showntha ta n intactcytoplasmi cmembran e isnecessary ,fo rcouplin gbetwee npyruvat e oxi dationan dnitrogenas eactivity . Inadditio ni twil lb e shown,tha treduce d flavodoxinca nac ta sa reductan tfo rnitrogenas e and thatreduce d ferredoxin cannot.Furthermor e itwil lb eshow ntha tflavodoxi n inintac tstructure s from thecel li sno tsoluble ,bu t ismore-or-les sburie dwithi nth emembrane .
Inpape r IVth epresenc eo fa membrane-boun dNAD(P)H : flavodoxinoxidoreduc - tasei sdemonstrated . Intha tpape r therol eo fth ecytoplasmi cmembran e in electrondonatio nt onitrogenas eo f Azotobaoter issummarized .
2.OXYGE NPROTECTIO N OFNITROGENAS E
Nitrogenfixatio ni sa nanaerobi cprocess ;th eargument s forthi spostulat e arereviewe db yYate san dJone s (38).Fo raerobicall ynitrogen-fixin g organisms severalprotectio nmechanism s againstoxyge nhav ebee nproposed .
1. Innodule s oflegumes ,leghemoglobi nplay s animportan trol e innitroge n fixation,a .Leghemoglobi nha sa hig haffinit y foroxyge n (39).Thi s facilitates ahig h fluxo foxygen ,a ta lowconcentration ,t oth ebac - teroids.b .Oxyleghemoglobi nincrease s theefficienc yo foxidativ ephos phorylationi nintac tbacteroid s (40,41).
2. Mostblue-gree nalga ewhic hfi xN „ inai rposses scharacteristi ccells , calledheterocysts .I ti sthough t thatthi s typeo fcel lprotect s the nitrogenase againstoxyge ni nth efollowin gways .a .Th e thick-wall ofth e heterocystspresent sa physica lbarrie r foroxyge n (42).b .Th eabsenc e ofth eoxygen-evolvin gphotosyste m IIi nheterocyst s (43),togethe rwit h ahig hrespirator y activity inth eheterocys t (44)ma yai d inkeepin g theoxyge nconcentratio nlow .
15 3. Freelivin gbacteri awhic h fixN 2 inai rals ohav eprotectin gmechanism s againstoxygen .Slim eproductio nma ypla ya rol e inreducin g the diffusiono fC L toth einsid eo fth ebacteri a (45,46). Inadditio n respiratory aswel l as conformationalprotectio nmechanism shav ebee n proposed(47) . a.RESPIRATIO N PROTECTION
Itwa s observed thatwhe n Azotobaotev was growna ta lowoxyge n input,th e cellswer eno tabl e tofi xnitroge n immediately aftera nincreas eo fth e oxygeninput .Followin ga perio do fadaptation ,however ,i nwhic hth e oxidationvelocit y increased,th ecell swer eabl e tofi xnitrogen .Thi s phenomenonwa scalle drespiratio nprotectio no fnitrogenase .Besid eth e influenceo foxyge ndurin ggrowt ho nth ecapacit y tofi xnitroge na ta hig h oxygeninput ,othe reffect so foxyge no nth ephysiolog yo f Azotobaotev are known.Whe n Azotobaotev cellsar eswitche d from lowt ohig h aeration,th e followingphenomeno nar eobserved .
1. Thewhol ecel lrespiratio n increases (48,49). 2. Therati oN ~ fixed/carbonsourc eoxidize ddecrease s(48) . 3. Thecompositio no fth erespirator ychai nchange s togiv ehighe r levels ofcytochrome s andNAD(P) Hoxidas eactivit y (48,49). 4. Theoxidativ ephosphorylatio nefficienc ya tsit e Ian d IIIdecrease s(49) .
Thesedat awer euse d inproposal s toclarif y thenatur eo frespiratio n protection in Azotobaotev (38,49,50).Unde rhighl y aerobiccondition snitro genase isinhibite dan drespiratio n ispromote dvi aa nelectro npathwa yt o oxygentha tha s alo wphosphorylatio nefficiency .Thes eevent s causea los s ofrespirator y controlassociate dwit h theNAD Hoxidatio nan dproduc ea decreased ratioo fATP/ADP.P . inwhol ecells .Sinc eke yenzyme s in A.vinelan- dii catabolismar estimulate db yAD Pan dAM Po r inhibitedb yATP ,th e expected lower intracellular energycharg ewoul d lead toth eobserve d increase inwhol ecel lrespiration .
Paper IIdeal smainl ywit h thequestio no frespiratio nprotection . Intha t paper itwil lb eshow ntha tth epropose dhypothesi s (50)i sno tcorrect . Thecompositio no fth erespirator y chaind ono tsho wa correlatio nwit h the intracellular energycharge .I ti sals opossibl e tosho w that Azotobaotev has respirationprotectio neve nwhe n therespirator ymembrane s areno tadapte d tohig hoxyge nconcentrations .Th eexperiment s indicate thatth eincreas eo f
16 wholecel lrespiratio n inincrease d oxygeninpu tdurin g growth isdu emainl y toa n increase insuga ruptak e andno tdu e toa chang e in intracellular energy charge or therati oo fNAD(P)H/NAD(P ).
b. CONFORMATIONAL PROTECTION
Itha s longbee nknow n thatnitrogenas e incrud eextract s of Azotobaater isoxygen-toleran twherea s incrud e extracts fromanaerobi cnitroge nfixers , the enzyme isoxygen-sensitiv e (51,52).Al lpurifie d nitrogenases,tha thav e been tested sofar,ar eoxyge nsensitive .Yate s (53)showe d thatDEAE-cellulose - purified nitrogenasebecome s oxygen sensitive,an dshowe d further thata crude preparationwit hNAD Hdehydrogenas e activity,ca nrestor e the oxygen stability ofpurifie d enzyme.Oppenhei me tal . (54)showe d that nitrogenase obtained from A. vinelandi-L by anosmiti cshoc kwa s freefro mmembrane swit h NADHoxidas e activity andmor eoxygen-sensitiv e thannitrogenas e obtained by disrupting thecell swit ha Frenc hpressur e cell.Thei rresult s suggest that thecytoplasmi cmembran e isi nsom ewa y concernedwit h the oxygen protection.Pape r IIIpresent s resultswhic h indicate thatnitrogenas e in Azotobaater isalway sprotecte d against oxygen,independen t onth ecel l rupturemethod .Purificatio no fth enitrogenas e complextil lDEA E cellulose chromatography gives anoxygen-stabl e nitrogenase complex. FollowingDEAE - cellulose-treatmentnitrogenas e isfoun d tob e oxygen-sensitive and it canno tb eprotecte d againstoxyge nb y cytoplasmicmembrane swhic hhav e highNAD Hdehydrogenas e activity.Pape r IVshow s thatth e rateo fsedimen tationo fnitrogenas e isindependen t of thecel lruptur emetho d and is the same asth esolubl epyruvat e dehydrogenase complex. Iti sals o demonstrated that aFe- Sprotei n stabilizeswithi n thecrud enitrogenas e complex against oxygen inactivation.
17 LITERATURE
1.Hellriegel ,H . andWilfarth ,H . (1888)Z.Ver.Riiberzucker-Industri e DeutschenReichs . 2. Carnahan,J.E. ,Mortenson ,L.E. ,Mower ,H.F . and Castle,I.E . (1960) Biochim.Biophys.Acta,4-4 ,520-525 . 3.Burris ,R.H . (1974)Plan t Physiol.54 ,443-449 . 4.Dilworth ,M.J . (1966)Biochim.Biophys.Act a 127,285-294 . 5.Burns ,R.C . andHardy ,R.W.F . (1975)Nitroge n fixation inBacteri a and higher Plants,Berlin ,Ne wYork ,Springer . 6. Quispel,A . ed. (1974)Th ebiolog y of nitrogen fixation,Amsterdam , NewYork ,North-Holland . 7. Stewart,W.P.P . ed. (1975)Nitroge n fixation byFree-livin g organisms, Cambridge Univ.press . 8. Shanmugan,K.T. ,Valentine ,R.C . (1975)Scienc eJ_87 ,919-924 . 9. Brill,W.J . (1975)Ann.Rev.Microbiol . 29,109-129 . 10. Cannon,F.C. ,Dixon ,R.A . and Postgate,J.R . (1976)J.Gen.Microbiol .93 , 111-125. 11.Dalton ,H . andMortenson ,L.E . (1972)Bact.Rev . 36,231-260 . 12. Zumft,W.G . andMortenson ,L.E . (1972)Biochim.Biophys.Act a 416, 1-52. 13.Winter ,H.C . and Burris,R.H . (1976)Ann.Rev.Biochem . 45,409-426 . 14. Proc. of I International symposium onN „ fixation,Pullman,Wash . Wash.StateUniv.press . 1975. 15.Proc . of II International symposium onN „ fixation,Salamanca , Spain,London, Academic Press. 1976. 16. Streicher,S.L . andValentine ,R.C . (1973)Ann.Rev.Biochem . 42,279-30 2 17.Dilworth ,M.J . (1974)Ann.Rev.Plan t Physiol.25 ,81-114 . 18. Schrauzer,G . (1975)Angew.Chem .J_4 ,514-522 . 19. Schrauzer,G.N. , Robinson,P.R. , Moorehead,E.L . and Vickrey,T.M . (1976) J.Am. Chem.Soc. 98,2815-2820 . 20.Mortenson ,L.E. ,Valentine ,R.C , Carnahan,J.E . (1963)J.Biol.Chem .238 , 794-800. 21. Shethna,Y.I. , Stombaugh,N.A . and Burris,R.H . (1971)Biochem.Biophys . Res.Commun. 42,1108-1116 . 22. Fisher,R.J . andWilson ,P.W . (1970)Biochem.J ._1_T7 >1023-1024 . 23. Mortenson,L.E . (1966)Biochim.Biophys.Acta , 127,18-25 . 24. Smith,R.V. ,Noy ,R.J . andEvans ,M.C.W . (1971)Biochim.Biophys.Act a253 , 104-109. 25. Tel-Or,E .an d Stewart,W.D.P . (1976)Biochim.Biophys.Act a43 ,189-195 . 26. Leach,C.K . and Carr,N.G . (1971)Biochim.Biophys.Act a 245,165-174 . 27. Bothe,H. ,Falkenberg , B. andNolteernsting ,K . (1974)Arch.Microbiol . 96, 291-304. 28. Bothe,H . (1970)Ber.Deut.Bot.Ges . 83,421-430 . 29. Benemann,J.R. , Yoch,D.C. ,Valentine ,R.C . and Arnon,D.I . (1971) Biochim.Biophys.Acta226 ,205-212 . 30.Wong ,P. ,Evans ,H.J. , Klucas,R . and Rusell,S . (1971)Plan t and Soil specialvolume ,525-543 . 31.Yoch ,D.C. , Benemann,J.R. , Valentine,R.C . andArnon ,D.I . (1969)Proc . Nat.Acad.Sci. 64,1404-1410 . 32. Yoch,D.C. , Benemann,J.R. , Arnon,D.I. ,Valentine ,R.C . and Russell, S.A. (1970)Biochem.Biophys.Res.Commun . 38,838-842 . 33. Bothe,H . andYates ,M.G . (1976)Arch.Microbiol . \0]_, 25-31. 34. Benemann,J.R. , Yoch,D.C .,Valentine ,R.C .an d Arnon,D.I . (1969) Proc.Nat.Acad.Sci. 64,1079-1086 . 35.Yates ,M.G . (1972)FEB S Letters, 2]_,63-67 . 36. Koch,B. ,Wong ,P. , Russell,S.A . andHoward ,R . (1970)Biochem.J . 118, 773-781. 37. Philips,D.A. , Daniel,R.M. ,Appleby ,C.A . and Evans,H.J . (1973)Plan t Physiol. 51_,136-138 . 38.Yates ,M.G . and Jones,C.W . (1974)Adv.Microbio l.Physiol .j_l_ ,97-135 . 39.Wittenberg ,J.B. ,Appleby ,C.A . andWittenberg ,B.A . (1972)J.Biol.Chem . 247,527-531 . 40.Appleby ,G.A. , Turner,G.L . andMacnicol ,P.K . (1975)Biochim.Biophys . Acta 387,461-474 . 41. Bergensen,F.J . and Turner,G.L . (1975)J.Gen.Microbiol . 9j_,345-354 . 42.Wolk ,C.P . (1973)Bacteriol.Rev . 37,32-101 . 43. Carr,N.G . and Bradley,S . (1973)Symp.Soc.Gen.Microbiol . 23,161-188 . 44. Fay,P . andWalsby ,A.E . (1966)Natur e 209,94-95 . 45. Hill,S . (1971)J.Gen.Microbiol . 67,77-83 .
19 46.Raju ,P.N. , Evans,H.J . and Seidler,R.J . (1972)Proc.Nat.Acad.Sci . 69, 3474-3478. 47. Dalton,H . and Postgate,J.R . (1969)J.Gen.Microbiol . 54,463-473 . 48. Drozd,J . andPostgate ,J.R . (1970)J.Gen.Microbiolog . 63,63-73 . 49. Jones,C.W. ,Brice ,J.M. , Whight,V . andAckrell ,B.A.C . (1973)FEB S Letters 29,77-81 . 50.Ackrell ,B.A.C . and Jones,C.W . (1971)Eur.J.Biochem . 20,29-35 . 51. Bulen,W.A . andL e Comte, J.R. (1966)Proc.Nat.Acad.Sc i56 ,979-985 . 52. Kelly,M . (1969)Biochim.Biophys.Act a 191,527-540 . 53.Yates ,M.G . (1970)FEB SLetter s 8,281-285 . 54. Oppenheim,J. , Fisher,R.J. ,Wilson ,P.W . andMarcus ,L . (1970) J.Bacteriol. 101,292-296 .
20 I Regulation of dinitrogen fixation in intact Azotobacter vinelandii
HUUB HAAK.ER, ARIE DE KOK and CEES VEEGER Departmentof Biochemistry, Agricultural University, DeDreijen 11, Wageningen (The Netherlands) (Received April 1st, 1974)
SUMMARY
1. In intact Azotobactervinelandii the influence of oxygen on the levels of oxidized nicotinamide adenine dinucleotides and adenine nucleotides in relation to nitrogenase activity was investigated. 2. The hypothesis that a high (NADH+NADPH)/(NAD++NADP+) is the driving force for the transport of reducing equivalents to nitrogenase in intact A. vinelandiiwa sfoun d to beinvalid .O nth econtrary , with adecreasin g ratio ofreduce d to oxidized pyridine nucleotides,th e nitrogenase activity of the wholecell sincreases . 3. By measuring oxidative phosphorylation and using 9-amino acridine as a fluorescent probe, it could be demonstrated that respiration-coupled transport of reducing equivalents to the nitrogenase requires a high energy level of the plasma membrane or possibly coupled to it, a high pH gradient over the cytoplasmic mem brane.Furthermor e nitrogenfixation i scontrolle d byth e presence of oxygenan d the ATP/ADP ratio.
INTRODUCTION
Since Mortenson [1] showed how reducing equivalents are transfered to the nitrogenase of Clostridium pasteurianum, it isgenerall y accepted that all anaerobic or photosyntheticnitrogen-fixin g organismsus ereduce dferredoxi n aselectro n donor for dinitrogen reduction. In these organisms ferredoxin is reduced by respectively the phosphoroclastic and photoreceptor systems. Since that time many investigators searched for the physiological electron donor in aerobic dinitrogen fixers [2-4]. In the current concept, proposed by Benemann et al. [5, 6], a high NADPH/NADP+ ratioi sth edrivin gforc efo r thetransfe r ofelectron sthroug h anelectro ncarrie rchain , thus obtaining the low-potential donor system needed to reduce Component II of thenitrogenase .Beneman ne tal . [5]i nthei rexperiment suse dcell-fre e extractssupple mented with isolated electron carriers. Thisproposa l wasteste di nintac t bacteria bymeasurin g theamount so f NAD+
Abbreviations:TTFB ,4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol ; HQNO,2-heptyl-4 - hydroxyquinoline-7V-oxide.
21 and NADP+ in relation to the nitrogenase activity under a variety of conditions. The relation between nitrogenase activity and energy level of the cytoplasmic membrane was simultaneously studied.
METHODS
Growth conditions of the bacteria Azotobacter vinelandii ATCC Strain OP was grown on a Burk's nitrogen-free basic salt medium [7].Th e sugar oxidation of Azotobacter depends strongly upon the oxygen input during growth [8]. During this investigation bacteria grown in batch cultures were used. The cultures became oxygen-limited during growth. Bacteria grown oxygen-limited had a low rate of sucrose oxidation (0.1-0.5 |/moIes oxygen • rnin-1 •mg -1 protein), but a high rate of acetate oxidation (1//mole oxygen •rnin -' •mg ~* protein). These cells, refered to as "oxygen-limited" cells were not able to reduce dinitrogen with oxygen present in the medium, with sucrose as carbon source. This type of cell can be converted into "oxygen-adapted" cells by exposing them to oxygen in the medium for a period of 3 h. This was done with sucrose as carbon source, in a New Brunswick Type C30 growth vessel, supplied with a standard New Brunswick oxygen probe. During the adaptation the sulphite oxidation rate was 110mmole s oxygen •1 _1 h~' at a bacterial protein concentra tion of 0.5 mg •ml -1. "Oxygen-adapted" cells had a rate of sucrose oxidation of 0.8-1.3 /imoles oxygen • rnin-1 •mg -1 protein (cf. ref. 9). The cells were harvested at 20 °C and then washed twice with distilled water; the cells were suspended in water at a protein concentration of 30-60 mg/ml. Each batch was tested for sucrose oxidation and dinitrogen reduction activity. The maxi mum oxidation velocity of a cell suspension and the influence of additions on the oxygen consumption were measured with an oscillating platinum electrode from Aminco Instruments Company. Fn all experiments the temperature was 24 °C.
Nitrogenase activity assay The standard nitrogenase activity incubation mixture contained: 25 mM Tris-HCl, 5m M phosphate, 2 mM EDTA, final pH 7.6. At / = 0 the cells were added to the standard incubation mixture in the cuvette and after 2 min 3 mM MgCl2. A closed gas phase consisting of 70 %argon , 10% acetylen e and 20 % oxygen was pumped with an adjustable airpump through the incubation mixture. When necessary, the oxygen and argon concentrations were varied. The total volume of the gas phase varied between 100 and 160 ml, in order to limit the decline in oxygen consumption of the gas phase to less than 1 %. The oxygen concentration in the incubation medium was measured with a standard galvanic oxygen electrode and oxygen analyser of the New Brunswick Scientific Company. In all experiments the oxygen concentration in the medium was measured. At suitable timeinterval s aliquots were removed from the gas phase to analyze for acetylene reduction with a Pye 104 gas chromatograph on a porapack R column.
Oxygen input assay The oxygen input into the reaction cuvette was either determined according to Cooper [10]o r calculated from the oxidation timeo f a known amount of dithionite.
22 For this determination the reaction cuvette contained a mixture of 25 mM Tris-HCl (pH 7.6),0. 1 mM benzylviologen, 40//g/m l catalase (Boehringer). The volume varied between 4 and 10ml . A known amount of neutralized dithionite was added, and the benzylviologen coloured the solution blue. The gas was bubbled through the solution tillth e bluecolou r disappeared. Then a further known amount ofdithionit e was added and the time of de-colourization measured. It was checked that in the ranges used, the oxygen input was linear with the partial oxygen tension in the gas phase and the gas flow through the cuvette. Determination of cofactors The cells were fixed by rapidly adding HC104 to the incubation mixture up to a final concentration of 4 % (w/v). After 10mi n at 0 °C the samples were neutralized + with solid KHC03 and stored at -20 °C.Th e levels of ATP, ADP, AMP, NAD and NADP+ were determined according to Williamson and Corkey [11]. Control experi ments show that no non-enzymatic breakdown occurs under these conditions (less than 5 %). Within 3 h NAD+ and NADP+ were determined; ATP, ADP and AMP within 30 h. Enzymatic assays were performed with an Aminco-Chance dual wave length spectrophotometer. Fluorescenceassays The total reduced pyridine nucleotide fluorescence was measured by placing the cuvette in an Eppendorf fluorimeter (filters: excitation, 313 and 366 nm; emission, an interference filter of 460 nm). By measuring the excitation and emission spectra of the oxidized and reduced cells, it was checked that the fluorescence changes were due to reduced pyridine nucleotides. In these experiments the oxygen input was changed by variation of the oxygen tension in the gas phase by a constant gas flow through the incubation mixture. The 9-aminoacridine fluorescence was measured with a primary filter 405+436 nm and a secondary filter of 500—3000 nm. Because the fluorescence of oxidized flavoproteins (transhydrogenase, lipoamide dehydrogenase) was also detected with the filter combination used, the changes in the bacteriological flavin fluorescence were registrated by experiments without the dye 9-aminoacridine. Where necessary the fluorescence emission was corrected as indicated. The fluorescence emission spectrum of 9-aminoacridine in the energized system was identical with that of the non-energized system. The difference was the much higher quantum yield in the energized system. Protein was determined with the biuret method as modified by Cleland and Slater [12]. Chemicals and gasses Acetylene and ethylene were purchased from Matheson, argon and oxygen from Loosco Amsterdam, 9-aminoacridine from Fluka and 2-heptyl-4-hydroxyquino- line-iV-oxide (HQNO) from Sigma. 4,5,6,7-Tetrachloro-2-trifluoromethylbenzimida- zol (TTFB) was a gift from Dr R. B. Beechey, Woodstock Agricultural Research Centre, Sittingbourne, Kent (U.K.).
RESULTS
NAD*, NADP+ levels and nitrogenase activity Fig. 1 shows the effect of an increasing oxygen input on the levels of NADH
23 Oxygen input (u.moles 02/min per ml) 0O9 024 047 0.59 0.81 0.93 ^ -. .. A . . ^0 6 .V6' 0 6* r-i i— c / 0.26+60 t E /i fi20| 40 £ ; u 1 E 0.13-^ g .3io| 2,'
< z ° 20 40 60 80 100 Time (min) Fig. 1. The effect of increasing oxygen input on the NAD(P)H level and nitrogenase of whole A. vinelandii cells. "Oxygen-adapted" cells (0.42mg/ml ; sucrose oxidation, 1.3//moles oxygen • min"1 -mg~' ) were suspended in 7m l standard incubation mixture with sucrose (20 mM) assubstrate . 10mi nafte r constant rateso facetylen ereductio n andoxyge n consumption were reached, the oxygen input into the reaction vessel waschange d by alteringth e oxygen concentration in the gas phase as indicated. At the end of the experiment the lowest (max. ox.) and highest (max. red.) possible level of NAD(P)H in the cells were determined by flushing with pure oxygen or argon, respectively. , NAD(P)H fluorescence; O-O, acetylene reduction; , oxygen input. plus NADPH compared with nitrogenase activity of whole A. vinelandii cells. This figure showsclearl ytha t ata highe r oxygeninpu t into thecel l suspension,th e amount ofintracellula r NADH plus NADPH decreases. Even at a low NADH plus NADPH level the cells are able to reduce acetylene. When the oxygen input into the cell suspension increases beyond the maximum oxidation capacity, oxygen becomes detectable in the medium and with a higher oxygen concentration in the medium the nitrogenase switches off, using the terminology of Dalton and Postgate [13]. With fluorimetric methods one cannot discriminate between NADH and NADPH levels. Theacid-sto p method was used to determine thelevel s of the oxidized pyridine nucleotides and adenine nucleotides in separate experiments with varying oxygen input (Fig.2) .Thi sfigure clearl y demonstrates that in whole bacteria the amounts of NAD+ and NADP+ increase with increased oxygen input. Thus under these condi tions the intrabacterial ratios NADH/NAD+ and NADPH/NADP+ decrease parallel with increased nitrogen-fixing activity of the whole cells. Between an oxygen input of 0.14 and 0.54^moles oxygen • min-1 • ml-1 incubation mixture there is anincreas eo f30 0% i nnitrogenas eactivity , but the ratioNADPH/NADP + decreases from the maximum to almost the minimum value. In the same oxygen-input traject the intracellular ATP concentration increases from approx. 1.1 to 1.8 mM, assuming 5-6fd intracellula r water per mgprotei n (cf. ref. 14).Sinc e no oxygen wasdetectabl e in the medium, it means that oxygen was used totally by the respiratory system(s) of the bacteria. Theflow rat e of electronsthroug h the respiratory chain can beinfluence d by a number of inhibitors such as HQNO (refs 15-17) and CN" (ref. 18). HQNO alone
24 0.2 0.4 0.6 OS Oxygen input (pmoles Oj/mirr' per mg~')
Fig. 2. Levels ofATP , ADP, NAD+ , NADP + and nitrogenase activity in intact ceilsof A. vinelandii with different oxygen supply. "Oxygen-adapted" cells (0.8 mg/ml; sucrose oxidation; l.l^moles oxygen • min"1 • mg-1) were suspended in 6m l standard incubation mixture with sucrose (20mM ) as substrate. 10 min after constant rates of acetylene reduction and oxygen consumption were reached at the oxygen input as indicated, the total incubation mixture wasfixed wit h HCIO*. After neutralization the cellextrac t wasanalyzed .Q-Q , ATP; O-O, ADP; •-•, NAD+ ; •-• , NADP+ x 10; A-A, nitrogenase activity. inhibits the sucrose oxidation only at high concentrations, 50% at a concentration of 100/iM. Low concentrations of CN" (10fiM) enhance the inhibitory effect of HQNO, for instance 85% inhibitio n at 10 fiM HQNO plus 10ji M CN". Theinhibi tion by HQNO and CN" (refs 18,19 )a sobserve d withisolate d membranevesicle so f A. vinelandii, is the same as observed here with intact bacteria. Due to the strong inhibition by the combined action of HQNO plus CN~, it wasimpossibl e to control therat eo foxidatio n byth ecells .Therefor e highconcentration s HQNO withoutC N"
40 80 120 HQNO (}iM) Fig. 3. The effect of HQNO on ATP, ADP, NAD+ , NADP+ levels and nitrogenase activity of intact A. vinelandii cells under constant oxygen supply. "Oxygen-limited" cells (1.4mg/ml ; sucrose oxidation, 0.19//mole soxyge n • min"1 • mg-1) weresuspende d in 10m lstandar d incubation mixture with sucrose (20mM) as substrate. The oxygen input was 0.15//moles oxygen • min"' -ml-1. The maximal oxygen consumption was0.2 6//mole soxygen-min" 1 • ml"1. 10 min after constant rateso f acetylene reduction and oxygen consumption werereache d at the HQNO concentrations asindicated , + the total incubation wasfixe d with HC104 and neutralized.D-D , ATP; O-O, ADP; •-•, NAD : •-•, NADP+ x 10; A-A, nitrogenase activity.
