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Aerobic Nitrogen Fixation in Azotobacter Vinelandii

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Aerobic Nitrogen Fixation in Azotobacter Vinelandii 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 j methyl-2-amino 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 e sourceo 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
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