25 Q Z < u o .9 ,s ei 0 ,-•> o 32 c - D '1 °? •a E co T o •o *- <^ «i e8 •o 0 2 z T3 N •0 0 00 0 0 0 e a wS t-- ci t~* r- r*" 5 eel .2 c. to 2 E CO o CO •* £ T3 J •o a> UJ a> _C«J O •o •3 c cu o ft! » ^- p- N -- in CA a S E
00 v> e § z P- g -) c oo t^ <- \£) VO 9y « w E >X , •* ^
26 UJ J o c SET X II s: » § I :> tt. 2 S - O *s «-• o ,'-' s O Q, **- t> >> 3 O Z H ft. .H ~ !~ V — T H r» oo o —• — «n O •o -p 5 E < Z S E O N » n N n ddddod UJ < + J2 ^ z Q o - < E so r^
27 were used to study the influence of inhibition of the respiration on the ATP, ADP, NAD+ and NADP+ levelsan d the nitrogenase activity (Fig. 3).A t a constant oxygen input, inhibition of the electron flow affects the nitrogenase activity considerably, while the ATP and ADP levels are much less influenced. Under the different condi tions decribed in Fig. 3 no oxygen was detectable in the medium. Therefore the decrease in nitrogenase activity could not be due to "switching off conditions" (cf. ref. 13).
Influence of uncoupleron nitrogenase activity In the obligate aerobe A. vinelandiisuga r is not taken up with group transloca tion, but is associated with the oxidatively energized cytoplasmic membrane [20]. It is clear that de-energization of the cytoplasmic membrane by uncoupler TTFB [21], has a direct effect on the sucrose and therefore on the oxygen uptake and intracellular ATP concentration (Table I). The same phenomenon has been observed by Postma [22] with the uptake and oxidation of succinate by A. vinelandii. The uptake of acetate is not energy-linked [23], thus an uncoupler will have less effect on the rate of oxidation of acetate. From the data presented in Table I it is clear that the acetate oxidation by the cells is not influenced at the concentrations of the uncoupler TTFB used. Table I also shows that the steady-state levels of ATP, ADP and AMP of cells which oxidize acetate are much less influenced by the uncoupler than those which oxidize sucrose. Therefore acetate was used as substrate to test the effect of low con centrations of uncoupler on the nitrogenase activity of whole cells. The results are shown in Table II. Notwithstanding the ATP and ADP levels are more or less constant, a dramatic decrease in nitrogenase activity with increasing uncoupler concentration is observed at a constant oxygen input. Since the concentrations of ATP and ADP, being substrate and inhibitor of the nitrogenase respectively, are practically constant, it means that at the saturating concentrations of acetylene used, the flow of reducing equivalents to the nitrogenase is inhibited by uncoupler. Furthermore the experiments show that this inhibition is accompanied by relative small changes of the NADPH/NADP+ and NADH/NAD+ ratios. The uncoupler, whose only known effect is to lower the energized state of the cytoplasmic membrane, inhibits the transfer of reducing equivalents indicating a direct relation between nitrogenase activity and energized state of the cytoplasmic membrane. Control experiments have shown in accordance with previous work of Hardy et al. [24] that in the concentration range used uncoupler has no effect on the nitrogenase activity with Na2S204 as electron donor.
9-Aminoacridine fluorescence and nitrogenase activity Fluorescence probes have beenuse d to detect changes in energy levelso f energy- transducing membranes [25-28]. The probe 9-aminoacridine itself has a low un coupling capacity and does not influence nitrogen reduction of wholebacteri a within the concentration range used. The mitochondrial inner membrane and the bacterial cytoplasmic membrane are thought to have the same polarity, while the chloroplast grana membrane is opposite in polarity (cf. ref. 29). Uptake [25] or binding [27] of the acridine dyes upon energization in chloroplasts causes a decrease of fluorescence. Therefore an increase in fluorescence, corresponding to a release of the probe upon energization of the bacterial cytoplasmic membrane is expected. That this is the case
28 anaerobiosis TTFB HQNO.CN" I H20 till
TTFB _. \. anaerobiosis \. N 1 Imin. ^ « *l A*_,'c ,—- t '.. ^~^^ H 2°2 "~' H202 *H202
Fig. 4. Enhancement of9-aminoacridin efluorescence b y whole A. vinelandii cells upon energization. "Oxygen-limited" cells (0.33mg/ml ; sucrose oxidation, OJS^moles oxygen-min"1 -mg-1) were suspended in 2m l standard incubation mixture with sucrose (20mM) , 9-aminoacridine (2//M), catalase (Boehringer, 20/ Oxygen input (fjmoles/min per ml) Fig. 5. Enhancement of9-aminoacridin efluorescence, ATP , NAD+ , NADP+ levelsan d nitrogenase activity of whole A. vinelandii cells on increasing oxygen input. "Oxygen-limited" cells (0.8mg/ml ; sucrose oxidation, 0.46/jmoles oxygen - min"' - mg-1) were suspended in 8m l standard incubation mixture containing sucrose (20mM ) and 9-aminoacridine (1 fiM). The 9-aminoacridine fluorescence enhancement is corrected for the bacterial fluorescence emission, as described in Methods. 10 min after constant rates of acetylene reduction and oxygen consumption were reached atth eoxyge ninpu t as indicated, the total incubation was fixed with HCIO4. After neutralization the cell extract was analyzed. •-•, ATP; •-•, NAD+ ; •-•, NADP+xl0; A-A, nitrogenase activity; x-x, fluorescence emission. 29 seen upon inhibition of the sucrose oxidation by HQNO plus CN~. Because in these experiments the sucrose oxidation wasinhibited , oxygen was present during the whole experiment. Thus the observed phenomena are not due to anaerobiosis. TTFB inhibits the formation of an energized state or pH gradient. Upon the addition of TTFB and acetate (Fig. 4) to an anaerobic cell suspension, the oxidation is normal (seen from the oxidation time of the H202) but the fluorescence enhancement is lower. The changes as given by the dotted line of Fig. 4 are the sum of the bacterial fluores cencechange s plusthos e of the dyeemission .I n otherword smuc hles stha nth echange s under energized conditions. If the oxygen input is lower than the oxidation capacity of a cell suspension, the fluorescence emission is not maximal. The amount of fluorescence emission was thus used as indicator for the energy level of the cytoplasmic membrane. Fig. 5 shows the dependence of the ATP level and nitrogenase activity on the energy level of the cytoplasmic membrane measured with the energy-induced fluorescence enhancement of 9-aminoacridine. It is clear from this figure that a relative high energy level of the membrane stimulates the nitrogenase activity of whole cells; When the oxygen input into the cell suspension gets near or exceeds the maximum oxidation velocity (0.31 ^moles oxygen •min" 1 • ml-1), the "switch off" phenomenon [13] is seen. The oxygen electrode registrates free oxygen in the medium (not shown). Under these conditions the ATP and either pH gradient or energized state are high. The data presented here cannot discriminate between inactivation of the nitrogenase by a conformational change or the oxidation of an electron donor or carrier. Fig. 6 shows the influence of uncoupler on the fluorescence of 9-aminoacridine in cells with an active nitrogenase. The initial maximum in the fluorescence emission in the presence of9-aminoacridin e isprobabl y due to theemissio n of oxidized flavoproteins. Thispea k is also seen in cell suspensions without 9-aminoacridine. That the flavoproteins of the gas off TTFB 4 none f- 1fjM ^ 2(uM V 3fjM rf\ 1 min gas on Fig. 6. The effect of TTFB on the fluorescence emission of 9-aminoacridine. "Oxygen-limited" cells (0.38mg/ml ; sucrose oxidation, 0.25,umoles oxygen • min-1 • mg_1) were suspended in 4m l standard incubation mixture with acetate (20mM ) and 9-aminoacridine (2//M). 10 min after a constant rate of oxygen consumption was reached (not shown), the gas-flow stopped. After anaero biosis for 5mi n the gas-flow restarted. TTFB was added in the concentration indicated. 8mi n after constant rates of oxygen consumption and acetylene reduction were reached, the total incubation was fixed with HC104. After neutralization the cell extract was analyzed. The results are given in Table III. The dotted line represents the fluorescence emission changes without added 9-amino acridine. 30 •o u c a 73 • o o 4: o> o O vi o\ m oo E o E x Z t/3 60 UJ D E O 2: ° UJ 5 E — r^r - r- TI- -J «S TT ci - z an z £ UJ Q a. H < a § vi UJ BO > £ UJ -J Q H E 0\ VO ^ s© 0\ Z < 3 O^ ^ ^ ON ~" < >• H H U < I sir UJ MO * < S > 2 « E *2 +•• « es *•» © oo © © © Z O *N Li. o *= o is o UJ O 3 z a 5 »5 w UJ c S 3 60 O * J ea -J E u. 03S © O © © © Z 31 bacteria are oxidized during the first minute is in agreement witn * lag that is seen in the oxidation rate. This is also found by Postma and Van Dam [14],wh o concluded that during the first minute of succinate oxidation the cells had build up a steady-state concentration of Krebs-cycle intermediates. As indicated in Fig. 6, TTFB lowers the fluorescence of 9-aminoacridine. Table III shows the nitrogenase activity and adenine nucleotide levels of the incubations of Fig. 6. It is clearly shown that there is a close relation between nitrogenase activity and height of 9-aminoacridine fluorescence emission. TTFB, at the concentrations used, does not influence the adenine nucleotide levels. DISCUSSION In the past decade several proposals have been put forward for the mechanism of electron donation to the nitrogenase in obligate aerobic organisms such as A. vinelandii. According to the recent hypothesis of Benemann et al. [5, 6], a high ratio NADPH/NADP+ is the driving force for electron transport to the nitrogenase in an electron transport chain consisting of NADPH ferredoxin oxidoreductase (added from spinach), ferredoxin and Shethna flavoprotein (azotoflavin). These experiments were done with cell-free extracts from A. vinelandii. From our experiments it is clear that an increase in nitrogenase activity in whole cells coincides with an increase in NADP+ content and thus with a lower ratio NADPH/NADP+ (Figs 2 and 5). Since in some of these experiments the ATP level increases concomitantly, it could be argued that ATP, being a substrate for nitro genase, is the cause of the increased activity. From the value of 5-6 n\ intracellular water per mg protein [14], it can be calculated that the ATP concentration in the respiring cells varies between 1.0 and 2.0 mM, well above the Km value of 0.1-0.3 mM (refs 30an d 31) of the isolated enzyme. Assuming a similar cooperativeinhibitor y rate of ADP as observed with the purified nitrogenase from C. pasteurianum [30], the large increase in nitrogenase activity (300 % or more) can be expected as consequence of the regulation by the intracellular ATP/ADP ratio. In these experiments acetylene is added in saturating amounts, therefore it can be concluded that under these conditions three possibilities exist: 1.Durin g the whole oxygen-input traject the nitrogenase activity is regulated by the ATP/ADP ratio. Under these conditions a lowering of the NADPH/NADP+ has no effect on the total nitrogenase activity. But according to the simple system proposed by Benemann et al. [5, 6], a high ratio NADPH/NADP"1" is required to decrease the redox potential of the electron carriers enough to give nitrogenase activity. Our results indicate that even at a very unfavourable ratio NADPH/NADP"1", the nitrogenase activity is maximal. Therefore it is unlikely that this simple system operates as the driving force for the donation of reducing equivalents to the nitrogenase in A. vinelandii.2. During the whole oxygen-input curve the overall nitrogenase activity is rate-limited by the generation of reducing equivalents. This possibility is also unlikely, because where initially thecell s are in a highly reduced state a lag in nitrogenase activity is observed. 3. Upon increase of the oxygen input the overall nitrogenase activity is initially regulated by the ATP/ADP ratio, but at the end of the curve where the nitrogenase activity is maximal, the generation of reducing equivalents israt e limiting. An increase in electron transport through the respiratory chain (Figs 2 and 5) stimulates the 32 overall nitrogenase activity, while a decrease in electron transport (Fig. 3) inhibits the nitrogenase activity. During an oxygen-input curve there is an increase in meta bolic activity or possibly a change in metabolic pattern and an increase in electron transport associated with the energized state of the cytoplasmic membrane. In order to discriminate between the production of a metabolite acting as electron donor for nitrogenase and a process connected with the driving force of the energized mem brane, the uncoupler TTFB was used. The oxidation velocity of acetate by A. vine- landiii s hardly influenced by addition of TTFB (Tables I and II) which creates a low energy state (Figs 4 and 6) together with a relative high ATP/ADP level (Tables I, II and III). Under these conditions where the metabolic activity and the ATP/ADP ratio are constant, the nitrogenase activity is totally inhibited by TTFB. Because no other effects of TTFB than uncoupling have been found, it means that TTFB inhibits the nitrogenase activity by lowering the energized state of the membrane. This is seen in Fig. 6. From this figure and Table III we can conclude that there is a direct relation ship between the generation of reducing equivalents and oxidatively generated mem brane energy. Whether dinitrogen reduction is directly coupled with the membrane potential or is coupled to an outwards proton movement through the membrane (pH gradient) cannot be derived from these data. In addition to regulation by the ATP/ADP ratio and electron transfer connected with membrane energization a third mechanism of switch off by oxygen (cf. ref. 13)i s clearly distinguishable. So far this process does not seem to be related with any of the parameters measured in this study. Work is in progress to characterize the interaction between the energized membrane and generation of reducing equivalents for the nitrogenase in A. vinelandii. ACKNOWLEDGEMENTS This work was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) under the auspices of the Netherlands Foundation for Chemical Research. (S.O.N.) We thank Mr Bery J. Sachteleben for preparing the figures. REFERENCES 1 Mortenson, L. E. (1964) Biochim. Biophys. Acta 81, 473-478 2 Yates, M. G. and Daniel, R. M. (1970) Biochim. Biophys. Acta 197, 161-169 3 Klucas, R. V. and Evans, H. J. (1968) Plant Physiol. 43, 1458-1460 4 Yates, M. G. (1972) FEBS Lett. 27, 63-67 5 Benemann, J. R., Yoch, D. C, Valentine, R. C. and Arnon, D. 1. (1971) Biochim. Biophys. Acta 226, 205-212 6 Benemann, J. R. and Valentine, R. C. (1972) Adv. Microbiol. Physiol. 8, 59-98 7 Newton, J. W., Wilson, P. W. and Burris, R. H. (1953) J. Biol. Chem. 204, 445-451 8 Drozd, J. and Postgate, J. R. (1970) J. Gen. Microbiol. 63, 63-73 9 Dalton, H. and Postgate, J. R. (1969) J. Gen. Microbiol. 56, 307-319 10 Cooper, C. M., Fernstrom, G. A. and Miller, S. A. (1944) Ind. Eng. Chem. 36, 504-507 11 Williamson, J. R. and Corkey, B. E. (1969) Meth. Enzymol. 13, 481-497 12 Cleland, K. W. and Slater, E. C. (1953) Biochem. J. 53, 547-556 13 Dalton, H. and Postgate, J. R. (1969) J. Gen. Microbiol. 54, 463-473 33 14 Postma, P. W. andVa n Dam, K. (1971) Biochim. Biophys. Acta 249, 515-527 15 Lightbown, J. W. andJackson , F. L. (1957) Biochem. J. 63, 130-137 16 Haas, D. W. (1964) Biochim. Biophys. Acta 92,433-43 9 17 Eilerman, L. J. M. (1973) Ph. D. thesis, University of Amsterdam, p. 53 18 Jones, C. W. and Readfern, E. R. (1967) Biochim. Biophys. Acta 143, 340-353 19 Ackrell, B. A. C. and Jones, C. W. (1971) Eur. J. Biochem. 20, 22-28 20 Barnes, E. M. (1972) Arch. Biochem. Biophys. 152, 795-799 21 Beechey, R. B. (1966) Biochem. J. 98,284-28 9 22 Postma, P. W. (1973) Ph. D. thesis, University of Amsterdam, p.9 6 23 Visser, A. S. and Postma, P. W. (1973) Biochim. Biophys. Acta 298, 333-340 24 Hardy, R. W. F. and Knight, E. (1966) Biochim. Biophys. Acta 122, 520-531 25 Schuldiner, S. and Avron, M. (1971) FEBS Lett. 14, 233-236 26 Eilermann, L. J. M. (1970) Biochim. Biophys. Acta 216, 231-233 27 Kraayenhof, R. (1970) FEBS Lett. 6, 161-165 28 Lee, C. P. (1971) Biochemistry 10,4375-438 1 29 Harold, F. M. (1972) Bact. Rev. 36, 172-230 30 Ljones,T . (1973) Biochim. Biophys. Acta 321, 103-113 31 Burns, R. C. (1969) Biochim. Biophys. Acta 171,253-25 9 34 II Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii Huub HAAKER and Cees VEEGER Department of Biochemistry, Agricultural University, Wageningen (Received September 30/December 15,1975 ) The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phospho- gluconate dehydrogenase) were determined in Azotobacter vinelandiicells , grown under 02- or N2- limiting conditions. It was concluded that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes a + c$, ban d ^differ incell sgrowt h either 02- or Nj-limited. It was possible to show that the respira tion protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized.Althoug h theleve lo fenerg ycharg e issomewha t dependent on thetyp eo f substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given. Obligate aerobic organisms which fix dinitrogen related by the adenylate kinase reaction, the so-called need a device to protect their nitrogen fixing system energy charge (cf. [5]),determine s the regulation. against oxygen. According to Dalton and Postgate [1] Since Jones and coworkers measured only ATP two protecting mechanisms operate in whole cells of levelsi n intact cells,w edecide d to check their proposal Azotobacter: firstly, a so-called switch-on/switch-off by determining the levels of the adenine and pyridine mechanism giving short time protection against nucleotides in relation to the ability of the growing moderate oxygenconcentrations ;secondly , adaptation cells to protect the nitrogen-fixing system against of the oxidation velocity to the 02 input. Drozd and oxygen. Because chemostat-grown cells are better Postgate [2] reported that the Q0l and cytochrome d defined than batch-grown cells we used a chemostat content increase when the p02 is increased during for culturing the bacteria. Two types of cells were growth. Jones and coworkers [3,4] investigated the used; cells whose growth rate was limited by Oa dependence of cytochrome content, oxidative phos (cells grown (Delimited), and cells grown with excess phorylation efficiency and whole cell respiration on oxygen and substrates, whose growth rate was limited the 02 input during growth. Isolated membranes from by the nitrogen fixation (cells grown N2-limited). cells grown at high oxygen input, showed a decrease Some properties of the two types of cells were com in phosphorylation efficiency, which correlated with pared in relation with the adaptation of the A. vinelan a decrease in ATP levels in whole cells. The increased dii respiration to the oxygen concentration during sugar respiration was attributed to this effect, namely growth and the cytochrome composition. In addition an activation of the key enzymes in sugar catabolism the oxidative phosphorylation of respiratory mem by the lower ATP level. In many cases however the branes isolated from the types of cells were compared. concentrations of all three adenine nucleotides, inter- Enzymes. Glucose-6-phosphate dehydrogenase or D-glucose- MATERIALS AND METHODS 6-phosphate : NADP+ oxidoreductase (EC 1.1.1.49); 6-phospho- gluconate dehydrogenase or 6-phosphogluconate : NADP+ oxido Growth Conditions of the Bacteria reductase (EC 1.1.1.44); 6-phosphogluconate hydro-lyase (EC4.2 . 1.12); 3-deoxy-2-keto-6-phosphogluconate aldolase is 3-deoxy-2- A. vinelandii ATCC strain O.P. was grown on a keto-6-phosphogluconate glyceraldehyde-3-phosphate-lyase (EC4 . Burk's nitrogen-free basic salt medium [6] in a New 1.2.14). Brunswick type C30 chemostat supplied with an oxy- 35 gen electrode. Two types of cells were used; cells Ackrell and Jones [12]. The membrane preparation whose growth rate was limited by 02 (oxygen input was used immediately. The assay mixture contained 1 1 32mmol 02 • l" • h" ; dilution rate 0.1 h~'), and at a final pH of 6.8: 40 mM piperazine N,N'-bis-2- cells whose growth rate was limited by the N2-fixation ethane sulphonic acid (PIPES) buffer, 10m M MgCl2, with 02 present in the medium, as discussed by Dalton 0.5 mM ADP, 5m M glucose, 5m M phosphate and 1 and Postgate [7] (oxygen input 90 mmol 02 T 10 U dialysed yeast hexokinase (Boehringer). Respira •h_1; dilution rate 0.1 h_1). Cells were harvested tory membranes (maximum oxygen consumption from the bulk of the growth medium in the chemostat 20 mmol 02/min at maximum amount of 20 ug, final rather than taking cells from the effluent. The culture volume 1.6 ml) were incubated 3mi n in the assay was centrifuged (10000 xg, 10 min) at 20 °C to main mixture. tain respiratory protection of nitrogenase. After The reaction was started by adding substrate. washing the cells twice with distilled water, they were Final concentrations: 1m M NADH, 4 mM D,L- suspended in water at 0 °C at a protein concentration malate, 4 mM ascorbate plus 0.1 mM 2,6-dichloro- of 20— 6 0 mg/ml. In all experiments the temperature indophenol or0. 1 mMtetramethyl-phenylene-diamine . was25°C. The reaction was stopped after 5mi n by transferring 1 ml of the reaction mixture into 0.1 M ice-cold HC104 40% (w/v). After removal of the protein Nitrogenase Activity Assay by centrifugation (3000xg , 20 min) the aliquot was and Determination of Nucleotides neutralized and its content of glucose 6-phosphate The standard incubation mixturecontained:25m M was determined. In the control experiments the sub strate was omitted. Tris-HCl, 1m M KC1 5m M MgCl2 and 10m M sub strate final pH 7.6. The cells were preincubated for 5mi n at an oxygen input of about 1/3 of the maximum Cytochromes oxidation capacity (independently determined) to restore the intracellular cofactor concentration. The Cytochromes c4 + c5, bt + o, at and d in mem oxygen input was then adjusted to the required level branes were analysed by measuring difference spectra and 10mi n after constant rates of acetylene reduction between oxidized membranes and membranes reduced and oxygen consumption were reached at the oxygen with dithionite; cytochrome o was analysed by mea input indicated, the total incubation mixture was suring difference spectra between membranes reduced fixed by rapidly adding HC104 up to a final concen with dithionite plus CO and membranes reduced with tration of 4% (w/v). After neutralization with KHC03 dithionite. Because of the contribution of cytochromes the adenine nucleotides and pyridine dinucleotides cA + c5 to the spectrum of cytochrome i: + 0, it was were determined as described earlier [8]. When the necessary to use a small slit (<0.6nm bandwith). oxygen input was zero the incubation mixture was The amounts of cytochrome c and b^ + o were fixed 2 min after anaerobic conditions had been calculated from the spectra according to Sinclear reached. The time sequence is important, firstly to and White [13]. Because cytochrome bx and 0 have enable the anaerobic equilibrium to be reached and their maximum absorbance at the same wavelength, secondly to avoid the breakdown of the adenine the net cytochrome At content was determined nucleotides [9]. by subtracting the concentration of cytochrome o from bt + 0. The molar absorption coefficients of Measurements of Oxygen Consumption cytochrome c4 + c5, bt + 0, d and o were used as re ported by Jones and Redfearn [14] respectively by Oxygen consumption was measured with a Clark Sinclear and White [13]. Cytochrome concentrations type electrode (Yellow Spring Instruments). Bacterial were expressed as nmol/mg protein. The spectra were cells were suspended in the standard incubation scanned on an Amino-Chance dual wavelength spec mixture with 10m M substrate at a protein concen trophotometer. tration of 0.01—0. 1 mg/ml. Oxygen consumption was measured during at least 10min . All rates are corrected for endogenous respiration. Oxygen input Preparation and Assays of Soluble Enzymes into thevesse lwa scalculate d according toCoope r [10]. Protein was determined with the biuret method as Freshly harvested cells (160— 21 0 mg protein in modified by Cleland and Slater [11]. 25 mM Tris-HCl and 1m M dithiothreitol, final vol ume 10ml , pH 7.6) were sonicated for 90 s in a MSE sonifier. After centrifugation of the broken cells Preparation of Respiratory Membranes (20000xg for 30min) , the supernatant was cen and Assay of Oxidative Phosphorylation trifuged 120 min at 200000xg. The resulting super The respiratory membranes from the chemostat- natant was dialysed for 2 h against 2 1 25 mM Tris- grown cells were prepared exactly as described by HCl and 1m M EDTA pH 7.6 at 4 °C. After dialysis, 36 1 raM dithiothreitol was added. Glucose-6-phosphate • mg~'). The observed differences in sucrose oxidation dehydrogenase, 6-phosphogluconate dehydrogenase, might be due to either a direct effect on metabolic 6-phosphogluconate hydro-lyase and 3-deoxy-2-oxo- rate or an effect on the rate of uptake of this 6-phosphogluconate aldolase were determined ac substrate. It is well known that ATP, ADP, NADH cording to Senior and Dawes [15]. and NADPH affect the activity of the enzymes of both the Entner Doudoroff and oxidative pentose phosphate cycle pathways in Azotobacter beijerinkii Incorporation of Radioactive Sucrose or Pyruvate li [15]. Still and Wang [17] showed that the Entner and C02 Assay Doudoroff pathway is the most important pathway The incubation mixture contained at a final for the degradation of sugar in Azotobacter species. pH 7.6: 25m M Tris-HCl, 10 mM substrate ([U-14C]- Though changes in metabolic rate may be induced sucrose or [2-14C]pyruvate). The reaction was started by these nucleotides, other possibilities are changes by adding A. vinelandii cells, 0.3— 0. 6 mg protein, in concentration of the enzymes catalysing rate- final volume 0.5 ml. Air was bubbled through the limiting steps or a change in kinetic pattern of solution to keep it aerobic and the reaction was individual enzymes in the two kinds of cells used. terminated by adding an 0.1 ml aliquot of the reac Therefore we studied the influence of growth condi tion mixture to 5 ml 10m M Tris-HCl and 10 mM tions on the kinetic parameters of the key enzymes of dithionite, final pH 7.5. The cells were collected on a the Entner Doudoroff pathway glucose-6-phosphate millipore filter (pore size, 0.45 um) and washed on dehydrogenase, 6-phosphogluconate hydro-lyase and the filter with 5 ml of the Tris-dithionite mixture. 6-phosphogluconate aldolase and the first enzyme of Postma [16]ha s shown that under anaerobic condition the oxidative pentose phosphate cycle, 6-phospho no leakage of Krebs-cycle intermediates occurs from gluconate dehydrogenase (Fig. 1,Tabl e 1). thecells ,althoug h they aretake n upenerg y dependent- The glucose-6-phosphate dehydrogenase in ex ly. The filters were transfered to 0.5 ml soluene tracts of A. vinelandii has unusual properties. Is has (Packard) and after adding scintillation liquid the about the same maximum velocity with NAD+ and radioactivity was counted. NADP+ but in contrast to other glucose-6-phosphate 14 Assays involving the liberation of C02 were dehydrogenases which are also active with both carried out in Warburg vessels. The reaction mixture pyridine nucleotides [18— 21 ] the plot of rate versus in the main vessel contained 25 mM Tris-HCl, 10 mM NADP+ concentration is not sigmoidal but biphasic labeled substrate and 0.3—0. 6 mg protein in a final (Fig.1) . The double reciprocal plot shows two linear volume of 0.2 ml and a final pH 7.6. The reaction regions, one presenting a site with a high affinity + was started by addition of cells and stopped by adding for NADP (Km{l) a 16 uM) and one a site with 0.1 ml 2.5 M H2S04 from the side-arm. The centre a low affinity (Km(2) ~ 0.17 mM). The double reci wall contained 0.05 ml 3.5 M KOH on a strip of procal plot of velocity versus NAD+ concentration + Whatmann paper. After shaking the mixture for 1 h is linear and gives Km (NAD ) of 0.12 mM. the filter paper was transfered to 0.5 ml soluene The specific activities of two of the enzymes of (Packard) and counted as described above. The the Entner-Doudoroff pathway are the same in ex respiration activity.o f the cells was determined under tracts from cells grown 02- or N2-limited (Table 1). the same conditions in parallel experiments with an However the Km (gluconate) of 6-phosphogluconate Clark type oxygen electrode. dehydrogenase is three times larger in cells grown N2-limited. In addition the specific activity of glucose- 6-phosphate dehydrogenase, the first enzyme of both Special Chemicals the Entner-Doudoroff pathway and the oxidative [U-l4C]Sucrose and [2-I4C]pyruvate were ob pentose phosphate cycle, shows a 3— 4 fol d increase in tained from the Radiochemical Centre (Amersham). extracts of cells grown N2-limited. Senior and Dawes [15] studied the regulation of glucose metabolism in A. beijerinckii and observed inhibition by ATP of glucose-6-phosphate dehydro RESULTS genase, 6-phosphogluconate hydro-lyase and 6-phos The rate of sucrose oxidation of cells grown phogluconate aldolase. Rather than using ATP alone, 1 02-limited is 20—14 0 nmol 02 • min" • mg~'; the we studied the effect of the energy charge (cf.[5] ) on oxidation rate pyruvate is considerably higher 300— the activities of these three enzymes. Fig.2 shows the 1 600 nmol 02min~' mg" . The large fluctuations influence of glucose 6-phosphate on the activity of in rate of sucrose oxidation depend strongly on small glucose-6-phosphate dehydrogenase at a saturating variations in growth conditions. In contrast cells concentration of NADP+ and at energy charges of + grown N2-limited oxidize sucrose and pyruvate at 0.3 and 0.9. Similar resultswer e obtained when NAD -1 + about the same rate (300—60 0nmo l 02 • min was substituted for NADP . It isclea r that at concen- . 37 O.b 1 ^__i—-—i ) 1.0 J*-~ ' A yS^ B » 0.4 0.8 y - 0.3 - f 0.6 - 0.2 J 0.4 - 0.1 j 0.2 - 0 , , 3 20 40 60 80 10 °3 1 2 3 [NADPl (|iM) [NADP*] (mM) 80 120 1/[Pyridine nucleotides] (mM j Fig. 1. Dependence of the activity of glucose-6-phosphate dehydrogenase from A. vinelandii on the concentration of pyridine nucleotides. (A) Plot of initial velocity versus NADP+ concentration for glucose-6-phosphate dehydrogenase in the presence of saturating concentrations (10 mM) of glucose-6-phosphate. Assays were carried out as described in Materials and Methods. Protein concentration of 2.8 ug/ml. (B) As in (A), but with higher NADP* concentrations. (C) Lineweaver-Burk plots of the effect of NADP* and NAD+ concentrations on the glucoses- phosphate dehydrogenase activity in the presence of saturating concentrations of glucose-6-phosphate (10 mM). The protein concentration was 2.8 ug •ml"' . (O O) NAD +, (• •> NADP + trations of glucose 6-phosphate lower than 0.7 mM No influence of energy charge was observed on the the enzyme is inhibited at this concentration of the activity of 6-phosphogluconate hydro-lyase and 6- adenine nucleotides however the energy charge has phosphogluconate aldolase. In a typical experiment little effect. Similar observations were made with the activity of the two enzymes was inhibited 10% saturating concentrations of glucose 6-phosphate and at an energy charge 0.3 and 11% at an energy varying concentrations of NADP+ (Fig.3) . Again charge 0.9, at a total adenine nucleotide concentration the enzyme is inhibited by adenine nucleotides, the of 1mM ; at a concentration of 2 mM the activity inhibition is especially marked at low concentrations was inhibited 18% at an energy charge 0.3 and 23% of NADP+ , but does not depend on the energy at an energy charge 0.9). charge. When NAD+ is substituted for NADP+, Jones et al. [4]studie d the decrease in P/O ratio of adenine nucleotides inhibit the enzyme competitively isolated A. vinelandiimembrane s occurring in changing and no effect of energy charge is observed. The results the growth conditions from low to high respiration. show that the NADP+-dependent glucoses-phos This was related to changes in cytochrome d level phate dehydrogenase shows two distinct types of and ATPconcentratio n inintac tcells .Th e intracellular binding sites.Th e low-affinity site ismainl y influenced ATP concentration was determined by removing by adenine nucleotides; the inhibition however com samples from the medium. Since the intracellular pletely disappears, similarly aswit h NAD+ , at saturat ATP concentration decreases very quickly after reach ing NADP+ concentration. Furthermore positive ing anaerobiosis [22]; the possibility cannot be cooperativity with respect to glucose 6-phosphate is excluded that the suspension of the sample trans- enhanced by adenine nucleotides. fered becomes anaerobic. Thus in our studies, under 38 Table 1. Kinetic parameters ofenzymes of Entner-Doudoroffpathway and 6-phosphogluconate dehydrogenase measured in a crude extract from 02- and N2-limited grown A. vinelandii Crude extract was prepared as described in Materials and Methods. Activity of all enzymes was measured as described in Materials and Methods. Protein concentrations of the enzymatic assays: glucose- 6-phosphate dehydrogenase, 5.7 ug/ml cell extract of cells grown 02-limited, 2.8 ug/ml cell extract of cells grown N2-limited. 6-phosphogluconate hydro-lyase dehydrogenase, 3-deoxo-2-oxo-5- phosphogluconate aldolase and 6-phosphogluconate hydrogenase: 17.1 |ig/ml cell extract of cells grown Orlimited, 14.0 ug/ml cell extract of cells grown N2-limited. For the definition of Km(1) and ^m(2> used by glucose-6-phosphate dehydrogenase see text and Fig.lC Enzyme Conditions of growth 2 3 02-limited N2-limited [Glucose 6-phosphate] (mM) Fig.2 . Effect of the energy charge on the activity of glucoses-phos Giucose-6-phosphate dehydrogenase nmol NADH phate dehydrogenase from A. vinelandii. Plot of velocity versus x min ' x tng ' glucose-6-phosphate concentration in the presence of a saturating concentration (3 mM) of NADP+ at a total adenine nucleotide K(NAD+) 371 1260 concentration of 1mM . Assays were carried out as described in K(NADP +) 274 910 Materials and Methods. Protein concentration of 4.5 ug/ml. (O O) Control; (x x) energy charge 0,3; (D Q) mM energy charge 0.9 Vs> glucose-6-P at NAD+ 3 mM 0.25 0.25 + Sro>3) glucose-6-P at NADP 3 mM 0.25 0.30 + Sm5) glucose-6-P at NADP 0.1 mM 0.5 0.4 + A:m{NAD ) 0.15 0.12 + Km(I)(NADP ) 0.011 0.016 + Km(2)(NADP ) 0.18 0.17 6-Phosphogluconate hydro-lyase dehydratase and 3-deoxo-2-oxo-6- phosphogluconate aldolase nmol pyruvate x min"1 x mg_1 V (apparent) 6-Phosphogluconate dehydrogenase nmol NADH x min^1 x mg~ K(NAD +) 23 15 K(NADP*) 90 95 120 160 200 1/lNADP'] (mM"') uM Fig.3 . Effect of the energy charge on the activity of glucose-6- phosphate dehydrogenase from A. vinelandii. Lineweaver-Burk + + Km 6-phosphogluconate (NADP ) 40 110 plots of the effect of NADP concentration on glucoses-phos + Km(NAD ) 90 100 phate dehydrogenase activity at a total adenine nucleotide concen AT„,(NADP*) 13 14 tration of 1 mM in the presence of saturating concentrations (10 mM) of glucose-6-phosphate. Assays were carried out as described in Materials and Methods. Protein concentration of 2.3 ug/ml. Since at high NADP+ concentrations the activities at an constant gas bubbling, to maintain the right oxygen energy charge 0.9 are nearly the same asa t an energy charge 0.3 these input, H004 was added to the cell suspension in points were omitted from the figure. (O O)Control ; (x x ) order to guarantee that no changes in the ATP con energy charge 0.3; (D D) energy charge 0.9 centration occurred. Fig.4 show sth e effect of different oxygen inputs on + N2-fixation, ATP, ADP, AMP, NAD and NADP+ intracellular ATP, ADP and AMP concentrations are levels in cells oxidizing sucrose or pyruvate and grown interrelated as energy charge [5] it is found that the 02- orN 2-limited.I t isclea r that no significant changes energy charge of cells grown 02-limited is somewhat in mutual ratios of ATP, ADP and AMP occur upon higher than that of cells grown N2-limited (Fig.5) . increasing the oxygen input between the two types of However, the effect of the oxidizable substrate is cells when the same substrate is oxidized. When the much more important. The increase in the energy 39 n medium (|iM) 0 0 0 14': * t 0 8 25 50 4 *-!-* *i35 0.5 0.6 Oxygen input (umol O2 • min • mg Fig.4. Effect of oxygen on the levels of adenine nucleotides, pyridine nucleotides and nitrogenase activity in intact cells of A. vinelandii. Cells were suspended in 5 ml standard incubation mixture with substrate. 10 min after constant rates of acetylene reduction and oxygen consumption were reached at the oxygen input as indicated, the total incubation mixture was fixed with HCI04. After neutralization the cell extract was analyzed. In all four figures the upper and lower horizontal axes represent the oxygen concentration in the medium and the oxygen input in the incubation respectively. The right and left vertical axesrepresen t thenitrogenas e activityan d theintrabacteria l amount ofcofactor s respectively. (• D) ATP; (o O) ADP; (A A) AMP; (• •) NAD+ ; IB •) NADP* x 10; ( x x ) nitrogenase activity. (A) Cells -1 -1 grown 02-limited (sucrose oxidation 0.13 umol 02 • min • mg ) were suspended at a protein concentration of 0.9 mg/ml with sucrose -1 -1 (20 mM) as substrate. (B) Cells grown 02-limited (pyruvate oxidation 0.45 umol 02 •min •mg ) were suspended at a protein concentra -1 tion of 0.82 mg/ml with pyruvate (20 mM) as substrate. (C) Cells grown N2-limited (sucrose oxidation 0.49 umol 02 •min" ' •mg ) were suspended at a protein concentration of 0.9 mg/ml with sucrose (20 mM) as substrate. (D) Cells grown N2-limited (pyruvate oxidation 0.35 umol 02 •mi n "' •m g'' ) were suspended at a protein concentration of 0.9 mg/ml with pyruvate (20 mM) as substrate. The arrows indicate the oxygen concentration detectable in the medium charge with oxygen input is larger with pyruvate than with sucrosean d themaximu m valuereache d ishigher . Regulation of sucrose degradation by energy charge thus seems possible only at low values of oxygen input. The total amount of intracellular adenine nucleo tides in Azotobacter varies with the conditions of incubation. Schramm and Leung [9] showed that after anaerobiosis AMP slowly degrades. Therefore in our experiments the cell suspension was first in 0.1 0.2 0.3 0.4 0.5 0.6 Oxygen input (utnol 02-min"1 •mg" 1) cubated aerobically during 10mi n to restore the pool Fig.5 . Influence of oxygen-input on the energy charge of different of adenine nucleotides after which it was further types of A. vinelandii cells. The cells were oxidizing sucrose or incubated for at least 10mi n at the desired oxygen pyruvate; conditions and data as described in Fig.4. Cells grown input. In the case of an anaerobic determination of 02-limited: (O O) sucrose; (• •) pyruvate. Cells grown cofactors the cell suspension was fixed 2 min after N2-limited: (• •) sucrose; (• •) pyruvate. The arrows anaerobiosis to eliminate as much as possible the indicate the first measurements at which 02 was detectable in the medium breakdown of the adenine nucleotides. We also found 40 1 X Table 2. Oxygen consumption, *C02 productionand incorporation of [U-^C] sucrose or [2~ *C]pyruvate in 02-or N2-limited grown A. vinelandii cells 14 Oxygen consumption, C02 production and incorporation of radioactive sucrose or pyruvate were measured asdescribe d in Materials and 14 Methods.Th ereaction swer estarte d withcell swhic hwer egrow na sdescribe d inMaterial san d Methods.Afte r 6mi nth erat eo f C02 produc tion, the oxidation velocity and amount of radioactivity incorporated wereconstan t and weredetermined . For thecalculatio n of the amount of oxygen necessary to oxidize sucrose or pyruvate completely to H20 and C02 the following equations were used: Sucrose: Ci2OuH22 + 120 2 -> 12C0 2 + 11H 20; Pyruvate2 C 303H4 + 50 2 -» 6C0 2 + H20 Type of cells Substrate Oxidation velocity C02 produced Calculated 02 Incorporated consumption radioactive substrate 1 1 nmol 02 nmol min ' • mg nmol - mg" • min-1 • mg'1 Cells grown 02-limited sucrose 22.4 22.7 22.7 31.4 pyruvate 380 235 196 45 Cells grown N2-limited sucrose 273 252 252 27.4 pyruvate 475 438 365 35.5 14 that the pool size of the adenine nucleotides depends and the rate of C02 production. However with on additional factors. For example a cell suspension pyruvate the correlation is poor. The measured rate incubated under aerobiccondition swithou t substrates, of oxygen consumption exceeds that calculated from 14 contained 8.2 nmol of adenine nucleotides (ATP the rate of C02 production. This effect indicates + ADP + AMP)/mg, with sucrose 12.5 nmol/mgand that pyruvate stimulates the oxidation of endogenous with pyruvate 11.2 nmol/mg; under anaerobic condi substrates; for example a high intracellular concen tions with sucrose or pyruvate 10.0 nmol/mg. tration of pyruvate together with a high oxaloacetate Fig.4 shows that cells grown N2-limited, thus with concentration lowersth erati o acetyl-CoA/CoA,whic h oxygen present in the medium during growth, are stimulates the degradation of poly-(3-hydroxybutyr- capable of fixing nitrogen with sucrose and pyruvate ate) [23]. Table2 shows that despite the much higher as substrate (Fig.4C, D). In contrast, cells grown fluxes of substrates through the metabolic pathways 02-limited fix nitrogen with oxygen present in the of the higher respiringcell sth e amounts of radioactive medium only with pyruvate as substrate (Fig.4 A , B). sucrose or puruvate incorporated are nearly the same in cells grown 0 - and N -limited. Byextractin g thecell swit h KOH instead of HC104, 2 2 NADH and NADPH were determined. Combined We were able to confirm the observation [3,4,12, KOH and HC104 extractions showed that under 24] that membranes isolated from cells grown N2- anaerobic conditions 5— 3 0% of the pyridine nucleo limited have a low phosphorylation efficiency at tides are in the oxidized form and that at saturating site I (Table 3). In spite of very careful attempts to oxygen concentrations noreduce d pyridine nucleotides prepare different cytochrome preparations of respira are detectable. It seems therefore that at saturating tory membranes under identical conditions the ab concentrations of oxygen the rate of sucrose oxidation solute P/O ratios varied between 0.1 and 0.4. On the in Azotobacter cells is not regulated by reduced pyr other hand the mutual ratios of P/O between different idine nucleotides. Surprising are the lower pyridine substrates were always the same. Cells grown N2- nucleotide levels in both types of cells oxidizing limited have no phosphorylation at site I (P/O with pyruvate. NADH minus P/O with malate). There is only phos The change in concentration of one of the key phorylation between ubiquinone and oxygen. The enzymesi ncarbohydrat e degradation, glucoses-phos phosphorylation as measured with ascorbate plus phate dehydrogenase (cf. Table 1) may account for dichloroindophenol observed incell sgrow n 02-limited part of the differences in oxidation rate of sucrose is very low in the N2-limited grown cells (Table 3). in the two types of A. vinelandiicells . Therefore the Ackrell and Jones [12] reported that oxygen uptake uptake and flux of [14C]sucrose and [14C]pyruvate and oxidative phosphorylation increase linearly with were studied and compared. Both kinds of washed respiratory membrane concentration over the range cells exhibit a lag period when they oxidize sucrose 0— 0.5 0 mg/ml. We observed deviation from linearity or pyruvate. The rates of oxidation and the amounts with malate or ascorbate plus dichloroindophenol as of radioactivity incorporated in the cell material and substrates at membrane concentration higher than 14 the C02 production were therefore determined after 50 ug/ml; at higher protein concentrations both the this lag, at linear rates (Table 2). With sucrose as rate of oxygen uptake and the P/O ratio decrease. substrate therei sa goo d correlation between therat eo f Therefore we used protein concentrations of 20 ug/ml oxygen consumption, measured polarographically, or lower in our experiments. In contrast to Jones et al. 41 Table 3. The effect of oxygen onthe properties ofK. vinelandii Respiratory membranes were prepared from cells grown 02- or N2-limited as described in Materials and Methods. P/O ratios, cytochrome content nmol/mg and dehydrogenase activities (uatom O • min-1- mg"1) of the respiratory membranes were measured as described in Materials and Methods. Ph(NMe2)2, tetramethylphenylenediamine Respiratory Dehydrogenase activity Cytochrome content isolated from NADH malate ascorbate ascorbate c4 + c5 b^ 4- o 0 *i d + dichloro- + Ph(NMe2)2 indophenol uatom 0 x. min-1 x mg"1 nmol/mg Cells grown 02-limited 1.7 1.1 2.3 3.0 4.9 3.7 4.3 0.9 Cells grown N2-limited 4.7 0.95 0.41 0.44 3.3 5.8 2.6 3.2 3.6 P/O NADH malate NADH Ascorbate plus malate plus dichloroindophenol Cells grown 02-limited (2.7 |ig protein/ ml incubation) 0.18 0.14 0.17 0.12 Cells grown N2-limited (7.3 ug protein/ ml incubation) 0.24 0.25 0.26 0.02 [4] we observed differences in the content of cyto phate cycle by lowering the intracellular NAD(P)H chromes c4 + c5 and by in the different types of cells. levels in N2-limited grown cells. Thus the proposal Cytochrome bt is higher in cells grown N2-limited [3,4], that the increase in respiratory chain dehydro while cytochrome c4 + c5 islower . We confirmed that genase activity and a decline in intracellular level of the amount of cytochrome d is higher in cells grown ATP is responsible for the increase in rate of sugar N2-limited than in those than in those grown 02- oxidation at high 02 concentration can be excluded limited [2,4]. Membranes isolated from cells grown byth e observation. N2-limited have a higher NADH dehydrogenase Similarly intracellular regulation by the energy activity and a higher content of cytochromes bt and d charge can be excluded. Our results show that there than membranes from cells grown 02-limited. The is no significant difference in energy charge at saturat reverse is observed for the ascorbate plus dichloro ing oxygen concentration between the different types indophenol oxidase activity and the cytochrome c4 of cells used. However, some differences in energy + c5 content (Table 3). charge can be induced by a change in substrate; the energy charge of both types of cells is higher with pyruvate than with sucrose. The decrease in phos DISCUSSION phorylation efficiency at sites I and III as measured The data presented in this paper show that under with isolated membranes, might be the cause of saturating concentrations of 02 in intact A. vinelandii a somewhat lower value of the energy charge in cells cells the ATP levels are maximum while the intra grown N2-limited. cellularpyridin enucleotide sar ealmos t completely oxi In addition, apart from a 4-fold increase inglucose - dized(Fig .4) .Therefor e anincreas ei nrespirator y chain 6-phosphate dehydrogenase activity and a 3-fold in dehydrogenase activity as was measured with isolated creased Km (gluconate) for 6-phosphogluconate de membranes doesno t contribute under these conditions hydrogenase no kinetic differences between the key to a lower intracellular level of reduced pyridine enzymes of the Entner-Doudoroff pathway isolated nucleotides because the generation of NAD(P)H by from cells grown 02- and N2-limited are detectable. catabolic pathways is slower than the conversion Changes in energy charge have little effect on the of NAD(P)H into NAD(P)+ by the respiratory chain activity of glucose-6-phosphate dehydrogenase and dehydrogenases. Thus it is unlikely that an increase the enzymes of the Entner-Doudoroff pathway. The in activity of the respiratory chain dehydrogenases total concentration of the adenine nucleotides is of influences the activities of the enzymes of the Entner- more importance for theactivit y of theseenzyme s than Doudoroff pathway and the oxidative pentose phos the variation in energy charge at a constant level of 42 total adenine nucleotides. Since the amount of intra depends on the growth conditions. Table 3show s that cellular radioactive material in the different types of cells grown 02-limited mainly contain cytochromes c4 cells does not vary significantly under steady-state + c5 and cytochrome o. In cells grown N2-limited conditions of respiration, it isobviou s that the sucrose apart from these cytochromes also cytochrome by translocator activity can be followed by the deter and cytochrome d are present. Nevertheless the pro mination of the rate of product formation. It follows blem of the composition of the branched respiratory from our data that in A. vinelandii cells under the chain and its involvement in oxidative phosphoryla conditions ofsaturatin g amounts ofoxyge n the sucrose tion in A. vinelandiineed s further investigation. respiration is mainly determined by the activity of the sucrose translocator, possibly coupled to the We wish to thank Mr Bery J. Sachteleben for drawing the figures, Mr C. van Dijk and Mr K. van der Heide for their help glucose-6-phosphate dehydrogenase activity. Our ex in some experiments and Dr A. de Kok and Dr S. G. Mayhew for periments cannot exclude however that the absolute their critical reading of the manuscript. The present investigation amount of sucrose translocator differs in the two types was supported by the Netherlands Foundation for Chemical of cells. The possibility exists that the activity of the Research (S.O.N.) and with financial aid from the Netherlands translocator is regulated by metabolites other than the Organization for the Advancement of Pure Research (Z.W.O.). adenine or pyridine nucleotides. The influence of the value of the energy charge and especially of the ratios REFERENCES NAD(P)H/NAD(P)+ on the activities of glucose-6- phosphate dehydrogenase and the enzymes of the 1. Dalton, H. & Postgate, J. R. (1969) J. Gen. Microbiol. 54, Entner-Doudoroff pathway can play an important 463-473. role at non-saturating oxygen concentrations in 2. Drozd, J. & Postgate, J. R. (1970)7. Gen. Microbiol. 63,63-73. preventing an accumulation of metabolites. 3. Ackrell, B. A. C. & Jones, C. W. (1971) Eur. J Biochem. 20, 29-35. Cells grown on sucrose have a high nitrogenase 4. Jones, C. W., Brice, J. M., Whight, V. & Ackrell, B. A. C. activity at least when they oxidize pyruvate. One could (1973) FEBS Lett. 29, 77-81. 5. Atkinson, D. E. (1968) Biochemistry, 7, 4030-4034. argue that the high value observed for the energy 6. Newton, J. W., Wilson, P. W. & Burris, R. H. (1953) J. Biol. charge under these conditions stimulates nitrogen Chem. 204, 445 -451. fixation, but the increase in nitrogenase activity with 7. Dalton, H. & Postgate, J. R. (1969) J. Gen. Microbiol. 56, increasing oxygen input at a nearly constant energy 307-319. charge, as discussed previously [8], is difficult to 8. Haaker, H., De Kok, A. & Veeger, C. (1974) Biochim. Biophys. Acta, 357, 344-357. explain in that case. A more likely explanation is 9. Schramm, V. L. & Leung, H. (1973) J. Biol. Chem. 248, that a more highly energized cytoplasmic membrane 8313-8315. in cells respiring pyruvate stimulates the transport of 10. Cooper, C. M., Fernstrom, G. A. & Miller, S. A. (1944) Ind. reducing equivalents to the nitrogenase (cf. [$}).Th e Eng. Chem. i<5, 504-509. 11. Cleland, K. W.& Slater. E. C. (1953) Biochem. J. 53, 547-556. observation that cells grown on sucrose under 02 12. Ackrell, B. A. C. & Jones, C. W. (1971) Eur. J. Biochem. 20, limitation and respiring pyruvate fix nitrogen with 22-28. oxygen present in the medium, proves that the re 13. Sinclear, P. R. & White, D. C. (1970) J. Bacterial. 101, 365- spiration protection of the nitrogen fixing system in 372. A. vinelandiidoe sno t depend on theoxidatio n capacity 14. Jones, C. W. & Redfearn, E. R. (1966) Biochim. Biophys. of the cells but on the substrate used. Therefore it is Acta, 113, 467-481. 15. Senior, P. J. & Dawes, E. A. (1971) Biochem. J. 125, 55-66. not necessary to modify elements in the electron- 16. Postma, P. W. (1973) Ph. D. Thesis, University of Amsterdam, donating or the nitrogenase system to fix nitrogen p. 101. in the presence of oxygen. The experiment with cells 17. Still, G. G. & Wang, C. H. (1964) Arch. Biochem. Biophys. 105, 126-132. grown 02-limited show that the oxidation capacity is high with pyruvate, while the much lower rate of 18. Hizi, A. & Yagil, G. (1974) Eur. J. Biochem. 45, 201-209. 19. Olive, C, Geroch, M. E. & Levy, H. R. (1971) J. Biol. Chem. sucrose oxidation is probably due to limitations in 24(5,2047-2057. translocation rate. The result of this limitation is a 20. Cancedda, R., Ogunmola, G. & Luzzatto, L. (1973) Eur. J. higher sensitivity of the nitrogenase to 02 and can Biochem. 34, 199-204. be overcome by adaptation of the sucrose metabolism. 21. Geisler, R. W., Mecluve, A. M. & Hansen, R. J. (1973) Bio chim. Biophys. Acta, 327, 1 — 10. One can only speculate on the relation between 22. Knowles, C. J. & Smith, L. (1970) Biochim. Biophys. Acta, *:he variation of the amounts of the different cyto 197, 152-160. chromes and the oxidative phosphorylation efficiency. 23. Senior, P. J. & Dawes, E. A. (1973) Biochem. J. 134, 225-238. Our data indicate that the composition of the respira 24. Jones, C. W., Ackrell, B. A. C. & Erikson, S. K. (1971) Bio chim. Biophys. Acta, 245, 54— 62 . tory chain as proposed by Jones and Redfearn [25] 25. Jones, C. W. & Redfearn, E. R. (1967) Biochim. Biophys. Acta, is at least doubtful. The composition of the chain 143, 340-353. H. Haaker and C. Veeger, Laboratorium voor Biochemie der Landbouwhogeschool, De Dreijen 11, Wageningen, The Netherlands 43 Ill Aerobic nitrogen fixation in Azotobacter vinelandii H.Haaker ,G .Schering san dC .Veeger ,Departmen t ofBiochemistry , Agricultural University,D eDreije n 11,Wageningen ,Th eNetherlands . INTRODUCTION Organismswhic h fixdinitroge n inth epresenc e ofoxyge nhav et oover cometw odifficulties :1 .Protec tth eoxygen-sensitiv e nitrogenase against irreversible inactivation. 2.Synthesiz e electroncarrier s with a lowredoxpotentia lnecessar y fromnitrogenas e activitywit h oxygen present inth emedium .Wit h regard toth efirs tproble m Daltonan d Postgate (1970)propose d arespiratio nan dconformatio n protection. Aboutth emechanism so fbot hprotectio n systems some informationi s available (Drozdan dPostgate ,1970 ;Jone se tal. ,1973 ;Haake ran d Veeger,1976 ; Yates, 1970).Electro n carriers sucha sferredoxi nan d flavodoxin, capableo fdonatin g reducing equivalents toth enitrogenas e (Benemanne tal. ,1969 ; Yoch,1969 ,197 2a ,b ;Yate s 1972),hav ebee n isolated from Asotobacter. Benemanne tal . (1971)propose d andelectro n transport chainbetwee nNADP Hvi aferredoxi nan dflavodoxi nt oth e nitrogenase.I nou ropinio nsom ereservation s mustb emade . 1.I nwhol e cellsnitroge n fixation takesplac ea tdifferen t ratioso fNAD(P)H/NAD(P ) (Haakere tal. ,1974 ;Haake ran dVeeger , 1976),whil e iti sdoubtfu l that the intracellular redoxpotential ofpyridin e nucleotides islo wenoug h togenerat e reducing equivalents forth enitrogenase .2 .Ferredoxin-MAD P oxidoreductaseha sneve rbee n found in Azotobacter. Inthi s reportw e present somerecen t results inrelatio nt oth eoxyge nprotectio n of,an d theelectro ndonatio n to,th enitrogenas eo f Azotobacter. MATERIALSAN DMETHOD S GROWTH CONDITIONSAN DENZYM E PREPARATIONS Azotobacter vinelandii OPwa seithe r growni na batc h culture,harves teddurin g thelogarithmi c phasean dstore da sdescribe d earlier (Bresterse tal. ,1975 )o rgrow ni na chemosta tN--limite dwit ha dilu - -1 tionrat eo f0. 1h ,harvested ,washe dan duse d directly (Haakeran d Veeger, 1976). 45 Crudeextract swer eprepare d intw oways .Mechanically ,usin g aManto n Gaulinhomogenize r (CEM)an db ya nosmoti c (CEO]treatmen ta sdescribe d bySha he t al. (1972). Nitrogenase complexwa spurifie d asdescribe db yBule nan dL eComt e (1972);dependin g onth eextrac t theywer ederive d from,th e terminology M and0 wa s added toth eterminolog y ofBule nan dL eComte .Th e following preparationswer e tested;crud eextrac t (CEMo rCEO )an dC 42-I( Man d0) . Thecrud ecomponent s I (crude I)an d II (crude II)wer eprepare d from C 42-1an d separated ona DEA Ecellulos ecolum na sdescribe db y Shahe t al. (1972).C 42-1wa s alsoelute d froma DEA Ecellulos ecolum na s a single fractionwit h9 0m MMgCl ~ (C-I-90).Chemostat-grow ncell swer e used toprepar ea Tris-EDTA-toluen epreparation . 100-200m gbacteria l proteinwa s suspended in5 0m MTris-HCL ,1 0m MEDTA ,0. 2m ltoluene , finalp H 8.0,fina lvolum e 20ml .Th e suspensionwa shel d anaerobically at0 °fo r 15min .Afte r centrifugation (10,000x g, 5min. )a t 4°Cth e pelletwa swashe dwit h 40m l 25m MTris-HC Lp H 7.4 and 5m MMgCl -an d centrifuged again.Th epelle twa sresuspende d inth esam ebuffe ran d stored at 0C . (TETpreparation) .A controlpreparatio nwa s prepared without adding toluene (TEpreparation) . ANALYTICALMETHOD S Acetylene reductionassay swer e runa t3 0 ,shakin g in6. 5 mlbottles , sealedwit hSubaseal ,whic hcontaine d amixtur e of 25m MTricin ebuffer , 2m MATP ,4 MgCl 2, 10mM ,creatin ephosphat ean d 5U .creatin ekinas e finalp H7.5 .Thi smixtur ewa s thouroughly flushed fora t least 30min . withpurifie d argonwhic hpasse da heate dBAS Fcatalyst .Th e acetylene (101)an d 20m Mdithionit ewer eadded .Th emixtur ewa s equilibrated for 10min .wit h thegasphas et oremov eth e lasttrace s ofoxygen ,afte r which thenitrogenas epreparatio nwa s addedan dth eethylen eproduce dwa s measured asdescribe d earlier (Haakere tal. ,1974) .Whe n otherreducin g agents instead ofdithionit ewer eused ,th e lasttrace s ofoxyge nwer e removedb y 10m M glucose,1 0U glucoseoxidas e and 40p gcatalase/ml , whichwa s added toth eassa ymixture . Pyruvatedehydrogenas ewa smeasure d inth eassa ymixtur edescribe db y Bresterse tal . (1975),bu tth eactivit ywa smeasure db y followingth e oxygenconsumptio ncause db y thepresenc eo fexces sNAD Hoxidas eactivit y using 5m Mpyruvat eplu s 5m Moxaloacetate .Th eNADH ,NADP Han dmalat e 46 oxidase activitieswer emeasure d according toAckrel l andJone s (1971), glucose-6-phosphate dehydrogenase according toSenio r and Dawes (1971). Acridine fluorescencewa smeasure d asdescribe db yHaake r et al. (1974) using 5p Matebri n instead of 9-amino-acridine. For aTE Tnrenaratio n thefluorescenc e assaymixtur e contained apyruvat e dehydrogenase assay mixture plus 0.2m g catalase,fina lvolum e 2ml .Th e fluorescence of a TEpreparatio nwa sperforme d ina mixture containing 20m MTris-HCL , 1m M MgCl2, 5m Mpyruvat e and 0.2m g catalase,fina lp H 7.4 finalvolum e 2ml . Photoreactionswer eperforme d inth enitrogenas e assaymixtur eunde r the following additions: 10o r 40p Mdeazaflavi n (3,10dimethyl-5-deazaiso - alloxazine), flavodoxinpurifie d according toYoc h (1972 a). The light 2 intensitywa s 15mW/c m measured between 400-500ran. Anaerobi c snectro- photometrywa sperforme dwit h "Subaseal"stoppere d cuvettes ona Cary-1 6 spectrophotometer finalvolum e 1ml .Oxyge n inactivationwa s followedb y exposingnitrogenas e toai ra sdescribe db y Kelly (1969)i na mediu m containing 25m MTris-HC Lp H 7.4 and 5m MMgCl ?, finalvolum e 2ml . 0.3 ml sampleswer e takenan d examined fornitrogenas e activity.Anaero bic gelelectrophoresi swa sperforme d according to Kedinger et al. (1974) except that thioglycolic acidwa s omitted and 2m MNa^S^O .wa s added to theuppe r buffer,afte r flushingwit h argon for 30min . Sampleswer e prior toapplicatio n diluted ina mediu m containing: 25m MTris-HCL , ? 2m M Na2S20., 5m MMgCl-,20 o sucrose and 0.04 mM indigosulfonic acid, finalp H 7.6.Abou t 50p go fprotei nwa s applied/gel. BIOCHEMICALS All enzymes andcofactor swer epurchase d fromBoehringer . Flavodoxin from P. elsdenii andD . vulgaris weregift s from S.G. Mayhew;deazaflavi n was agif tfro mV .Masse y and S.Ghisla . 3. RESULTS AND DISCUSSION OXYGEN PROTECTION Oppenheime t al. (1970)an dDroz d andPostgat e (1970)suggeste d that a nitrogenase-cytoplasmic membrane complexo r thepresenc e of cytoplasmic membranes isresponsibl e fora noxygen-toleran tnitrogenase .Whe n Azotobaater cells arerupture db y osmotic shock,th ecel l free extract isrelativel y freeo fcytoplasmi cmembrane s and thenitrogenas e was oxygen sensitive (Oppenheim et al. 1970). this contrastswit h acel l 47 exposuretim e(min ) Fig. 1 Oxygen stability of different nitrogenase preparationso f A.vinelandii. Conditions as described inMaterial s andMethods . For every tested preparation anitrogenas e activity ofabou t 100nmole s ethylene produced permin .wa s used. • 1,CEM ;0 0,CEO ;® »,CE0 + 0.1 MKC L • •, C42-I-M ;D D, C 42-1-0;-* %-C 42-1-90 ; A A, crude I+ crud e II (1:6); A ACrud e I+ crud e II (1:6)plu s cytoplasmicmembrane s 3.6m g extractprepare d after disruptionwit h aFrenc hpressur e cell. Fig.1 gives acompariso no fth eoxyge nsensitivit y ofnitrogenas epreparation s preparedb y different cell-lysismethods .Additio no fo. 1 M KCL toth e cell-free extracto fosmoticall y lysedcell s increases theoxygen-sensi tivity considerably.Sinc e inth eexperiment spresente db y Oppenheim et al. (1970)i nth eosmoticall y lysed cellextrac t 0.1M KCLwa spresent , we think thatth eobserve d oxygen-sensitivity isdu e toth e ionicstrengt i andno tt oth emetho d ofcell-rupture .Fig . 1als oshow s thatdurin g the purificationo fth enitrogenas e complexaccordin g toBule nan dL eComt e (1972)th eoxyge nstabilit y isrelativel y constant.Afte relutin g the nitrogenase asa singl efractio nwit h 90m MMgC l froma DEAE-cellulos e column,th enitrogenas e ismor eoxygen sensitiv e thanth eorigina lcom plex.Th enitrogenas e complexprepare db ymixin g thecrud e components is extremelyoxyge nsensitiv e andonl ya sligh tstabilizatio nagains t oxygen inactivationca nb eobtaine db y addingwashe d cytoplasmicmem - 48 branes.Thi s incontras twit h theeffec to f acrud eo rpurifie d NADH dehydrogenase reportedb yYate s (1970). Fig. 1als oshow s thatth e oxygen inactivation isbi-phasic .Th e firstphas e shows arapi din - activation; theexten t depends on the typeo fpreparation . The second phase isslowe ran d occurs inal lpreparations . Fig. 2 Anaerobic non-denaturating poly- acrylamide gel electrophoresis of "C 42-1" nitrogenase prepara tions.Gel swer e prepared and runa sdescribe d inMaterial s andMethods .Pattern s are from the following preparations: A, C 42-1; B. crude component I; C. Crude component II. 49 Fig.2 show s anaerobicpolyacrylamid ege lelectrophoresi s ofdifferen t nitrogenasepreparations .Th enitrogenas e componentsar eeasil ydetec table; thesemai nband swer e shownt ob eFe-containin gb yreactio nwit h dipyridyl reagent accordingt oBril le tal . (1974). Inn ocas eprotei n was retainedo nth estackin g gel,therefor eth eoxygen-protectin g pro teinsmus tb edetectabl eo nth egels .Th efirs tpatter ni stha to fa nitrogenase complexpurifie d accordingt oBule nan dL eComt e (1972)u p toth esecon dM gprecipitation ,whic hha shig hNAD Hdehydrogenas eacti vity (NADHoxidas e activity alsowa smeasurable) .Th eactivit yo fth e complexi sstabl efo ra tleas t3 month sa t0 C unde rAr .Whe n thispre parationwa s appliedt oa DEAE-colum nan delute dsuccesivel ywit h0.2 5M and0.3 5M NaCL ,th eeluate s showedpattern s 2an d3 .Thes e eluateso r combinations haven oNAD Hoxidas eo rNAD Hdehydrogenas e activityan d uponstorin gth ecomponent s 2-3week s separately,n onitrogenas eacti - vily couldb einduce db ycombinin g them.Arrow s inFig .2 Aindicat eth e proteinswhic har eonl ypresen ti nth eoxyge nstabl e complexan dth epro teinsma yb einvolve di nstabilizatio nan doxyge nprotectio no fth enitro genase.Th eresult spresente di nFig .1 an d2 allo wu st oconclud e that oxygenstabilit yo f Azotobaoter nitrogenasei sno texplaine db yth epre senceo fcytoplasmi cmembrane sbu ti scause db ya hig hmolecula rweigh t protein factor thatform smor eo rles sa comple xwit hth enitrogenase . Themetho do fcell-ruptur e determines onlypartl yth eformatio no fsuc h a complex. PROPERTIESO FTOLUENE-TREATE DAZOTOBACTE R CELLS Jacksonan dDemos s (1965)showe d thatlo wconcentration so ftoluen e makesbacteria lmembrane s permeablet osmal lmolecules .Sinc eth e possibility exists thattoluen eattack sth epermeatio nbarrie ran ddoe s notinfluenc eth eorigina lstructure ,suc ha preparatio nma yb eusefu l forstudyin g thegeneratio no freducin gequivalent sfo rth enitrogenas e in Azotobaoter. Table Igive s some informationabou tth einfluenc eo ftoluen eo nth e permeationo fimportan tmetabolite s andcofactors .Al lcofactor s tested likeATP ,TPP ,CoASH ,NAD +,NADP + permeate intoa TE Tpreparation . Probablyth ediffusio n limitfo rmolecule swit ha mol .wt .les s than 1000i srelativel y small;however ,sinc ecytoplasmi c enzymesd ono t leak intoth emedium ,enzyme swhic har eadde dexternall yprobabl yd o 50 Table I. Some enzymatic properties ofA.vinelandi i cells after a toluene treat ment.TE Tpreparatio nwa s prepared and enzyme activitieswer emeasure d as described in thetext . .- 1 -] The activity isexpresse d asnmole smi n mg nitrogenase TET sonicated TET complete 1.6 3.0 no-ATP regenerating system 0.3 0 no-dithionite 0 0 pyruvate dehydrogenase complex complete 105 66 no-pyruvate 4.0 7.3 no-TPP 6.0 7.8 no-CoASH 5.3 6.9 no-NAD+ 0 0 glucose-6-phosphate dehydrogenase complete 410 440 no-NAD+,NADP + 0 0 no-glucose-6-P,NA D 13 12 no-glucose-6-P,NAD P 17 4 no-glucose-6-P 30 100 oxidases NADH-oxidase 347 659 NADPH-oxidase 95 280 malate-oxidase 200 350 51 notpas s themembranes .Sinc e thediffusio n limitation forsmal lmole cules isals ooccurrin gwit h theproducts ,on emus tb e carefulwit h the interpretation ofth eobserve dvelocitie s ina TE Tpreparation . Nitrogenase activityproduce sADP ,a stron g inhibitor (Ljones,1973) . SinceAT Pha s tob e regenerated on the other sideo f themembrane ,th e diffusiono fAD P outo f theTE Tvesicl eprobabl y determines themaxi mumnitrogenas e activity ina TE Tpreparation .Th e increase inoxidas e activity isdu e toa membran edestructio nb y sonication. Freezing and thawing of Azotdbactev membranes also increases theoxidas e activities (Jones andRedfearn , 1966).A s showni nTabl e Iglucose-6-phosphat e and other substrates like isocitrate andNAD(P ) haven o diffusion limit into aTE Tpreparatio n inspit eo f thever yhig h enzymeactivities . Another aspect ofa TE Tpreparatio n is theenzym eorganization . Ina TETpreparatio n oneca nexpec t ahighe r degree oforganizatio n thani n ruptured cellpreparations .Th epyruvat e dehydrogenase overallreaction , measuredb y theoxyge nuptak evi aNAD Hoxidase ,i sa nexampl e of this higher degree ofenzym eorganizatio n ina TE Tpreparation .Becaus eNAD H and especiallyAcCo A strongly inhibit theoveral l reaction (Bresters et al., 1975 b), it isnecessar y togenerat eCoAS Han dNA D efficiently toobtai n amaximu m steady-state activity.Becaus e iti sunlikel y that enzymes such asth epyruvat e dehydrogenase complex orcitrat e synthase are inactivatedb y sonication,a highe rdegre e ofenzym e organization mustb e thereaso nfo r thehighe r activity ofth ecombine d actiono f pyruvate dehydrogenase complex,citrat e synthase andNAD Hoxidas e ina TETpreparatio n ascompare dwit h asonicate dTE Tpreparation .However , onecanno t exclude thepossibilit y that,i nth e caseo fnitrogenase ,th e higher degreeo f organizationwithi n themembran e leads tolowe ractivi ty,a sdiscusse d ontheoretica l groundsb y Goldmanan d Katchalski (1971). REACTION OFFLAVODOXIN SWIT H NITROGENASE Complete reductiono fflavodoxins ,especiall y theflavodoxi n of Azotobaoter, has longbee na problem .Photoreductio n offlavodoxi n in thepresenc e ofEDTA ,acetat e andTricin ewa sno tpossibl ebu twit h catalytic amounts of deazaflavin iti spossibl e tophotoreduc e flavo doxins fastan dcompletel y (V.Massey ,persona lcommunication) .Wit h this system,w ewer e able toreduc e different typeso fflavodoxin s com pletely and studied theirreactivitie s aselectro ndono rfo r thenitro genase. 52 Fig. 3 Timecours eo fenzymati can dnon-enzymati coxidatio no f photo-reduced electroncarriers . Flavodoxinsar efro mth efollowin gorganism sA , A.vinelandii (0.3mg/ml) ;C , P.elsdenii (0.2mg/ml) ;D . D.vulgaris (0.2mg/ml ;B ,o fmethylviologe n(0. 2mM) .Reaction swer e performedwit h 10y Mdeazaflavi na sdescribe d inth etext . Differentamount so fenzym eC 42-1 ,12. 5mg/m lwer eadde d atth etime sindicated . Reducedflavodoxin sfro m A.vinelandii, P.elsdenii and D.vulgaris weretreate dwit hdifferen tamount so fpartl ypurifie dnitrogenas ean d rateso felectro ntransfe rwer emeasure dspectrophotometricall ya sshow n inFig .3 .Thes erate sdiffe rwidel yan don eca nobserv etha to fth e threetype so fflavodoxi nth e Azotobaotev flavodoxini sb yfa rth ebes t electrondonor .Continuou silluminatio nwit ha fixe dlimitin gamoun t ofcarrier ,dependin go nth emol .wt .21-3 2pg/m la non-rate-limitin g amounto f1. 4nmol./m lnitrogenas ecomple x(0.2 4mg/ml )an d20 % CM~ presentgav erate so f2 ,0. 3an d0. 2nmole sethylen eproduce dmi n . nmol offlavodoxin ,respectively .O nth eothe rhan dw ehav eobserve d thatwhe n P.elsdenii flavodoxin(0.4 2mg/ml )wa scontinuousl yphoto - reducedb ychloroplast san da cellfre eextrac to f A.vinelandii was added (withendogenou selectro ncarrier spresent )rate so f(^^-reduc tionapproache dtha to fth edithionite-drive nreaction . A.vinelandii flavodoxin0.5 5mg/m li shardl yactiv e(101 )i nthi ssyste mbecaus ei t 53 isno t reducedbeyon d thesemiquinon estat e (c.f.Beneman ne t al., 1969). Sopresumabl y theredo xpotentia l aswel l as the "fit"wit h the nitrogenase are important indeterminin g therat e ofelectro n transfer mediatedb y aflavodoxin .Lastly ,th e importance ofredo xpotentia l is especially clearwhe n theexten to foxidatio no freduce dmethylviologe n by nitrogenasewa s comparedwit h thato freduce dbenzylviologen .Benzyl - viologen,photoreduce d as fara spossibl ewit h oursystem ,ca nb eoxidi zedb ynitrogenas e only slightlywherea s Fig.3 Bshow s that reduced methylviologen canb eoxidize d toa hig hextent .B y continuous illumi nation,however ,n odifferenc e inreactio nrate swit heithe rmethyl viologeno rbenzylviologe n ascarrier s canb e observed. LOCALISATION OFFLAVODOXI N INA TET PREPARATION As shown inFig .3 Afully-reduce dflavodoxi n from Azotobaoter actsa s a very good electrondono r forpurifie d nitrogenase.Unde r constant illuminationphoto-reduce d flavodoxinha s fora t least 20min . the same activity asdithionit e aselectro ndono r forth enitrogenase ;als o ina crud e extract (Fig. 4B). Fig. 4A,B shows the lownitrogenas e activitywit hphotochemicall yre duced deazaflavin ina TE Tpreparatio n incontras twit h the activity ofa sonicate d TETpreparation .Wit h apurifie d nitrogenase complex, photochemicallyreduce d deazaflavinha s anactivit y ofabou t 101o fth e dithionite activity.Thi smean s that theobserve dnitrogenas e activity withdeazaflavi n ina sonicate d TETpreparation ,abou t 501o f thato f thedithionit e activity,i sdu e to thepresenc e ofendogenou s flavo doxin,ferredoxi n oranothe runknow n low-potential electrondonor . As already showni nTabe l Idithionit eca nac ta s electrondono r for nitrogenase ina TE To r sonicated TETpreparatio n (Fig.4) .Th esam e is true forphotochemicall y reducedbenzylviologen . Ina TE T preparation therate-limitin g step inth eoveral lnitrogenas e activity isprobabl y not thedonatio no felectron sb y thereducin g agentbu t the diffusion of thestron g inhibitiorAD Pou to fth evesicle ,becaus e therat eo f ethyleneproductio n isnearl y thesam ewit hdithionit e aswit h reduced benzylviologen.Wit ha purifie d nitrogenase complex therat eo f ethylene productionwit h reducedbenzyl -o rmethylviologe n (seeals o Fig.3 )i s about 1/3 of thato f thedithionite -o rreduce d flavodoxindrive nnitro genase activity.Th e observed activitywit hbenzylviologe n ina soni - 54 10 time(min ) Fig. 4 Effect of different electron donors on the nitrogenase activity intoluene-treate d A.vinelandii cellso r sonicated toluene-treated k.vineXandii cells.Preparation swer e made and activitiesmeasure d as described inMateria l andMethods . A, 0.93 mgTE Tpreparatio nwa s suspended in the acetylene assaymixture .Electro ndonors :0 0,dithionite ;X X,light , 40y Mdeazaflavin ,4 0u Mbenzylviologen ; A A, light,4 0u M deazaflavin;D Qlight, 40u Mdeazaflavin , 3.8 nmoles A.vinelandii flavodoxin.B , 1.1m g sonicated TET preparation was suspended in the acetylene assaymixtur e same symbols asA . 55 catedTE Tpreparatio n iso f course thesu mo fth eactivit ywit hphoto - chemically reducedbenzylviologe nplu s thatwit h thephotochemicall y reduced endogenous electroncarriers . Theresult s indicate thatbenzylviologe n anddithionit e react inth e TETpreparatio ndirectl ywit h thenitrogenas e ora sit eclos e toit . Furthermore the lack ofactivit y ofth eTE Tpreparatio nwit h deazaflavin indicates thatflavodoxi nan d ferredoxinar eburie d inside themembran e and thus areno t reducable.Th e lack ofactivit y ofa TE T preparation inth epresenc eo fdeazaflavi nplu s flavodoxini sexplaine db y thefac t that the flavodoxindoe s notpenetrat e through themembrane . ELECTRON TRANSPORT SYSTEM TONITROGENAS E IN AZOTOBACTER Benemanne t al. (1971]suggeste d thatreduce d pyridine nucleotides donate electrons via ferredoxinan d flavodoxin toth enitrogenase .W e tried torepea t theirexperiment swit hextract so fmechanically-rupture d cells,bu twher eunsuccessful .Th ereaso n for the failureo f theseex periments inou rhand s isno t known,bu t itmigh tb e connectedwit h the facttha t theredo xpropertie s of flavodoxin fromth estrai no f Azotobaeter usedb y Benemanne tal . (1974)diffe r fromthos e offlavo doxinfro m thestrai nuse db yu s and others (Yoch, 1975). Because aTE Tpreparatio n certainly hasorganize d structureswhic h could provide anelectro ndonatin g system fornitrogenase ,w e tested suchpre parations foracetylen e reductionwit h organic substrates as theulti matesourc eo freducin gequivalents .However ,i nth epresenc eo fth e substrates and cofactorsuse db y Benemanne tal . (1971)th erat eo f -1 -1 ethyleneproductio nwa sver y low (0.1nmoles .mi n .m g ).I nearlie r publications (Haakere t al.,1974 ;Haake r andVeege r 1976)w e proposed thats ocalle d energized stateo fth ecytoplasmi cmembran e is important innitroge n fixationb ywhol e cells andpossibl y coupled toth egene rationo freducin g equivalents.W e therefore investigatedwhethe r iti s possible toenergiz e thecytoplasmi cmembran e ina TE Tpreparatio nb y using thechang eo facridin efluorescenc e asa prob e (Haakere tal. , 1974). Intact cells (TETpreparation ) showa hig hnitrogenas e activity withpyruvat ea ssubstrat e (Haakeran dVeeger ,1976 )an dunde r aerobic conditions they causea large increase inatebri n fluorescence indica ting thatth ecytoplasmi cmembran e ishighl y energized (Fig.5) . 56 Fig. 5 Effect of toluene on the enhancement of the atebrinfluores cence upon energization of A.vinetandii cells. A.vineland'Li cellswer e treated with toluene and fluorescence wasmeasure d as described inMaterial s and Methods.0 0, 8 mgTE T preparation in 2m l medium; t •, 8 m g TETprepara tion in 2m lmedium .A t the arrow t5 u M atebrinwa s added, and fluorescence followed ,TE T preparation; ,T E preparation.A t arrow t0.3 4 umoleH„ 0wa s added toa TE T pre paration and 3.4 ymoleH„ 0 to aT E preparation.A t +anaero - biosiswa s reached.Th e fluorescence of both preparations in the absence of atebrin are given in the corresponding • • and 0 0 curves.Th e corresponding arrows indicate the time ofH- 0additio n (corresponding amounts) and anaerobiosis. Table Ian dFig .5 sho w thatTE Tpreparation s alsooxidiz e pyruvate,bu t thatther e isn oconcomitan t energizationo fth emembrane .Thi s lacko f energization isno tsurprisin g sinceTE Tpreparation sar eno tselectivel y permeablefo rsubstrate san dar ethu spermeabl efo rprotons . This couldb eth ereaso nwh yn onitroge n fixationca nb eobserve daero - bicallyo ranaerobicall y ina TE Tpreparatio nwhil eth econdition sfo r nitrogenfixatio nar eoptimal :organi c substrateswhic h introduce high rationso freduce d tooxidize d pyridinenucleotides ,togethe rwit ha high ratioo fAT Pt oADP .Suc hcondition sar eno tpossibl e eveni nwhol e cells. 57 A furtherattemp twa smad e toobtai na preparatio n inwhic h theoxida tiono forgani c substrates couldb e coupled tonitrogenase . Since it was observed thatcrud enitrogenas epreparation s becomever y sensitive the oxygen inth epresenc e ofreducin g agents (dithionite or oxidizable substrates),w eworke d under anaerobicconditions .Pyruvat e is chosen as substratebecaus e iti sfoun d thatunde ranaerobi ccondition spyru vateoxidatio nca nb e coupled tonitrogenase ,althoug h theactivit y is low (H of theactivit ywit hpyruvat eunde r optimal aerobic conditions). Cells arebroke ngentl yb y treating themwit h lysozyme inth epresenc e ofTris-EDTA . During cell-lysis theactivit ywit hpyruvat e decreases to zero indicating thateve nthes emil d conditions finally causecomplete ly loss ofcouplin gbetwee npyruvat e oxidationan dnitrogenas e activity. Coupling isno trestore db y addingo fa variet y ofcofactors ,bu t full nitrogenase activity isobserve dwit hdithionit e andATP .However , there issom e evidence for intermediate structureswhic h respond to substrates thatar e thought toinfluenc e therati oAcety l CoA/CoASH. During thecours eo f the lysis reaction oxaloacetate causea 2-fol d stimulation ofactivit y and 6-hydroxy-butyrate and acetylphosphate causea ninhibition .Non e of thesecomponent sha s anyeffec to nth e activity ofwhol e cells.I tappear s therefore that,a ta n intermediate stageo f lysis,th ecel lbecome spermeabl e to lowmol .wt .compounds . These observationemphasis e thestron g dependence ofnitrogenas e acti vitywit hphysologica l substrates ona n intactcytoplasmi cmembran e and suggest thatth erati oAcety lCoA/CoAS Hca nb e important inth ecoup lingwit hpyruvat eoxidation . ACKNOWLEDGEMENTS Wewis h tothan kDr .S.G . Mayhewfo rhi sver yusefu l comments during this investigationan dMis s L.A.M.Wolber s for thehel p insom eexperi ments.W ewis h tothan kV .Massa y forpermissio nt oappl yhi smetho d of flavodoxinreductio nprio r topublication .Th epresen t investigation was supportedb y theNetherland s Foundationfo rChemica l Research (S.O.N.)wit h financialai d fromth eNetherland s Organization for the Advancement ofPur eResearc h (Z.W.O.). 58 LITERATURE Ackrell,B.A.C . and Jones, C.W. (1970)Eur . J. Biochem., 20,29-35 . Benemann,J.R. , Yoch, D.C., Valentine, R.C. and Arnon, D.I. (1969) Proc. Nat.Acad . Sci. 64, 1079-1086. Benemann, J.R., Yoch, D.C., Valentine, R.C. and Arnon, D.I. (1971) Biochim.Biophys . Acta 226, 205-212. Bresters,T.W. ,D e Abreu, R.A., De Kok,A. , Visser, J. and Veeger, C. (1975) Eur. J. Biochem., 59, 335-345. Brill,W.J. , Westphal, J., Stieghast,M. ,Davis ,L.C . and Shah,V.K . (1974) Anal.Biochem. , 60,237-241 . Bulen,W.A . and Le Comte,J.R . (1972)Meth .Enzym .XXIV , 256-470. Dalton,H . and Postgate, J.R. (1970) J. Gen. Microbiol., 54, 463-473. Drozd, J. and Postgate, J.R. (1970) J. Gen.Microbiol. , 63,63-73 . Goldman, R. and Katchalski,E . (1971) J. Theor. Biol., 32.,243-257 . Haaker, H., De Kok,A . and Veeger, C. (1974) Biochim. Biophys. Acta 357, 344-367. Haaker,H . and Veeger, C. (1976) Eur. J. Biochem., 63,499-507 . Jackson, R.W. and Demoss,J.A . (1965) J. Bacteriol., 90, 1420-1425. Jones,C.W. ,Brice ,J.M. , Whight,V . and Ackrell, B.A.C. (1973) FEBS Lett. 29,77-81 . Jones,C.W . and Redfearn, E.R. (1966) Biochim. Biophys. Acta U3, 467-481. Kedinger, C, Gissinger, F. and Chamban,P . (1974)Anal . Biochem., 60, 237-241. Kelly, M. (1969) Biochim. Biophys. Acta 191,527-540 . Ljones,T . (1973) Biochim. Biophys. Acta 321, 103-113. Oppenheim,J. , Fisher, R.J., Wilson, P.W. and Marcus, L. (1970) J. Bacteriol., 101,292-296 . Senior, P.J. and Dawes,E.A . (1971) Biochem. J., 125,55-66 . Shah,V.K. , Davis,L.C . and Brill,W.J . (1972) Biochim. Biophys. Acta, 256, 498-511. Yates,M.G . (1970) FEBS Lett. 8, 281-285. Yates,M.G . (1971) Eur. J. Biochem., 24,347-357 . 59 Yates,M.G . (1972)FEB S Lett. 27,63-67 . Yoch, D.C., Benemann,J.R. , Valentine, R.C. and Arnon, D.I. (1969) Proc. Nat.Acad . Sci. 64_, 1404-1410. Yoch,D.C . and Arnon, D.I. (1972 a) J. Biol. Chem. 247,4514-4520 . Yoch,D.C . (1972 b) Biochem. Biophys. Res. Commun., 49,335-342 . Yoch,D.C . (1975) Arch. Biochem. Biophys., 170,326-333 . 60 IV Involvement of the cytoplasmic membrane in nitrogen fixation by Azotobacter vinelandii H. Haaker and C.Veeger ,Dep .o f Biochemistry,Agricultura l University, DeDreije n 11,Wageningen ,Th eNetherlands . SUMMARY 1. Azotobaater vinelandii cellswer e rupturedb yusin ga Frenc h pressure cell,b ylysozym e treatment,o rb yosmoti c shock.Th erate so fsedimen tationo fnitrogenas e from theseextract swer ecompare dan drelate dt o therate so fsedimentatio no fmarke r enzymes.N oevidenc ewa sfoun dfo r •th eso-calle d particulate nitrogenaseo ra ninteractio nbetwee nnitroge nasean dcytoplasmi c membrane vesicles.Nitrogenas e sediments asa com plexan dth erat eo fsedimentatio n iscomparabl ewit h thato fpyruvat e dehydrogenase complex. 2. Theoxyge n stabilityo fnitrogenas e from Azotobaater isno tcause db y thepresenc eo fcytoplasmi cmembrane s inextract sbu tb ya complexatio n ofth enitrogenas e componentswit ha nFe- Sprotein .Recombinatio nexperi ments showed thatmaximu m stabilization againstoxyge nwa sonl y obtained at certainratio so fth eFe-S-protei nan dnitrogenas e complex. Iti s proposed thatth eswitch-of f statei n Azotobaater cells iscause db yth e oxidationo fflavodoxi nhydroquinon e rather thanb ya reversibl e inactivationo fth enitrogenase . 3. Amembrane-boun d NAD(P)H-flavodoxinoxidoreductas ewa sdetected . Evidencewa sobtaine d thatflavodoxi nmigh t alsob emembrane-bound . 4. Aproposa l isgive nfo relectro ndonatio nt onitrogenas e in Azotobaater. Inthi sproposa l thegeneratio no freducin g equivalents fornitrogenas e is localized inth ecytoplasmi cmembran ean dmediate db yth eNADH-flavo - doxinoxidoreductase .Th ep Hgradien twhic hi sgenerate db ymembran e energization,i suse dt oreduc e flavodoxint oit shydroquinon e formi n ordert oachiev ea redoxpotentia l lowenoug ht oreduc enitrogenase . INTRODUCTION Iti sgenerall y accepted thatnitroge nreductio ni sa nanaerobi cprocess , and thatorganism swhic h requireoxyge nt oobtai nenerg yfo rnitroge n fixation havemechanism s thatprotec tnitrogenas e fromth edeleteriou s effectso f oxygen.Tw oprotectio nmechanism s havebee npropose d for Azotobaater spp (1), 61 both involving thecytoplasmi c membrane;augmente d rateso frespiratio nt o scavenge excess oxygen (respirationprotection )an da conformationa l stateo f nitrogenase thatprevent s damageb yC L (conformational protection). Some information isavailabl e about respirationprotectio n (2-5),bu tth elitera tureabou tth econformationa l protectiono fnitrogenas e issomewha t confusing. Forexampl e itha sbee n assumed thatafte r ruptureo f Azotobaatev cellswit h a Frenchpressur e cell,th enitrogenas e inth eresultin g crude extracti s oxygen tolerant (6-8)an dparticle-boun d (9,10).O nth eothe rhan d Oppenheim et al. (11)showe d thatafte rruptur eo fth ecell sb yosmoti c shock,nitroge nase isoxygen-labil ean dsoluble .Thes e authors,a swel la sDroz dan dPost - gate (2),suggeste d thatth eparticle-boun d nitrogenasema yrepresen tth e conformational protectednitrogenase .I ncontras tt oOppenhei me tal . (11), Reede tal . (12),reporte d thatth esedimentatio nbehaviou ro fth enitroge nase isindependen to fth emetho do fcel lrupture .A furthe r functiono fth e cytoplasmic membrane inobligat e aerobicnitrogen-fixin gorganism sma yb eth e generationo freducin g equivalents forth enitrogenase .Yate san dDanie l(13 ) andBiggin san sPostgat e (14)reporte d thati nmembran epreparation slo w nitrogenase activity inth eabsenc eo fartificia l electroncarrier s couldb e observed.W ehav e shown (15)tha tth eenergize d cytoplasmicmembran e isin volved inth egeneratio no freducin g equivalents forth enitrogenas e inintac t cellso f Azotobaatev vinetandii. Inthi s studyw erepor to nth einteractio no fth ecytoplasmi cmembran ewit h thenitrogenas ewit hrespec tt oth elocalisatio no fnitrogenas ean dth erol e ofth ecytoplasmi c membrane inth eprotectio nagains t oxygenan dth eelectro n donationt oth enitrogenase . MATERIALSAN DMETHOD S Growth conditions andenzym e preparations Azotobaatev vinelandii ATCC47 8wa sgrow ni na batc h cultureo f250 0liter , harvested duringth elogarithmi c phasean dstore da sdescribe d earlier (16). Crude extractswer eprepare d inthre eways . 1. Mechanically,usin ga Manto nGauli nhomogenizer ,a Frenc hpressur e cell. 2. Enzymicallyb yincubatin g 1.0g o fbacteria lprotei ni n4 0m l1 0m M EDTA-Tris,p H8.0 ,wit h4 0m g lysozyme.Afte r 120mi na t24 °C ,MgCl 2 was addedt o1 1mM .Th epH ,whic hdroppe d to6 ,wa sbrough twit hTrizm a base (Sigma)t op H7. 4an d0. 1mg/m ldeoxyribonucleas ewa sadded .Afte r a further3 0mi nth epreparatio nwa suse dfo rcentrifugatio n experiments. 62 3. Osmoticallyb yth emetho ddescribe db ySha he tal . (17). Centrifugation experimentswer eperforme dwit ha nMS E7 5ultracentrifug e using an8 x 3 5m lrotor .Afte r each centrifugation stap,th esupernatan t plus fluffy layerwa suse dfo rth enex t following centrifugation step.Th efollowin g centrifugation schemewa sused : 20,000x g ,3 0min ,P, ;100,00 0x g ,3 0min , P2; 200,000x g ,6 0min ,P_ ;200,00 0x g ,24 0min ,P 4an d S^. Alloperation s were doneunde rargon ,pellet swer e suspended in2 5m MN-tri sQvydroxymethy l methyl-2-aminoethaneI)sulfoni c acid-KOH (TES-KOH),1 m MMgCl 2,p H7.4 .Th e P1 pelletswer ewashe d twicewit h2 5m MTES-KOH ,1 m MMgCl 2,p H7.4 . Nitrogenase complexwa spurifie da sdescribe db yBule nan dL eComte ,til lth e secondMgCl -precipatio n (C42-1 ) (18).Th enitrogenas e complex,C 42-1 ,wa s separated into3 fraction sb yDEA E cellulose chromatography. Thefirs tfrac tionwa selute dwit h0.1 5M NaCl,th esecon d fractionwit h0.2 7M NaC lan d a third fractionwit h0. 4M NaCl.Ferredoxi nan dflavodoxi nwer epurifie da s describedb yYoc han dArno n (19).Fres hgrow ncell swer euse dt oprepar e toluene treated Azotobaoter cells. 100-200m gbacteria l proteinwa ssuspende d in5 0m MTris-HCl ,1 0m MEDTA ,0. 2m ltoluene ,fina lp H8.0 ,fina lvolum e 20ml .Th esuspentio nwa shel d anaerobically at0 C fo r1 5min .Afte rcentri fugation (10,000xg ,5 min. )a t4 C ,th epelle twa swashe dwit h 40m l2 5m M Tris-HClp H7. 4an d5 m MMgCl- ,an dcentrifuge d again.Th epelle twa sresus - pended inth esam ebuffer ,store da t0°C ,sonicate d ortreate dwit h 1mg/m l lysozymea t3 0C fo r6 0min . Analyticalmethod s Acetylenereductio nassay swer eru na t3 0, shakin g in6. 5m lbottle s sealed with Subaseal,whic h contained amixtur eo f2 5m MTricin ebuffer ,1 m MATP , 2m MMgCl 2, 10m Mcreatine-phosphat ean d5 U creatin e kinase finalp H7.5 . Thismixtur ewa sthorougl y flushedfo ra tleas t3 0min .wit hpurifie d argon whichpasse da heate d Basfcatalyst .Th eacetylen e (10%)an d2 0m M dithionite were added.Th emixtur ewa sequilibrate d for1 0min .wit hth egasphas et o removeth elas ttrace so foxygen ,afte rwhich th enitrogenas e preparation was addedan dth eethylen eproduce dwa smeasure da sdescribe d earlier(15) . Several amountso fa nitrogenas epreparatio nwer e analysed toexclud ea n underestimationo fth enitrogenas e activity causedb ylo wnitrogenas e concen trations. Photoreactionswer eperforme d inth enitrogenas e assaymixtur ewit h4 0y M deazaflavin (3,10dimethyl-5-deazaisoalloxazine) ,1 0m Mglucose ,1 0U glucose 2 oxidasean d4 0y gcatalase/ml .Th eligh t intensitywa s2 0mW/c m measured between 400-500nm .Th eNAD Han dNADP Hoxidas e activitieswer emeasure da s 63 describedb yAckrel lan dJone s (3). Acridine fluorescencewa smeasure d atp H 7.5 ina 2m l incubationmixtur e containing 25m MTES-KOH , 1m M MgCl2, 10y gcatalase ,2 m M NADHan d 5 \M9-amino-6-chloro-2-methoxyacridinehydro - chloride.Pe r incubation 0.5 -1. 0m gbacteria lprotei nwa s added.Th e fluorescencemeasurement swer eperforme dwit h aPerki n Elmer fluorimeter, excitationwavelengt h 410nm ,emissio nwavelengt h 490nm .Th e amount ofquench ing obtainedupo nenergizatio n of the cytoplasmicmembran ewa s calculated from thedifferenc ebetwee n the fluorescence after allo f the oxygenha dbee ncon sumed,an dth efluorescenc eunde raerobi c conditions.A t theprotei nconcen trations andwavelength s used, the contribution to the fluorescence of bacterial componentswa snegligible . A highly effectiveNAD(P) Hregeneratin g systemwa s obtainedwit h 25m MTES-KOH , 5 mMMgCl , 10m Mglucose-6-phosphate , 20ug/m lglucose-6-phosphat e dehydro genase,0.2 2 mg/ml transhydrogenase,0.2 5 mMNAD P and 0.25 mMNA D ,fina l pH 7.5.Pur e transhydrogenase of Azotobaater vinelandii wasuse d (21). NADH-flavodoxinoxidoreductas ewa smeasure d in2 5m MTES-KOH , 1m MMgCl, , 10m Mglucose ,0. 5 mg/mlglucos eoxidase ,1 0ug/m lcatalase ,2 m MNAD(P) H and 15y M flavodoxin,fina lp H 7.4.Th eabsorbanc e increase at 580n mwa s monitored. For the calculationsw euse d the following molar extinction coefficients for Azotobaater vinelandii flavodoxin:E.rrOX ^ 10,600M cm , Er„0semiquinone= 5.540M cm" (22). Transhydrogenase wasmeasure d according toKru l (21), pyruvate dehydrogenase according toSchwart zan dRee d (23),glucose-6-phosphat e dehydrogenase according toSenio r andDawe s (24)an dNAD Hdehydrogenas e according to Yates (25)usin gbenzylviologe n asth eelectro nacceptor . Nitrogenasewa s exposed toai r in2 5m MTES-KOH ,p H 7.5. Prior tooxyge n inactivation,crud ecomponen t Ian dcrud e component IIan d stabilizing factor werebrough t togetherunde r argon.Complexatio nwa s completeafte r 30mi na t room temperature.Th e incubationmixtur ewa s thenrapidl y aeratedb yusin g a whirlmixer .Test s showed thatwithi n 5se c the incubationmixtur ewa s satu ratedwit h air.Afte r 1min ,a n0. 1 ml samplewa s examined for nitrogenase activity.Anaerobi c DEAEcellulos e chromatography wasperforme d according to Shahe tal . (17).Th eeluate swer e anaerobically concentrated and desalted with anAmico nultrafiltratio n cellusin g aU M 10filter .Anaerobi c gel electrophoresiswa sperforme d asdescribe d previously (20)exep t that thioglycolic acidwa s replacedb y o.o4m M indigosulfonic acid and 2m MNa2S2C> 4 was added to theuppe rbuffe r afterflushin gwit h argonfo r3 0min . About SO ugo fprotei nwa s applied/gel. 64 Biochemicals All enzymes andcofactor swer epurchase d fromBoehringer .Deazaflavi nwa s a gift fromV .Massa y and S.Ghisla . RESULTSAN D DISCUSSION LOCALISATION OF NITROGENASE Thesedimentatio nbehaviou ro fnitrogenas ewa s determined after exposure A.vinelandii cells tothre edifferen tbreakag eprocedure s and correlated with thesedimentatio nbehaviou r ofa numbe r ofothe r enzymeactivities(Fi g 1). Theknow ncharacteristic s ofth emarke r enzymes allowed the fragmentation, causedb y eachcel lruptur emethod ,t ob e assessed. NADHoxidas ewa suse d asa marke r enzyme for thecytoplasmi cmembranes . Its activity correlatedwel lwit h theamount s ofcytochrome s present ina particu latepreparation .Th emutua l ratios of thecytochrome swer e the same inal l membranepreparation s indicating thatthe y arepart s of thesam emembrane . Fig. 1clearl y shows thatdurin gbreakag eo f thecell swit h aFrenc h pressure cell,th ecytoplasmi cmembran edegrade s intoa largenumbe r ofpieces ,an d thatenzymi cattac ko nth epeptidoglyca n layerha s asimila r effect.Durin g lysozymetreatmen tn omechanica l orosmoti cforce s areexerte d on thecells , only therigidit ydu et oth epeptidoglyca n layer is lost.Thi s effect is probably enough tocaus eth e formationo fvesicle s and fragments fromth e cytoplasmicmembrane .Th eosmoti cshoc k isa relativel ymil d cell rupture method,whic hha s littleeffec to nth epeptidoglyca n layer,sinc emos t of thecytoplasmi cmembran e remainsattache d toi t (11).Thes e experiments show thatth esedimentatio nbehaviou r ofcytoplasmi cmembran evesicle s and fragments isdetermine db y theefficienc y ofruptur eo fth e peptidoglycan layerdurin g cellbreakage .Th eNAD Hoxidas e activity observed in cytoplasmic membranes prepared afterbreakag eo fth ecel lwit h aFrenc hpressur e cell is remarkablyhigh .A n explanationma yb e thehighe r degree of inside-out orientation ofthes emembrane s comparedwit h themembrane s preparedb y lyso zyme treatmento rosmoti cshoc k (26).Sinc eth ecytoplasmi cmembran e isimper meable forNADH ,on eca nexpec ta highe rNAD Hoxidas ewit h theconverte dvesi cles. Ingenera l acridinefluorescenc e canb euse da sa prob e for the energized stateo fa numbe r ofcytoplasmi cmembranes ,includin g Azotobaeter membranes (27).Upo nenergization ,th edy ebind s toth emembran e and the fluorescence 65 0) „ (u • aw— u CeoU N^ CcoU CGU 4-co1 •- ct) 4CJ aC*r4c0UCU>*.cflcU >»cU4-icuct)cGrnco N -H Ll Li ct) oo •H 66 isquenched .W e checked thatunde rou rcondition s thequenchin g ofth efluores cence isno tdependen t onth eprotei nconcentration ,bu to ncouplin gbetwee n oxidationan denergization ;additio no fa lo wconcentratio no funcouple r caused aretur no ffluorescence .Cytoplasmi cmembrane s canb edeplete d of ATP-aseb y incubating themembrane s ina mediu m of lowioni cstrength . Such ATP-asedeplete dmembrane s dono tsho wquenchin g ofacridin e fluorescence uponenergization ;bu t this canb e restoredb y adding crudeATP-as e (27, 28). Weuse d this fluorescencemetho d todetermin ewhethe r themetho d ofcel l ruptureremove d tightlyboun d enzymesuc ha sth emembrane-boun dATP-as e and todemonstrat e closedvesicle s inmembran epreparation s inwhic h oxidation canb e coupled toenergization .Contro lexperiment s show,ATP-as e activity inpreparation swher e acridine fluorescencequenchin g isobserved .ATP-as e remainsboun d toal lcytoplasmi cmembrane spreparation s exceptP ,an d S. obtained fromosmoti cshocke d cells;th eP.. ,P -an dP ,pellet s obtained by thisruptur e method are,however ,wel l coupled. Some small membrane vesicles arepresen t inS .derive d froma Frenc hpressur e cellan d lysozyme ruptured cellextract ,sinc edurin g centrifugation thefluff y layers are takenwit h thesupe rnatants . Inmos torganism s transhydrogenase ismembrane-boun dan d theproductio n of NADPHfro mNAD Hca nb e drivenb y energy. Iti spossibl e tosolubiliz e theen zymebu t iti s thenunabl e tocatalys e theenergy-linke d reductiono fNADPH . In Azotobaatev, transhydrogenase isreadil y solubilized,bu twhe n the cells areopene dmor e gently (lysozymean dosmoti c shock)transhydrogenas e is found inP . andP 2 (Fig- !)• Inthes emembran epreparation s energy-linked transhydrogenase activity ismeasurabl e (29,H .Haake runpublishe d observation). Purified transhydrogenaseha s aminimu mmolecula rweigh t of 54,000bu t in thepresenc e ofCaCl 2 theenzym eaggregate s (21).Therefor e iti sno tpossibl e todiscriminat ebetwee nmembran eboun d transhydrogenase orhighl y polymerized transhydrogenase inth eP ,an dP .sediments .Fro m theresult spresente d in Fig. 1w epropos e tous e transhydrogenase asa marke r for loosely membrane- boundenzymes . The isolatedpyruvat edehydrogenas e complexfro m Azotobaeter (minimummol . weight 1.10 ,c.f.ref . 16)sediment s after 4h . 200.000 xg.Th e sedimentation behaviouro f thecomple x inou rpreparation s issimila r indicating that it doesno t forma nassociatio nwit h thecytoplasmi cmembrane .Therefor e the pyruvate dehydrogenase complex canb euse d asa marke r forenzym ecomplexes . Theglucose-6-phosphat e dehydrogenase sedimentationcharacteristic s are thoseo f arelativel y smallenzym e (mol.weigh t + 100,000). Glucose-6-phos- 67 phate dehydrogenase canb euse d asa marke r forsmal l solubleenzymes . With thisse t ofmarke r enzymes,w e think iti spossibl e toanswe r the questionwhethe rnitrogenas e in Azotobaoter issolubl e orparticulat e and whether itsdistributio n inthes e fractionsdepend s onth emetho duse d to rupture thecells . Fig. 1clearl y shows thatth enitrogenas e sedimentationbehaviou r isindepen dento f thecel lruptur emetho d andtha tal lnitrogenas e activity occurs in thepelle tafte r centrifugation ofth ecel l extracts 4hour s 200,000xg . It isals oclea r thatwhe n thecell s arerupture db y amechanica lmetho d the cytoplasmicmembran e desintegrates,an d fragments sediment togetherwit h the nitrogenase,producin g theso-calle dparticulat e nitrogenase. Some association withmembran e fragmentsma y occurbu t ifs o iti sonl y awea k interaction. Thisconclusio n issupporte db y experimentswhic h confirm thoseo fRee d et al. (12)wh o showed thatwhe na nitrogenase-containin g sediment,i nou r caseP. , isbrough t ona linea rsucros e density gradient,nitrogenas eactivi tyseparate s from fragments of thecytoplasmi cmembrane .Nitrogenas e activity isfoun dnea r the topo fth egradient , (0.6M sucrose)an d thecytochrome s in themiddl e of thegradient , (around 1.6M sucrose).Anothe r indication that inth eso-calle dparticulat enitrogenase ,ther e isn o interactionbetwee n nitrogenase and thecytoplasmi cmembran e isth eobservatio n thatnitrogenas e activity isonl yfoun d inth eP .sediments .Th e activity found inth eothe r sediments canb eremove db ywashin g thesediments ,an d further cytochrome analysis indicates thatal lsediment s arepart s ofth esam e cytoplasmic membrane. Some importantaspect shav e tob ementione d inorde rt ocom et oa prope r interpretation ofth esedimentatio nbehaviou r of thenitrogenase . 1. Thenitrogenas e concentrationo fth eextract s iso fgrea t importance, especiallywhe n thecell s arerupture db y osmotic shock.Lo wprotei nan d highsal tconcentration s causedissociatio n of thenitrogenas e complex. Theseparate d components areunstable ,mor e oxygensensitiv e and therat e ofsedimentatio n islowe rtha ntha to f thecomplexe dcomponent s (30).T o compare therat eo fsedimentatio n of thenitrogenas e inextract s preparedwit hdifferen tcel lbreakag emethods ,th econcentratio n of nitrogenase inth eextrac tmus tb e of thesam eorder .Therefor ew euse d theosmoti c shockmetho d asdescribe db y Shahe tal . (17)an dno t the osmoticshoc kmetho d asdescribe db yOppenhei man dMarcu s (31)whic h introduces ahig h saltconcentratio n andextrem edilution . 68 2. Cellruptur eb y lysozymeo rb y osmotic shock isno t complete and a largequantit y ofun-lyse dcell s occurs inth e firstpellet . Sonification of thesepellet s indicates that theamoun tmigh tvar ybetwee n 10-30%. Therefore theenzym e activitieswhic h canb edetecte d inth ewashing s from evenextensivel ywashe d pellets,ma y come fromresidua l un-lysed cells. 3. During ourstudie sw e found that thep Hals odetermine s therat e of sedimentation ofnitrogenase .A t lowp H (pH=6.0)al lnitrogenas e activity issedimente dafte r 120min . 200,000 xg.Sinc e rupturing the cells, especiallywit h aFrenc hpressur e cell, lowers thep Ho f theextract , iti snecessar y toreadjus t thep H to 7.4.Thi sp Heffec t might explaindifference s inth erat e ofsedimentatio n of the nitrogenase whichhav ebee nreporte d inothe rpublication s (9,10). Whenartifact s due topH ,hig h concentrations ofsalt s orextrem e dilution areavoided ,w e canconclud e from Fig. 1,tha t tightlymembrane-boun d enzymes such asATP-as e remainboun d toth ecytoplasmi cmembrane ; transhydrogenase canb econsidere d asa loosely-bound membrane-enzymei n Azotobaoter; therat e ofsedimentatio n ofnitrogenas e is independent ofth emetho d used toruptur e thecell san d similar totha to f thepyruvat e dehydrogenase complex. Nodirec tassociatio nwit h thecytoplasmi cmembran e canb eobserved ,bu t our experiments cannotexclud e thepossibilit y that in vivo eithernitrogenas e orth epyruvat edehydrogenas e complexar eweakl y associated with thecyto plasmicmembrane ; it is likely thatnitrogenas e sediments asa comple x thati s larger thana simpl e 1:1 complexo fcomponen t Iplu s component II (mol. weight =300,000) . Therat eo fsedimentatio n ismuc h faster thantha t of glucose-6-phosphatedehydrogenas e (mol.weigh t =100,000 )an dabou t the same asth epyruvat edehydrogenas e complex (mol.weigh t 1.10) .Ou r experiments confirm the ideatha tnitrogenas e in Azotobaoter ispresen t as a complex (18,32 )whic h aswil lb e shownbelo w exists ofa t least three proteins (component Ian d IIplu s an Fe-Sprotein ),whic hperhap s ispresen t in vesicularmembrane s (12). OXYGEN STABILIZING FACTOR Oppenheime tal . (11)suggeste d that themetho d ofcel l rupture is important indetermin gwhethe r orno tnitrogenas e incrud eextract s issensitiv e to oxygen.Base d onthes e experiments,Droz dan dPostgat e (2)speculate d that the cytoplasmicmembrane smigh tb e involved inth eprotectio n ofnitrogenas e against oxygen. Ina previou spape r (20),w e showed thatth emetho d ofcel l 69 \ Fig. 2. Anaerobic non-denaturing polyacrylamide gel electrophoresis of nitrogenase complex before and after treatment onDEA E cellulose. Gels are prepared asdescribe d inMethods .Pattern s are from the following preparations.A ,nitrogenas e complex (C 42-1); B, 0.15 M NaCl fraction; C, 0.27 M NaCl fraction;D , 0.40 M NaCl fraction. rupturei sno timportant ,bu ttha tth esal tconcentratio nan dth econcen trationo fnitrogenas e determine theoxyge n labilityo fpreparations .Yates(30 ) showed thata fractio nwit hmembrane-boun d NADHdehydrogenas e activityprotect eda partiall y purified preparationo fnitrogenas e against oxygen inactivation. Wehav e re-examined thisquestio nusin ga sstartin gmateria la noxygen-stabl e nitrogenase complex (C42-1) ,prepare d according toBule nan dL ecomt e (18) from extractsmad ewit ha Frenc hpressur e cello rb ya nosmoti c shock.Thi s 70 complexshow sa t least9 protei nband safte relectrophoresi s ina polyacryl - amidegel ,indicatin g thatsuc ha nitrogenas e complex isfa r frompure . Severalenzym eactivitie sar edetectabl e e.g.NAD Hoxidase ,0.2 1 ymoles -1 -1 -1 -1 NADH.mi n ,m g ;NADP Hoxidase ,0.1 6 ymolesNADP Hmi n .m g ;transhydro - -1 -1 genase,3 nmole ss-NADH .mi n .m g ;NAD Hdehydrogenase ,0.1 9 ymolesbenzyl - -1 -1 viologenreduce dmi n .m g ;NADP Hdehydrogenase ,1 6nmole sbenzylviologe n -1 -1 reducedmi n .m g ;NADH-flavodoxi noxidoreductase ,1 0nmole s flavodoxin semiquinonemi n .m g ;NADPH-flavodoxi noxidoreductase ,0. 9 nmoles flavo- -1 -1 doxinsemiquinon emi n .m g andnitrogenas eactivity , 125nmole sethylen e . -1 -1 mm .m g . Whenth ecomple x isadsorbe do na DEA Ecellulos ecolum nan delute dwit h Tris-HClbuffe rcontainin g successively 0.15M , 0.27 M and0. 4M NaCl,th e elutionpatter nobserve d issimila r totha tdescribe db yBule nan d LeComt e (18).Analysi so feac hfractio nb ypolyacrylamid e gel electrophoresis showtha tal lo f thecomponent s inth e initialcomple xca nb e accounted for (Fig.2 b,c,d) ,bu ttha ta fewne wband sappea r (band3a ,9a ,b,c) . It mustb enote dtha tcrud enitrogenas ecomponen t II (Fig.2D )contain s large amountso fcomponen t I,eve nafte rextensiv ewashin go fth eDEA Ecellulos e columnwit h 0.27M NaCl;evidentl y thisconcentratio no fNaC ldoe sno tcom pletelydissociat e thenitrogenas ecomplex .Recombinatio no fcrud ecomponen t I withcrud ecomponen t IIgive sgoo dnitrogenas eactivit y (0.132m gcrud e I +0.0 7 mg crude IIpe rm l gavea nactivit yo f35. 8nmole s C^H,forme dpe r min). Asknow nfro mth e literaturean d showni nFig .3 ,suc ha reforme d complexi sextremel yoxyge nlabile .However ,i tca nb e stabilizedb y addition ofth efractio nelute d fromth eDEAE-cellulos ecolum nwit h 0.15 MNaC l (Fig.3) .Ther e isa noptima lconcentratio no fthi sfractio ntha tgive s maximumprotectio nagains toxygen .Th elarges tprotei ncomponen t inthi s fraction istha tcorrespondin gwit hcomponen t 8i nth eorigina lcomple x (compareFig . 2aan d 2b); component 8i suniqu et oth e0.1 5 MNaC lfractio n and therefore itseem s likelytha tthi scomponen t isresponsibl e forth esta bilizing effect.Furthe revidenc e forthi s isobtaine db y testinga further fractioni nwhic hth erati oo fcomponen t 8t ocomponent s 1,3,4 and 5wa s different fromtha t inth efractio nuse d inth eexperimen to fFig .3 .Th esta bilizing effect isrelate d toth econcentratio no fcomponen t 8an dno tt oth e othercomponent s inth efraction .I nadditio nt o itseffec to nth eoxyge nsta bilizationo fnitrogenase ,th e0.1 5M NaClfractio nals oprotect sth eenzym e against inactivationtha toccur sa t lowenzym econcentrations .Th eamoun to f thisfractio ntha tgav emaximu mstabilizatio n isth esam ea stha trequire dfo r 71 0.15 0.30 0.45 0.60 mgstabilizin gfacto r mg nitrogenasecomple x Fig.3 .Influenc e ofstabilizin g factoro nth eoxyge n stability ofa nitro genase complex reconstituted from crude components,Oxyge n inactiv- ationwa sperforme d asdescribe d inMethods .0.12 0m gcrud e component I plus0.0 3m gcrud e component IIwer emixe d withvariabl e amounts ofth e0.1 5M NaC l fraction; ina volum eo f1 ml. stabilization against oxygen.Thes e resultsan dou rearlie r results (20)strong lysugges t thatprotectio no fth enitrogenas e against oxygeni n Azotobacter is notcause db yth epresenc eo fcytoplasmi c membrane fragmentsbu tb ya com - plexationo fth enitrogenas eprotein s I+ I Iwit h component 8,producin gth e so-called conformational-protectednitrogenas e (1).W ehav eals o shown that nitrogenase in A.vinelandii isfoun dt ob eoxyge ntoleran t (20)independen t onth ecel lruptur emethod .Th eoxyge nstabl ecomple xha sa hig h activitywit h flavodoxinhydroquinone .Thu sw epropos e that in vivo nitrogenase isprotecte d against oxygen inactivationb ycomplexatio nwit h component 8(Fig .2) .Sinc e flavodoxinhydroquinon e ishighl y oxidizableb yCL ,w epropos e thatth e switch-off statei n Azotobacter iscause db yth eoxydatio no fflavodoxi n hydroquinone rather thanb ya reversibl e inactivationo fth enitrogenas ea s proposedb yDalto nan dPostgat e(1) . Wehav e triedt ocharacteriz e component 8.Whe nth e0.1 5M NaC l eluateo fth e DEAEcellulos e columni sapplie do na Sephade xG7 Scolum nan delute d with 25m MTris-HC1 ,p H7. 5severa l fractionsar eobtained .Th emai n fraction was analysed. ItsE.P.R . spectrum,optica l absorption spectruman dcircula r dichroismspectrum ,sho wi ti sa nFe- Sprotei n (33).It smol.weigh ta sdeter minedb ysodiu mdodecylsulphat e gelelectrophoresi s andwit ha Sephade xG10 0 72 column is 24,000.Thos echaracteristic s indicate that component 8i s Azotdbaatev Fe-Sprotei n IIa s isolatedb y Shethnae t al.(34) . G. Scherings inou r laboratorium (33)ha s shown that inth e nitrogenase complex,C 42-1, the Fe-Sprotei n (component 8,Fig . 2A)ma yhav e functions in additiont o itsrol ei noxyge nprotectio no fnitrogenase . Itseem s to regulate thenitrogenas e activity. Table I.Distributio n ofNADH-flavodoxi noxidoreductase , transhydrogenase and NADHdehydrogenas e over cytoplasmicmembran e preparations and soluble cell fraction after cell rupture. Azotobaoter cells,con taining 2.7 g of proteinwer edisrupte d with aManton-Gauli nhomo - genizer and centrifuged asdescribe d inMethods .Th e preparations were dialysed against 25m M TES-KOH, 1m MMgC l ,p H 7.4.Th e enzyme activities weremeasure d asdescribe d inMethods . Centrifugation NADH-flavodoxin Transhydrogenase NADHdehydro oxidoreductase genase (umoles.min ) (ymoles.min ) (umoles.min ) 30min , 100,000x g pellet 3.25 10.1 598 120min , 200,000x g pellet 0.47 138.5 1587 120min , 200,000x g supernatant 151.3 224 ROLE OF CYTOPLASMICMEMBRAN E INELECTRO N DONATION FOR NITROGENASE Thephysiologica l electrondonatin g system tonitrogenas e inobligat e aerobessuc ha s Azotobaoter, Myaobaaterium flavum or Rhizobium, isstil lun known.Ther e arereport s inwhic h lownitrogenas e activity is demonstrated inmembran epreparation swit hNAD Ha selectro ndono r andwithou t artificial electroncarrier s (13,14).W e showed lowactivitie swit hpyruvat e assub strate (20),bu t a few intactcell s remaining inth emembran e preparations couldhav ebee nresponsibl e forth eactivity .W ehav epreviousl y obtained evidence that inintac tcell s of Azotdbaatev thecytoplasmi cmembrane , especially itsenergize d state,is important forelectro ntranspor t tonitro genase (15).Beneman ne tal . (35)showe d thatwit h theelectro ncarriers , ferredoxin and flavodoxin anda crud eenzym epreparation ,lo wnitrogenas e activity couldb e obtainedwit hNADP Ha selectro ndonor .Sinc e theirprepara tioncorrespond s toou rP . fractionb y Frenchpressur e rupture,i tseem s highly likelyt ou s thatcytoplasmi cmembrane swer epresen twhic h couldhav eplaye d 73 a role inthei robserve d activity. Byusin g thereducin gpowe r of illuminated spinachchloroplasts ,Arno nan d co-workers showed that ferredoxin andflavo - doxinca ncoupl ebetwee n chloroplasts andnitrogenas e (36,37). Further, Yates (38)demonstrate d thatsubstrat e amounts ofdithionite-reduce d flavo- doxinact sa sa nelectro ndono r fornitrogenase .Thes e latterexperiment s can becritisize d fromth efac t thatflavodoxin-hydroquinon e canb e oxidizedb y 2- SO, forming SCL radicals (39)whic h intur nca nb eresponsibl e for reduction ofth enitrogenase . Inorde r toavoi d thiscomplicatio nw e reduced substrate amounts offlavodoxi nphotochemicall ywit hdeazaflavi n (20)an d showed that even higher activities thanwit hdithionit e canb e obtained. Substrate levels of photochemically reduced Azotobactev ferredoxin (c.f.ref . 19)ar eno t able toac ta sa reducin g agent fornitrogenase . Table Ishow s thata NADH-flavodoxi noxidoreductas e is localizedmainl y in thephosphorylatin g membranes.Th e activity is lowi nsmalle r membrane vesicles andno tdetectabl e inth esolubl e cell-fraction. TheNADH-flavodoxi n reductase activityma yb e asid ereactio no fothe renzymes .Tw opossibl e candidates,transhydrogenas e andNADH-benzylviologe noxidoreductas e were tested andca nb e excluded (TableI) . Tabel IIshow s the influence ofth eelectro ndonatin g systemo nth e activity ofNADH-flavodoxi n oxidoreductase and onth erati oo f flavodoxin semiquinone to flavodoxinoxidize d afterreachin g equilibrium.NAD H isa bette r electron donor thanNADP Hfo rNAD(P)H-flavodoxi noxidoreductase . Byusin g thepurifie d transhydrogenase from Azotobactev, coupledwit h glucose-6-phosphatean d glucose-6-phosphate dehydrogenase aver y efficient NADPHan dNAD H regenerating system canb e obtained.Thi s systemdoe sno t increase the initialrat e and doesno t reduce flavodoxinbeyon d itssemiquinon e state.G . Scheringsha s shown (33)tha tnitrogenas e activity isobserve d onlywhe nth erati oo f flavodoxinhydroquinon e toflavodoxi nsemiquinon e ishigh . Iti sth e therefore clear that thereduce dpyridin enucleotide svi a themembrane-boun dNADH-flavo doxinoxidoreductas e areno tcapabl e togiv e anysignifican t nitrogenase activity.W e have tried toenergiz e cytoplasmicmembrane s of Azotobactev withATP ,usin g thefluorescenc e ofacridin e asa prob e for energization, butw e didno t succeed.Th e couplingbetwee nAT Phydrolysi s and energization isprobabl yto o loose in isolated cytoplasmic membranes.Th e cytoplasmic membrane canonl yb eenergize d withhig hrate s ofelectro n transport through therespirator y chain. 74 Table II.Influenc eo fth eelectro ndonatin g systemo nth eNAD(P)H-flavodoxi n oxidoreductase activityan dth eamoun to fflavodoxi n semiquinone formed.A membran e preparation ?2 wasprepare d after cell rupture witha Frenc hpressur e cella sdescribe d inMethods .Th emembran e preparationwa sdialyse d against 2x 1lite r 25m MTES-KOH , 1m M MgCl„,p H7.4 .0.4 9m gmembran e proteinwa sincubate d asdescribe d inMethods ,a spectru mwa sscanne d andth ereactio nwa sstarte db y adding flavodoxint oth esampl e cuvet.Whe nth eabsorbanc e at58 0 was constant,a furthe r spectrumwa sscanne d andth eamoun to fflavo doxin semiquinonewa scalculated .Th eNAD(P) H regenerating system isdescribe d inMethods . Electrondonatin g Initial velocity,nmole s Ratio flavodoxin semi- system flavodoxin semiquinone quinone/oxidized after formed.min-'.m g reachinga nequilibriu m NADH 4.9 0.57 NADPH 1.8 0.52 NAD(P)Hregeneratin g system 5.8 0.72 Toluene treatmentcause sbacteria l cellst obecom epermeabl efo rsmal lmole cules (40),an denzym esystems ,suc ha sth eDN Areplicas e systemo f E.coli (41),wher e labile interactionswit hth ecytoplasmi cmembran ear enecessar y formaintenanc eo factivity ,remai nactiv eafte ra toluen e treatment.W euse d a toluenetreatmen tt oinvestigat ei flabil e interactions existbetwee n cytoplasmicmembranes ,flavodoxi nan dnitrogenase ,whic hma yb elos tdurin g cellbreakage . Fig.4 ashow stha ti ntoluene-treate d Azotobaater cellsgoo dnitrogenas e activitieswit hdithionit ean dAT Pca nb eobtained .Th ephotochemica l reducing systemdeazaflavi nwit hmethylviologe na selectro ncarrie rals osup portshig hnitrogenas e activity.Thes e activities indicate thatnitrogenas e isfull yactiv ei ntoluene-treate d Azotobaater cells.Ver ylo wactivitie sar e observedwit hth ephotochemica l reducing systemalon ean di nth epresenc eo f added flavodoxin (Fig.4A) .Sinc ephotochemicall y reduceddeazaflavi nb yitsel f isa poo relectro ndono rfo rpurifie dnitrogenase ,hardl yan yactivit yi n toluene-treated cellsca nb eexpected ,b ydirec t reductiono fth enitrogenas e withinth ecell .Howeve ri nth ecas eo ftransferrin git selectron svi aendo genouscarrier snitrogenas eactivit yca nb eexpected .Adde d flavodoxindoe s not stimulatedth eactivit ypresumabl ebecaus ei tcanno t enterth ecell . Whenth ecytoplasmi cmembran e isrupture db ysonicatio no rb ya lysozym e treatment therei sa nincreas ei nnitrogenas e activitywit hdithionit ea s 75 0 5 10 15 0 5 10 15 t(min) t(min) Fig. 4.Effect s of different electrondonor s on the nitrogenase activity in toluene-treated A.vinelandii cells,sonicated-toluene-treate d A.vinelandii cells and lysozyme-toluene-treated A.vinelandii cells. Preparations weremad e and activitiesmeasure d asdescribe d inMethods . A, 1.1m g toluene-treated A.vinelandii cells,wer e suspended in 0.5 ml of theacetylen e assaymixture ,electro ndonors :o-o ,dithio - nite; x-x, light,4 0u M deazaflavin, 40y Mmethylviologen ;D-D , light, 40y M deazaflavin, 3.8 nmoles A.vinelandii flavodoxin,A-A , light, 40u Mdeazaflavin . B, 1.3m g sonicated toluene-treated A.vinelandii cells were suspended in0. 5 ml acetylene assaymixture . C, 1.2m g lysozyme-toluene-treated A.vinelandii cellswer e suspended in0. 5 ml acetylene assaymixture . B and C, same additions and symbols asA . reductant (Fig.4B) .Severa l explanationsar epossible .I ntoluene-treate d cellsth ediffusio no fth estron g inhibitorAD Pou to fth evesicl emay b e rate limitingo rth ediffusio no fdithionite ,bu tno tdiffusio no fth emor e lipid solublemethylviologen ,t oth ereducin g siteo fnitrogenas emay b e hindered.Th enitrogenas e activitywit h endogenous electroncarrier s reduced photochemically,increase s dramatically after rupturing (Fig.4B,C )th ecyto plasmicmembrane .Fig .4B, Cals o shows thatlo wamount so fflavodoxi n added toth esonicate d toluene-treated Azotobaater cells increaseth erat eo fnitro genaseactivit y considerably.Th eresult spresente d inFig .4 sho wtha ti n thepresenc eo fdeazaflavi nalone ,befor e ruptureo fth ecytoplasmi cmembrane , thelo wpotentia l electroncarrier scanno tb ereduce dan dthu smediat e reducing equivalents tonitrogenase ,indicatin g that theyar epresumabl y burried inth ecytoplasmi c membrane.Thi svie wi ssupporte db yth eobservatio n thatth eactiv e siteso fth enitrogenas ear econstantl y attainablea sth e activitywit hdithionit ean dwit h reduced methylviologen shows. 76 A combinationo fou rearlie rwor ki nwhic hw eshowe d thata nenergize d cytoplasmicmembran ei snecessar yfo relectro n transportt onitrogenase ,an d theobservation s presented inthi spaper ,tha t Azotobaoter cells containsa membrane-bound NADH-flavodoxinoxidoreductas ean dtha tth elo wpotentia l electroncarrier sma yb eburrie di nth emembrane ,allo wu st opropos eth e scheme showni nFig .5 .Th eelectro ndonatin g systemconsist so fthre ecompo nents.A membran eboun dNADH-flavodoxi n oxidoreductase;a bindin g sitefo r flavodoxino nth eNADH-flavodoxi n oxidoreductase; flavodoxin. Inadditio nw e didno tge tevidenc efo rth epresenc eo fa NAD(P)H-ferredoxi n reductase withinth ecells . Iti sgenerall y accepted thatdurin goxidativ ephosphorylatio na p Hgradien t isforme d overth ecytoplasmi cmembrane; a differenc eo ftw op Hunit s over anenergize d cytoplasmicmembran ei sno tuncommon .Th ep Ho nth einsid eo fth e cytoplasmicmembran e couldb e9 whil eth ep Hi nth emediu mi s7 . Atp H9. 0th eredo xcoupl eNADH/NAD +ha sa midpoin t potentialo f-38 0m V (Fig.5 )NADH-flavodoxi noxidoreductas e isreduce dan delectron sar e transferred toth eflavodoxin-reducin gsite ,wher e flavodoxinsemiquinon e isbound .Proto ntransfe r throughth emembran e coupledwit helectro n transport throughth erespirator y chain,lower sth ep Ha tthi ssite .A tpH= 5th emid pointpotentia lo fth eredo xcoupl e flavodoxinhydroquinone/flavodoxi nsemi - Nase(red) Nasefox) Fdhh) Em=-495(mV) ^-^ pH>7.0 NADH NAD' u>H9f> NADH-FLAVODOXIN OXIDOREDUCTASE Fig.5 .Proposa l forelectro n transport tonitrogenas e in Azotobaoter vinelandii. 11 quinonei s-38 0m V (22,42),an dflavodoxi n semiquinoneca nb ereduce db y NADH-flavodoxinoxidoreductase .Th efull yreduce d flavodoxindoe sno tbin dan d diffuses intoth ecytoso lwher eth ep Hi shighe rtha n 7.0.A ta p H> 7. 0 themidpoin t potentialo fth ecoupl e flavodoxinhydroquinone/flavodoxi nsemi quinonei s-49 0m V (c.f.ref .42 )lo wenoug ht oreduc enitrogenase . Essentialt othi shypothesi s isth eide atha tth ecytoplasmi c membrane createsa differenc ei np Ha ttw osite so fNADH-flavodoxi n oxidoreductase e.q. pH=9a tth einpu tsit e (NADH),pH= 5a tth eoutpu t site (flavodoxin). Ifthi si spossible ,the nNAD Hca nac ta sa nelectro ndono rfo rnitrogenas e in Azotobaotev. ACKNOWLEDGEMENTS Wewis ht othan kDr .S.G .Mayhe wfo rhi sver yusefu l commentsdurin g this investigationan dhi scritica l readingo fth emanuscript .L.A.M .va nZeeland - -Wolbersan dS .Zwanenbur g forth ehel pi nsom eexperiments .Th epresen t investigationwa ssupporte db yth eNetherland s Foundationfo rChemica l Research (S.O.N.)wit h financialai dfro mth eNetherland s Organizationfo r theAdvancemen to fPur eResearc h (Z.W.O.). 78 LITERATURE 1.Dalton ,H . andPostgate ,J.R . (1969)J.Gen.Microbiol . 54,463-473 . 2.Drozd ,J . andPostgate ,J.R . (1970)J.Gen.Microbiol . 6J3,63-73 . 3.Ackrell ,B.A.C . and Jones,C.W . (1971)Eur.J.Biochem . 20,29-35 . 4.Jones ,C.W. , Brice,J.M. , Whight,V . andAckrell ,B.A.C . (1973)FEB S Lett._29 ,77-81 . 5.Haaker ,H . andVeeger ,C . (1976)Eur.J.Biochem . 63,499-507 . 6.Kelly ,M . Klucas,R.V . and Burris,R.H . (1967)Biochem.J .J_05 ,3c-5c . 7.Kelly ,M . (1969)Biochem.Biophys.Act a 191,527-540 . 8.Wong ,P.P . andBurris ,R.H . (1972)Proc.Nat.Acad.Sci . 69,672-675 . 9. Bulen,W.A. ,Burns ,R.C . andL e Comte,J.R . (1964)Biochem.Biophys.Res . Commun. ]]_>265-271 . 10.Hardy ,R.W.F . andKnight ,E . (1966)Biochim.Biophys.Act a 132,520-531 . 11. Oppenheim,J. , Fisher,R.J. ,Wilson ,P.W . andMarcus ,L . (1970)J.Bact . 101,292-296 . 12. Reed,D.W. , Toia,R.E . and Raveed,D . (1974)Biochem.Biophys.Res.Commun . ^8, 20-26. 13.Yates ,M.G . andDaniel ,R.M . (1970)Biochim.Biophys.Act aJ97. > 161-169. 14. Biggins,D.R . andPostgate ,J.R . (1971)Eur.J.Biochem .j^ , 408-415. 15.Haaker ,H. ,D eKok ,A . andVeeger ,C . (1974)Biochim.Biophys.Act a357 , 344-357. 16.Bresters ,T.W. , De abreu,R.A. , DeKok ,A. ,Visser ,J . and Veeger,C . (1975)Eur.J.Biochem . 59,335-345 . 17. Shah,V.K. ,Davis ,L.C . andBrill ,W.J . (1972)Biochim.Biophys.Act a256 , 498-511. 18. Bulen,W.A . andL e Comte,J.R . (1972)Meth.Enzym. ,XXIV ,456-470 . 19.Yoch ,D.C . andArnon ,D.I . (1972)J.Biol.Chem . 247,4514-4520. 20. Haaker,H. , Schering,G . andVeeger ,C . (1976)Proc.I I International symposium onNitroge n fixation,Salamanca ,Spain ,Academi cpress . 21. Krul,J . (1975)Thesis ,A study of theNAD(P ) transhydrogenase from Azotobaoter vinelandii, Agricultural University,Wageningen . 22. Edmonson,D.E . and Tollin,G.(1971 )Biochemistry , 10,133-145 . 79 23. Schwartz,E.R . and Reed,L.J . (1970)Biochemistr y % 1434-1439. 24. Senior,P.J . andDawes ,E.A . (1971)Biochem.J . 125,55-66 . 25.Yates ,M.G . (1971)Eur.J.Biochem . 24,347-357 . 26. van Thienen,G . andPostma ,P.W . (1973)Biochim.Biophys.Act a 323,429-440 . 27. Eilermann,L.J.M . (1970)Biochim.Biophys.Act a 2J_6,231-233 . 28. Nieuwenhuis,F.J.R.M. ,Kanner ,B.I. ,Gutnick ,D.L. , Postma,P.W . and VanDam ,K . (1973)Biochim.Biophys.Acta , 325,62-71 . 29.Morgan ,T.V . andDerVartanian ,D.V . (1976)Abstracts , 10th International Congress of Biochemistry, p.328 . 30.Yates ,M.G . (1970)FEB S Lett.8 ,281-285 . 31. Oppenheim,J . andMarcus ,L . (1970)J.Bacteriol .J_0J_ ,286-291 . 32. Burns,R.C. , Stasny,J.T . andHardy ,R.W.F . (1975)Proc. I International symposium onNitroge n fixation,Pullma nWashington ,Washingto n UniversityPress . 33. Scherings,G.S . andVeeger ,C . submitted forpublication . 34. Shethna,Y.I. ,DerVartanian ,D.V . and Beinart,H . (1968)Biochem.Biophys . Res.Commun. 3J_»862-868 . 35. Benemann,J.R. , Yoch,D.C. ,Valentine ,R.C . andArnon ,D.I . (1971) Biochim.Biophys.Acta, 226,205-212 . 36. Benemann,J.R. , Yoch,D.C. ,Valentine ,R.C . and Arnon,D.I . (1969) Proc.Nat.Acad.Sci. 64,1079-1086 . 37. Yoch,D.C. , Benemann,J.R. , Valentine,R.C . and Arnon,D.I . (1969) Proc.Nat.Acad.Sci. 64,1404-1410 . 38. Yates,M.G . (1972)FEB S Lett. 27,63-67 . 39.Mayhew ,S.G. , Abels,R. , Platenkamp,R. , unpublished results. 40. Jackson,R.W . and Demoss,J.A . (1965)J.Bacteriol . 90,1420-1425 . 41.Moses ,R.E . and Richardson,C.C . (1970)Proc.Nat.Acad.Sci . 67_,674-681 . 42. Barman,B.G . andTolli nG . (1972)Biochemistr y 11,4755-4759 . 80 Discussion ELECTRON TRANSPORT TO NITROGENASE A. ELECTRON DONORS FOR NITROGENASE Isolatednitrogenas eha sful lactivit ya tp H8. 0a ta redo xpotentia lo f -500m Vbu tha sn oactivit ywhe nthi spotentia lbecome shighe rtha n-42 0m V (1,2).Therefor ea nefficien telectro ndono rfo rnitrogenas emus thav ea midpointpotentia lo fa tleas t-42 0m Va tp H8.0 .I nanaerobi cbacteri a severalenzym esystem sar eknow nt ocatalys ereaction stha tproduc ereducin g equivalentsa ta sufficientl ylo wpotential : 1. Theso-calle dphosphoroclasti creactio ni scatalyse db ypyruvat edehydro genase,ferredoxi nan dhydrogenase : ^° + ^° CE,- C -C ' +GoAS H+ H ->-CH ,- C +CO ,+ H 9; 0 0 * ^SCoA l l Em = -51 0m Va tp H7. 0 r(3) . 2. Theformat edecarboxylatio nreactio ni scatalyse db yformat edehydrogenase , /° + ferredoxinan dhydrogenase :H-C/_+ H -+H 2+ C0 7;E =-42 0m Va t pH7. 0(3) . ° Inthes esystem sa ferredoxi no rflavodoxi nfunction sa sth edirec t electronaccepto ran dca nsubsequentl ydonat eelectron st ohydrogenas e forth ereductio no fprotons ,o rt onitrogenase .Th ereductio no fproton s isreversibl es otha tmolecula rhydroge nca nals ob euse dt oreduc e ferredoxinan dultimatel ynitrogen . 3. H,+ 2 ferredoxi n(ox )• +2H ++ 2 ferredoxin(red) ;E =-42 0m Va tp H7.0 . Inphotosyntheti corganisms ,ferredoxi nha sa simila rnoda lfunctio ni ntrans ferringreducin gequivalent sa tlo wpotential ,bu ti nsuc horganism sth e reducingequivalent soriginat ei nphotosyste mI wit ha redo xpotentia llowe r than-70 0m V (4).N osimila rferredoxin-linke doxidativ eenzym esystem swit h asufficientl ylo wredo xpotentia lar eknow ni naerobi co rsymbioti c 81 nitrogen-fixingorganisms ;th epyruvat eoxidizin g system in A.vinelandii for example iscouple d toth ereductio no fNA D (E (pH7.0 ) =-32 0mV )an dno tt o thereductio no fferredoxin . Thermodynamically iti spossibl et oreduc enitrogenas ewit h thereduce d pyridinenucleotides ,b y coupling thepyridin enucleotide s toa mor eo r less irreversible reaction.Fo rexample :whe n 5m Mglucose-6-phosphat e and0. 2 mM NADP isbrough t inequilibriu ma tp H 8.0wit h6-phosphogluconat ean dNADP H by adding glucose-6-phosphatedehydrogenase ,an d taking intoaccoun tth emeas - sured equilibriumconstant ,fo rthi sreactio n (K=60c.f.ref.5) ,a tt = 2 5C a ratiofo rNADPH/NAD P of999. 2ca nb ecalculated .Fro mthi srati oan dth e Nernstequatio nth etheoretica lredo xpotentia l is-0.4 4mV , lowenoug ht o givea lownitrogenas e activity. Indeedwit h glucose-6-phosphate,NAD P , glucose-6-phosphate dehydrogenase,an dNAD P-ferredoxin eoxidoreductas eplu s ferredoxint ocoupl ebetwee nNADP Han dnitrogenase ,a lownitrogenas eactivit y canb eobtaine d (6-8). But iti sdoubtfu lwhethe rsuc hhig hratio so freduce d tooxidize dpyridin enucleotide s everexis ti nlivin gorganisms .Fo r Azotobaater wehav eshow ntha tunde rcondition s ofmaximu mnitrogenas e acti vity,th erati oo fNAD(P)H/NAD(P) +varie sbetwee n0.25-0.7 5 (paper II,III) and thusequivalen twit h redoxpotentials ofaroun d-31 0mV . Amor edefinit eproo ftha tth ereducin gpowe rnecessar y fornitroge nfixatio n isgenerate d ina proces s othertha ntha tvi aa hig hrati oo freduce d to oxidizedpyridin enucleotides ,i sgive n inpape r I.A possibl eenerg ysourc e which couldgenerat e lowpotentia lreducin g equivalents fornitroge nfixation , isNAD Hoxidatio nvi ath ecytoplasmi cmembran ecouple d tomembran eenergisation . Inth eexperiment s toinvestigat e therol eo fth eenergize d cytoplasmicmem brane inelectro ndonatio nt onitrogenase ,acetat ewa suse d assubstrate . Acetatewa suse dbecause ,i ncontras twit h substrates likesucros eo rsucci nate,energ y isno trequire s for itsuptak e into Azotobaater vinelandit (9). When Azotobaater cellsar erespirin g acetate,lo wconcentration s of uncouplerd ono t influenceth eoxyge nuptake ,indicatin g thatth erate so f themai nmetaboli cprocesse s areconstant .Thi s facilitates interpretation ofexperimenta l results considerably.A s showni npape r I,lo wconcentration s uncouplerd ono t influenceth eintracellula r ratioo fATP ,a substrat efo r nitrogenase,an dADP ,a ninhibito r ofnitrogenase ,bu tth esam econcentratio n ofuncouple r inhibits thenitrogenas eactivit ycompletely .Assumin g thatth e acridinefluorescenc e canb euse da sa marke r for theenergize d state,w ehav e showntha ta hig henerg y levelo fth ecytoplasmi cmembran e in Azotobaater 82 isnecessar y togenerat ereducin g equivalents for thenitrogenase .W e tried toprov e thishypothesi swit h isolated cytoplasmicmembrane s andnitrogenase , butn onitrogenas e activity couldb eobserved .A nexplanatio n for this failure couldb e thefac t thatth ecytoplasmi cmembrane s of Azotobacter areno t energizablewit hATP ,onl ywit ha noxidizabl e substrate and oxygen.Anothe r indicationtha t theenergize d cytoplasmicmembran e isnecessar y forelectro n transport toth enitrogenas e isgive n inpape r III,i nwhic h it isshow ntha t a toluene treatmentmake s Azotobacter cellspermeabl e forsmal lmolecules . Normally Azotobacter hasgoo dcouplin gbetwee npyruvat eoxidatio nan dnitro genaseactivit y (paper II).Afte ra toluene treatmentn opyruvat e oxidation isobserved .Th epyruvat e oxidationca nb erestore db y adding theappropri atecofactors ,bu tn oassociate d nitrogenase activity isfound . Sincew e haven o evidence thatenzyme s leakou to fth ecel lenvelop eunde r these conditions,a membran ewhic h isenergizabl ema yb e themissin g linkbetwee n pyruvate oxidationan dnitrogenas e activity. B.ROL EO FFERREDOXI NAN D FLAVODOXIN INNITROGE N FIXATION Foursmal lan dsolubl enon-hea miro nelectro ntransfe rprotein swit h "labile sulfur"hav ebee nisolate d from Azotobacter (10,11).Th e ferredoxins isolated by Shetnae t al. (10)canno teithe r replacespinac h ferredoxin inth e photoreduction ofNAD P by illuminated chloroplasts orsubstitut e clostridial ferredoxin innitroge nfixatio ni na ferredoxin-fre eextrac to f C.pasteurianum. Incontras thowever ,th etw oferredoxin s isolatedb yYoc han dArno n (11,12) arebiologicall yactiv e inth eabov ementione d tests.Thes e ferredoxins can alsotransfe rth ereducin gpowe ro f illuminated spinachchloroplast s tonitro genaseo f Azotobacter. Thispropert y led toth epostulat e thatferredoxi n isa physiological reductant fornitrogenas e in Azotobacter (2).I nou rvie w however iti sunlikel y thatth eferredoxi na s isolatedb yYoc h (11)fro m Azotobacter playsa rol e innitroge nfixation .Firstly ,substrat e levels of reduced ferredoxinar eno tcapabl eo ftransferrin g electrons tonitrogenas e (G.S.Schering s tob epublished) . Secondly theamoun to fferredoxi nextract - able from Azotobacter grownunde rnitrogen-fixin gcondition s isver y low comparedwit h anotherelectro ncarrie r- flavodoxi n- isolate db y the same method (20m g ferredoxinan d 500m g flavodoxinfro m 1k gcel lpaste) . Shethnae tal . (13)isolate da flavodoxi n from Azotobacter, which differs fromflavodoxin s fromothe r organisms.Firstl y iti sno t induciblea tlo w concentrations ofF e inth ecultur emedium .Secondly ,i ti sa poo r replacent 83 forferredoxi ni nth epyruvat ephosphoroclasti creactio nan di nth ephoto chemicalreductio no fNAD P byspinac hchloroplast s (14).Thirdl yth e couplingcapacit yo fflavodoxi nbetwee nilluminate dchloroplast san dnitro - genasei spoo r (12).Yate s (15)an dw ehav eshow n (paperIII )tha tsubstrat e amountso fflavodoxi nhydroquinon eac ta sa ver yefficien telectro ndono rfo r Azotobacter nitrogenase.I npape rII Iw ehav eshow ntha tther ear esevera l interactionsbetwee n Azotobacter flavodoxinan dnitrogenase . 1. Highactivit ywit hsubstrat eamount so fflavodoxi nfro m Azotobacter comparedwit hflavodoxin sfro m P.elsdenii and D.vulgaris andals ocom paredwit hth eactivitie so fdithionit ean dreduce dmethylviologen . 2. Nitrogenaseactivit yi sindependen to fth eredo xpotentia lo f Azotobacter flavodoxinove ra lon grange . 3. Azotobacter flavodoxinprevent sdissociatio no fth enitrogenas ecomple x atlo wprotei nconcentration ,whic hwa sals oshow nb yYate s(22) . Theseobservation stogethe rwit hth einabilit yo freduce dferredoxi nwithou t areducin gsyste mt oreac twit hth enitrogenas ean dth elo wamoun to fferre doxinextractabl efro m Azotobacter suggesttha tflavodoxin ,an dno tferre doxin,i sth ephysiologica lelectro ndono rfo rnitrogenas ei n Azotobacter. C.ELECTRO NTRANSPOR TCHAI NT ONITROGENAS EO RTH EFLAVODOXI NREDUCIN GSYSTE M Asdescribe di npar tA o fthi sdiscussion ,w ethin ktha tth ereducin gpowe r fornitrogenas ei n Azotobacter isgenerate di nth ecytoplasmi cmembrane . Wehav etrie dt oisolat eth eelectro ndonatio nsyste mt onitrogenase ,bu tw e didno tsuccee dwit han yo fth ecell-ruptur emethod stha twer eused .A n illustrationo fth elabilit yo fthi ssyste mi sgive ni npape rIII .Unde r anaerobicconditions ,Azotobacter cellshav elo wnitrogenas eactivit ycouple d withpyruvat eoxidation . Azotobacter cellslys espontaneousl yafte ra lyso - zymetreatment .Durin gthi sautolysi sa decreas ei nnitrogenas eactivit y coupledwit hlos so fpyruvat eoxidatio nactivit yan da nincreas ei ndithionit e -mediatednitrogenas eactivit yha sbee nobserved .Th epyruvat edrive nnitro genaseactivit yi sno trestore db yaddin gth eappropriat ecofactor sfo r pyruvateoxidatio nan da nAT Pregeneratin gsystem . Despiteou rfailur et oisolat eo rreconstitut eth ephysiologica lelectro n donatingsyste mt onitrogenas ei n Azotobacter, wehav efoun dtw oindication s thatth ecytoplasmi cmembran ean dflavodoxi nar einvolved . 84 1. Flavodoxin seems tob eburrie dwithi n themembran e oftoluene-treate d cells (paperIII) . 2. A NAD(P)H-flavodoxinoxidoreductas e ispresen t inth ecytoplasmi c mem brane (PaperIV) . Based onthes e observations,w e give inpape r IVa proposa l how flavodoxin can be reduced toth ehydroquinon e formb y thecytoplasmi cmembrane . Inthi shypo thesis differences inp Hcreate d energy-linked,ar e thedrivin g force for the reduction of flavodoxinb yNADH . Possibly asimila rphenomenon ,a nenergy-linke d electrontranspor t from NADH to ferredoxinoccur s in Clostridium pasteurianum. Jungermann et al. (16)hav e shown thatAcety lCo A isnecessar y forelectro n transport fromNAD Ht o ferredoxin. It ispossibl e that the energypresen t inAcety l CoA isuse d to transport electrons fromNAD Ht o ferredoxin,a carrie rwit h alowe r redox potential.N o further informationabou t thisclostridia l system isavailable . OXYGEN PROTECTION OF NITROGENASE A Respiration protection Itwa s longknow n thatoxidatio nb y Azotobaater cells depends onth e oxygen inputdurin g growth.Dalto nan dPostgat e (17)suggeste d that augmented respiration in Azotobaater keeps theenvironmen t ofth enitrogenas e oxygen- free,an dprotect s theoxygen-sensitiv e nitrogenase. They called thispheno menon therespiratio nprotection .Jone s andco-worker s havedescribe d the changeswhic h occurwithi n therespirator y systemo f A.vinelandii upona n increase inoxyge n inputdurin ggrowth . 1.Increase d NAD(P)Hdehydrogenas e activity. 2. Increased concentration ofcytochrom e d. 3.Los s ofenerg y conservation at site I (18,19). Inou ropinio n theexperiment s ofAckrel l andJone s (18,19)ar equestionable . Firstly,the y grow thebacteri a ina batc h culture,whic h ingenerall y yields cells ofa non-define d physiological state.Secondly ,the yd ono t take into accountmutua l spectral overlapo fcytochrome s c.+ c, .an dcytochrome s b +0 and furthermore theyd ono tdiscriminat e betweencytochrom eb and0 . Weuse d cells grownunde r specified conditions ina chemosta tan duse d the method ofSinclea r andWhit e (20)t ocalculat e theamount s of cytochromes present ina membran epreparation .Th e following observations havebee nmade . 85 When A.vinelandi'L cellsar egrow nunde roxygen-limite dcondition smainl y cytochromes c. + Cran do ar edetectable .Couplin gsite sar efoun dbetwee n NADHan dCo Q (site I), betweenmalat ean doxyge nan dbetwee ncytochrom e c.+ C r andoxygen .Whe nth ecell sar egrow nwit ha nexces s ofoxygen , beside adecreas e incytochrom ec .+ c^ ,a nincreas e incytochrome sb and d isobserved .Onl ybetwee nCo Qan doxyge nphosphorylatio n isdetectable . Our resultssuppor tth esuggestio no fJone s andcoworker s (18,19)tha tcell s grownoxygen-limited ,hav ecytoplasmi cmembrane swit h ahig h oxidative phosphorylationcapacity ,whil ecell sgrow n inexces s oxygenhav emembrane s with a lowoxidativ ephosphorylatio ncapacity .Bu t incontras t tothei r suggestions ourresult s inPape r IIclearl ysho wtha tth echange swithi nth e respiratory systemar eno tresponsibl e forth eobserve dphenomeno no fenhance d whole cellrespiratio n (seediscussio nPape r III). Itturne dou t thatth e uptake ofsuga r into Azotobaater isth erate-limitin g step insuga roxidation . Wehav eals oshow ntha tth echange s inth erespirator y systemar eno trelate d toth ephenomeno no frespiratio nprotectio no fth enitrogenase ,b yusin g substrates likepyruvat e oracetate .Pyruvat ean dacetat ear e takenu p into Azotobaater without energy incontras twit hsugar san d thetricarboxyli c acid cycle intermediateswhic huptak e isenergy-dependen t (9).I tma yb epossibl e thatonl ydurin ggrowt ho nsubstrate swhic har etake nu penergy-dependent , changes inth erespirator y systemoccu ra sa regulatin gmechanis mfo rsub strateuptake .Mor eexperiment sar enecessar y togiv ea physiologica l explanation for thechange s observed inth erespirator y systeman dth e regulationo fthes echange s in Azotobaater. CONFORMATIONAL PROTECTION Nitrogen-fixingsystem shav ebee nextracte d from Azotobaater ina so-calle d particulate formwhic h isreasonabl e stable inair .Dalto nan dPostgat e (17) proposed aconformationa lprotectio no fnitrogenas e inwhic hth eoxyge n sensitivesite s ofth enitrogenas ear eprotecte d againstoxygen .Cytoplasmi c membrane fragments,presen t ina particulat enitrogenas epreparatio n (22)o r a crudeNAD Hdehydrogenas epreparatio n (22)ar ethough tt ointerac twit hth e nitrogenase tofor mth eoxyge nstabl econformational-protecte dnitrogenase . Theexperiment s ofOppenhei me tal . (23)confirme d thisview .The y ruptured Azotobaater cellswit ha Frenc hpressur e cellan dnitrogenas ewa s found particulate andoxyge nstable .Whe n A.vinelandii was rupturedwit ha nosmoti c shock,nitrogenas ewa s found solublean doxyge nlabile .Howeve r inPape rIII , 86 wesho wtha tth enitrogenas ecomple xisolate dfro m Azotobaater aftercel l ruptureb ya nosmoti cshock ,i soxyge nstable ,i nspit eo fth efac ttha tth e nitrogenasei nthes eextrac ti snon-particulate .Hig hsal tconcentration san d extremedilutio nmake snitrogenas efro m Azotobaater oxygenlabile .W eals o showtha tmembran efragment swit ha hig hNAD Hdehydrogenas eactivit ywhic h arepropose dt oprotec toxyge nsensitiv esite so fth enitrogenas e (24)i n factd ono tprotect .I npape rIV ,w edemonstrat etha ton ecanno tspea ko fa particulatenitrogenase .Nitrogenas ei sfoun dt ob ea solubl eenzym ecomple x comparablewit hth epyruvat edehydrogenas ecomplex .Dependin go nth ecell - rupturemethod ,cytoplasmi cmembran efragment ssedimen ttogethe rwit hth e nitrogenase.W eals osho wtha tth eoxyge nstabilit yo fnitrogenas efro m Azotobaater isdu et oa complexatio no fth enitrogenas ecomponent swit ha Fe- S protein.Thi sFe- Sprotei ni sidentifie da sferredoxi nII ,isolate dfro m Azotobaater byShethn ae tal .(10) . Withou rresult si ti spossibl et ore-evaluat eth emechanis m ofth eswitch-on , switch-offphenomeno no fwhol e Azotobaater cells (17).Sinc enitrogenas ei s presentactiv ean doxygen-protecte di n Azotobaater, theobserve dinhibitio n ofwhol ecel lnitroge nfixatio nupo nincrease doxyge ninput ,mus tb edu et o oxidationo felectro ncarrier sinvolve di nelectro ntranspor tt onitrogenas e andno tb edu et oa chang eint oconformationa lprotecte dnitrogenase ,oxyge n stabilebu tinactive . 87 LITERATURE 1.Evans ,M.C.W . andAlbrecht ,S.L . (1974)Biochem.Biophys.Res.Commun . 61, 1187-1192. 2.Watt ,G.D . and Bulen,W.A . (1975), International Symposium on Nitrogen Fixation,Newton ,W.E .an dNyman ,C.J. ,eds. ,Pull ,Wash ,Washingto n StateUniversit y Press,248-256 . 3.Krebs ,H.A ., Romberg , H.L. and Burton,K . (1957)Energ y transformations in livingmatter ,Springer ,p . 186-281. 4.Kok ,B. ,Rurainski ,H.J . andOwens ,O.V.H . (1965)Biochim.Biophys.Act a 109,347-356 , 5.Bergmeyer ,H.U . (1970),Methode nde rEnzymatische nAnalyse ,p .1981 . 6. Benemann,J.R. , Yoch,D.C. ,Valentine ,R.C . andArnon ,D.I . (1971) Biochim.Biophys.Acta 226,205-212 . 7.Bothe ,H . (1970)Ber.Deut.Bot.Ges . 83,421-432 . 8.Bothe ,H . andYates ,M.G . (1976)Arch.Microbiol . \07_, 25-31. 9.Visser ,A.S .an dPostma ,P.W . (1973)Biochim.Biophys.Act a298 ,333-340 . 10. Shethna,Y.I. ,DerVartanian ,D.V . and Beinert,H . (1968)Biochem.Biophys . Res.Commun.21 . 862-868. 11.Yoch ,D.C . andArnon ,D.I . (1972)J.Biol.Chem . 247,4514-4520. 12.Yoch ,D.C , Benemann,J.R. , Valentine,R.C . andArnon ,D.I . (1969) Proc.Nat.Acad.Sci. 69,1404-1410 . 13. Shethna,Y.I. ,Wilson ,P.W . and Beinert,H . (1966)Biochim.Biophys.Act a 113,225-234 . 14.Hinkson ,J.W . and Bulen,W.A . (1967)J.Biol.Chem . 242,3345-3351 . 15.Yates ,M.G . (1972)FEB S Letters 27.,63-67 . 16.Jungermann ,K. ,Kirchinawy ,H. , Katz,N . and Thauer,R.K . (1974) FEBS Letters 43,203-206 . 17.Dalton ,H . and Postgate,J.R . (1969)J.Gen.Microbiol . 54,463-473 . 18.Ackrell ,B.A.C . and Jones,C.W . (1971)Eur.J.Biochem . 20.29-35 . 19.Jones ,C.W. , Brice,J.M. , Whight,V . and Ackrell,B.A.C . (1973) FEBSLetter s 29,77-81 . 88 20. Sinclear,P.R . andWhite ,D.C . (1970)J.Bacterid . 101,365-372 . 21. Kelly,M . (1969)Biochim.Biophys.Act a 191,527-540 . 22.Yates ,M.G . (1970)FEB SLetter s 8,281-285 . 23.Oppenheim ,J. , Fisher,R.J. ,Wilson ,P.W . and Marcus,L . (1970) J.Bacetriol. 101,292-296 . 24.Drozd ,J . andPostgate ,J.R . (1970)J.Gen.Microbiol .63 ,63-73 . 89 Summary I ELECTRON DONATION TO NITROGENASE Paper Ishow s that thehypothesis ,tha ta hig hrati o of (NADH+ NADPH) / (NAD +NAD P )i s the sourceo freducin g power fornitrogenas e inintac t A.vine landii, is invalid.O n thecontrary ,wit ha decreasin g ratio ofreduce d tooxi dizedpyridin e nucleotides,th enitrogenas e activity ofwhol e cells increases. The experiments described inpape r I,indicat e that thereducin g power necessary fornitroge n fixation in A.vinelandii is generatedwithi n thecytoplasmi c mem brane. Iti sdemonstrate d that transport ofreducin g equivalents to thenitroge nase requires ahig h energy level of thecytoplasmi cmembrane .Th e energy level of thecytoplasmi cmembran ewa smeasure d by the intracellular ATP concentration andb yusin g 9-aminoacridin e asa fluorescent probe.Othe r regulatinpfactor s of thenitrogenas e activity in A.vinelandii are shown tob e the intracellularATP / ADP ratio and thepresenc e ofoxygen . PaperII Ishow s thattoluen emake s A.vinelandii cellspermeabl e for small molecules butno t forenzymes .I ntoluene-treate dcells ,enzym e activities can bemeasure d by adding theappropriat e cofactors and substrates. Iti spossibl e torestor e theoxidatio no forgani c substratesbu tn o concomitant nitrogenase activity canb eobserved .W e suggest thata nobserve d lacko fenergizatio n of the cytoplasmicmembrane s is themissin g linkbetwee noxidatio nan d generationo f the reducing equivalents fornitroge n fixation. Inpape r IIIan d IVw e show that theendogenou s lowpotentia l electroncarrier s intoluene-treate d cells areno t reduced. Inpape r IVa membrane-boun d NAD(P)H-flavodoxin oxidoreductase isdemon strated and aproposa l isgive n inwhic hNAD H is theelectro ndono r fornitroge nase.Th e electroncarrie r flavodoxin isreduce d by themembrane-boun d NADH-fla- vodoxinoxidoreductas e at alo wpH ,tha t isdevelope d ina nenergy-linke dprocess . II OXYGEN PROTECTION OF NITROGENASE Inpape r IIth e source ofrespiratio nprotectio n is investigated.Experi - 91 mentswit hradioactiv epyruvat ean dsucros esho wtha tth erat eo fsucros eoxi dationb y A.vinelandii isassociate dwit hth esucros etranslocato ractivity .W e showtha tth erespiratio nprotectio no fth enitrogen-fixin gsyste mi n A.vinelandii isdependen to fth eoxyge ninpu tdurin ggrowth .Th eoxidatio ncapacit yintrins icallydepend so nth etyp eo fsubstrat ean dca nb epartl yadapted . Membranesric hi ncytochrome sc +c ando an dwit hphosphorylatio n betweenNAD Han dCoQ ,Co Qan doxyge nan dcytochrom ec ,+ c .an doxygen ,ca nb e isolatedfro m A.vinelandii grown0„-limited .Cytochrome sb an dd ca nb edetecte d inadditio nwhe n A.vinelandii cellsar egrow nN „limited .Th eactivit yo fth e NADHoxidas esyste mi sincrease di nsuc hcell san dphosphorylatio ni sonl yob servedbetwee n CoQan doxygen .Unde rsaturatin goxyge nconcentration sth etyp e ofrespirator ymembrane swa sno tobserve dt oinfluenc eth eintracellula renerg y charge. Inpape rII Ian dI Vth emechanis mo fth econformationa lprotectio no f nitrogenasewa sinvestigated .I ti sshow ntha tnitrogenas eca nb eisolate da sa n oxygen-stablecomple x form A.vinelandii independento fth ecel lruptur emethod . Alson oinfluenc eo fth ecel lruptur emetho do nth erat eo fsedimentatio no fth e nitrogenaseca nb eobserved .Th erat eo fsedimentatio no fth enitrogenas ei s foundt ob econcentratio nan dp Hdependent .A tpH=7. 4th erat eo fsedimentatio no f thenitrogenas ecomple xi scomparabl ewit htha to fth epyruvat edehydrogenas e complex. Noevidenc ewa sfoun dfo ra particulat enitrogenase ,i ti sdemon stratedtha tth eoxyge nstabilit yo fnitrogenas ei ncrud eextract si scause db y complexationo fth enitrogenas ecomponent swit ha nFe- Sprotein .A nalternativ e proposalfo rth eswitch-o nswitch-of fphenomeno ni nwhol e Azotobaatev cellsi s given..Nitrogenasei spresen ti nviv oa sa nactiv ean doxyge ntoleren tcomple x butnitroge nfixatio ni nwhol ecell si sinhibite db yth eoxidatio no fflavodoxi n hydroquinone. 92 Samenvatting De experimentenwelk ei ndi tproefschrif tbeschreve ne nbediscussieer d zijn,hebbe nall ebetrekkin go pd estikstoffixati e in Azotobacter vinelandii. Hetonderzoe kka nworde nonderverdeel d intwe ehoofdrichtingen .D ebeschermin g vand enitrogenas e tegen zuurstofe nhe tgenerere nva nreductie-equivalente n voord enitrogenase . I BESCHERMINGVA NHE TSTTKSTOFFIXEREN D SYSTEEM TEGEN ZUURSTOF Debeschermin g tegen zuurstofvind tplaat so ptwe eniveau's .O phe tnivea u vand ehel eee l (ademhalingsbescherming)e no penzyma.tisc hnivea uconformatie - bescherming). a) Ademhalingsbescherming Ind etweed epublicati e isnade ro pd eademhalingsbeschermin g ingegaan. Aangetoond isda td eaanpassin gva nd eademhalingssnelhei dva nhel e cellenaa n het zuurstofaanbod tijdensd egroe iberus to pee naanpassin gva nd eopnamesnel - heidva nd ekoolstofbron .Doo r substratenaa nt ebiede n (pyruvaate nacetaat) , waarvand eoxidati ed esnelheidsbeperkend e stappenva nd esuikerademhalin gover - slaat,i saangetoon dda tvoo rhe tzuursto fvrijhoude nva nhe tstikstoffixeren d systeemi nintact e Azotobactev, ergee nspecial e aanpassingenva nhe tcytoplasma - membraane nhe tstikstoffixeren d systeem,noodzakelij k zijn.Di ti stegenstrij - digme t wati nd eliteratuu r gesuggereerd is. Naas the tverschi l insuike r translocator activiteit,zij nafhankelij kva nd ehoeveelhei d zuurstof tijdensd e groei,d evolgend e fysiologische verschillen tussentwe e typencelle n aangetoond. Zuurstofbeperkend gegroeide cellenbevatte nee nademhalingskete nme tee nhog e oxidatieve fosforyleringsefficientie. Fosforylering isaangetoon d tussenNAD He n CoQ,tusse nCo Qe nzuursto fe ntusse ncytochroo mc ,+ c .e nzuurstof .D ebelang - rijkstecytochrome nzij ncytochroo mc ,+ c encytochroo m o.Wannee rd ecelle n inovermaa t zuurstof gegroeid zijn,worde nademhalingsmembrane naangetroffe nme t 93 een lageoxidatiev e fosorylerings-efficientie. Fosforyleringword tallee nwaar - genomentusse nCo Qe nzuurstof .D eademhalingsmembrane nbevatte nminde rcyto - chroomc ,+ c .e no e nmee rcytochroo mb e nd .Oo kneem the tNAD Hoxidas e systeem sterkto ei nactiviteit .D everminderd eoxidatiev e fosforylerings-efficientie vand egeisoleerd emembranen ,veroorzaak t geen lagere intracellulaire energie- lading.Gee nverschille n inkinetisch e eigenschappenva nglucose-6-fosfaa t dehy drogenase,6-fosfogluconaa thydro-lyas ee n3-deoxy-2-keto-6-fosfogluconaa t aldo lasei nruw eextracte nva nd etwe e typencelle n zijnwaargenomen . b) Conformatiebeschernring Inartike l IVword taangetoon dda tnitrogenas eui t Azotobacter inte - genstelling totd eliteratuur ,onafhankelij kva nd emethod ewaarme ed ecelle no - pengebroke nworden ,sedimenteer tal see ncomplex .Gedurend ed ezuiverin gva ndi t complexm.b.v . selectieveprotaminesulfaat-precipitati e enee nMgCl_-precipita - tie,blijf tdi tcomple x zuurstofstabie l (artikel III).N ascheidin gva ndi t complexi n3 fractie sm.b.v .DEAE-cellulos echromatografie ,i sdoo rmidde lva n reconstitutie-experimentenaangetoon dwel k eiwiti nhe toorspronkelijk e complex denitrogenas e componententege n zuurstofinactivatiebeschermt .Di teiwi theef t eenmolecuulgewich tva n24.00 0e nbeva tal skarakteristiek e groepee nFe- S kluster. Daari nartike l 3i saangetoon dda the tzuursto f stabiele nitrogenase complexme tflavodoxin ehydrochino n alsreducti emidde lee nhog eactivitei tver - toont,i she tduidelij kda td ereversibel e remmingva nd estiksto fbindin gbi j Azotobacter (switch-on-switch-off)berus to pee noxidati eva nflavodoxin ehydro chinon. II HETGENERERE NVA NREDUCTIE-EQUIVALENTE N VOORD ESTIKSTOFFIXATI E Inaeroo b stikstofbindendemicro-organisme n zonder foto-systeem zijn geenenzymsysteme naangetoond ,waarbi j redoxcarriers zoalsferredoxin eo fflavo doxinevolledi g gereduceerd kunnenworden .I nartike l Ihebbe nw eaangetoon d voor intacte Azotobacter cellenda tvoo rhe tgenerere nva nreductie-equivalente n voor de stikstofbinding energienodi g is,verkrege ndoo relectrone n transportnaa r zuurstofdoo rd eademhalingsketen . Inhe tderd eartike lword the tterminal egedeelt eva nd eelectronen - transportketennaa rd enitrogenas ebehandeld .Hierbi j isgebrui k gemaaktva nd e 94 methode omm.b.v .fotoreducti eva n 3,10 dimethyl-5-deaza-isoalloxazine flavo- doxine tereduceren .Aangetoon d is:ee nhog e activiteit enee nspecial e fittus - senflavodoxin e ennitrogenas eui t Azotobaoter vergelekenme tflavodoxine s uit P.elsdenii en D.vulgaris. Met fotochemischgereduceer d flavodoxineui t Azotobao ter isd enitrogenase-activitei t over eengroo tgebie donafhankelij kva nd ere - doxpotentiaal integenstellin g totfotochemisc hgereduceer dmethylviologe nal sre - ductiemiddelvoo rd enitrogenase . Experimentenme t tolueenbehandeld e Azotobaoter cellengeve naa nda tfla vodoxine in intactestructure nnie treduceerbaa r isvoo rhe t fotochemischreduc - tiesysteem (artikel IIIe n IV). Inartike l IVword the tbegi nva nd eelectronen - transportketennaa rd enitrogenas ebeschreven .Enig eeigenschappe nva nee nmem - braan-gebondenNADH-flavodoxin eoxidoreductas eworde ngegeven .Teven sword t in hetvierd e artikel eenvoorste l gedaanho evi a eenpH-verschi l tussend eplaat s waarNAD HNADH-flavodoxin eoxidoreductas e reduceert enwaa rdi tenzy melectrone n overdraagt op flavodoxine,NAD H (E (pH7. 0 =-32 0mV ) instaa tgeach tmoe twor den flavodoxine semichinont ereducere n totflavodoxin ehydrochino n (E (pH 7.0) = -495 mV). 95 Curriculum vitae De schrijverva ndi tproefschrif ti sgebore no p2 4jul i194 6t eBadhoeve - dorp.I n196 5behaald ehi jhe tHBS- Bdiplom aaa nhe tPiu sX Lyceu mt eAmsterdam . Inhetzelfd e jaarbego nhi jzij nstudi echemi eaa nd eUniversitei tva nAmsterdam . InSeptembe r 1968wer dhe tkandidaatsexame nS 1 behaalde ni njun i 1971he tdoc - toraal examen,cu mlaud e (hoofdvakbiochemie ,bijva kdierfysiologi ee nspecial e richting fysische-organisch e chemie). Vanaf 1augustu s 1971to thede ni sd eauteu rwerkzaa mbi jhe tLaboratoriu m voorBiochemi eva nd eLandbouwhogeschoo l teWageningen .To t1 januar i 1975wa s hiji ndiens tbi jd eStichtin g Scheikundig Onderzoek Nederland (S.O.N.)welk ege - subsidieerdword tdoo rd eNederlands eOrganisati evoo r ZuiverWetenschappelij k Onderzoek (Z.W.O.). 96 fi H U- O