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Studies on the Iron-Sulfur Clusters of Hydrogenase, Sulfite Reductase, Nitrogenase and the Prismane Protein

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Studies on the Iron-Sulfur Clusters of Hydrogenase, Sulfite Reductase, Nitrogenase and the Prismane Protein Studies on the Iron-Sulfur clusters of hydrogenase, sulfite reductase, nitrogenase and the prismane protein t,p^ Promoter: Dr. C. Veeger hoogleraar in de Biochemie Co-promotor: Dr. W.R. Hagen Universitair hoofddocent, vakgroep Biochemie AJ. Pierik Studies on the Iron-Sulfur clusters of hydrogenase, sulfite reductase, nitrogenase and the prismane protein Proefschrift ter verkrijging van de graad van doctor in de landbouw- en milieuwetenschappen op gezag van de rector magnificus, Dr. CM. Karssen, in het openbaar teverdedige n op maandag 18oktobe r 1993 des namiddags te vier uur in de Aula van de Landbouwuniversiteit te Wageningen !Sn 58QCQ2 BlBUUlKhi.rv ESKBB0UWUNIVERSEEfci4 EAGENINGEM Aan mijn ouders en grootouders A)/U0?2o', l^°l STELLINGEN 1. Superclusters en Superspins bestaan. Hoofdstuk 5, 7 en 8 van dit proefschrift. Chan, M.K., Kim, J. & Rees, D.C. (1993) Science260, 792-794. Bolin,J.T. ,Ronco ,A.E. ,Morgan ,T.V. ,Mortenson ,L.E .& Xuong ,N.-H .(1993 ) Proc. Natl. Acad.Sci. (USA) 90, 1078-1082. Interpietatie van spectroscopische resultaten van eiwitten met meerdere redox toestanden is alleen zinvol na bepaling van de redox halfwaarde potentialen. Hoofdstuk 2, 5, 8 en 9 van dit proefschrift. Hetrhombisch eEP Rsignaa lme tg=2.07,1.9 6e n 1.89 isee nartefak t veroorzaakt door de aktivering van het zuurstof-stabiele ijzer-hydrogenase uit Desulfovibrio vulgaris (Hildenborough). Hoofdstuk 2 van dit proefschrift Patil, D.S., Moura, J.J.G., He, S.H., Teixeira, M., Prickril, B.C., DerVartanian, D.V., Peck, H.D., LeGall, J. & Huynh, B.-H. (1988) J. Biol. Chem.263, 18732-18738. Het koper-bevattend nitriet reductase is net als aconitase een kannibalistisch enzym. Masuko, M., Iwasaki, H., Sakurai, T., Suzuki, S. & Nakahara, A. (1984)/ . Biochem. 96, 447-454. Kennedy, M.C., Emptage, M.H., Dreyer, J.-L. & Beinert, H. (1983) J. Biol. Chem. 258, 11098-11105. Libby, E. & Averill, B.A. (1992) Biochem.Biophys. Res. Commun. 187, 5129-1535. Het door Fu et al. beschreven stereo- en regioselectieve karakter van de halohydrin- vorming uit 2,3-dehydrosiaalzuur wordt niet veroorzaakt door chloorperoxidase. Fu, H., Kondo, H., Ichikawa, Y., Look, G.C. & Wong, C.-H. (1992) /. Org. Chem. 57, 7265-7270. 6. Het humane glutathion reductase is geen goed model voor de voorspelling van de structuurva n de actieve plaats van het humane lipoamide dehydrogenase. Jentoft, J.E., Shoham, M., Hurst, D. & Patel, M.S. (1992) Proteins: Struct.Funct. Genet. 14, 88-101. De zuivering en karakterisering van het produkt van de nifN en nifE genen had moetenworde nuitgevoer dme tee nwild-typ esta me nnie tme tAzotobacter vinelandii UW45. Paustian, T.D., Shah, V.K. & Roberts, G.P. (1989) Proc. Nat. Acad. Set. USA 86, 6082-6086. Serine en threonine kunnen in tegenstelling tot wat Frausto da Silva en Williams vermelden als liganden optreden in Mg^-eiwit-nucleotide complexen. The Biological Chemistry of the Elements. Fnuistod a Silva, J.J.R. & Williams, RJ.P. (1991) Clarendon Press, Oxford. Pai, E.F., Krengel, U., Petsko, G.A., Goody, R.S., Kabsch, W. & Wittinghofer, A. (1990) EMBO J. 9, 2351-2359. Met de llnwording vand e landen vand e EEGmoe td e sluiting vand e Engelsepub s verlaat worden tot66a uur. 10. Onbeschermdi n 't veld is men snel hethaasje . Stellingen behorende bij het proefschrift 'Studies on the Iron-Sulfur clusters of hydrogenase, sulfite reductase, nitrogenase and the prismaneprotein ' Wageningen, 18 oktober 1993 A.J. Pierik Voorwoord Voor het tot stand komen van dit proefschrift is in tegenstelling tot wat de titelpagina en omslag suggereert meer dan een persoon betrokken geweest. Ik zali n dit voorwoord proberen de directe en indirecte bijdrage van al deze personen te specificeren, hoewel ik hiermee het risico loop (ongewenst) iemand te vergeten. Allereerst wil ik mijn promotor professor C. Veeger en mijn co-promotor Fred Hagen bedanken. Door de zeer open houding ten aanzien van het onderzoek en het stimuleren van het bezoek aan (internationale) congressen en cursussen kon ik me verdiepen in allerlei facetten van de bio-anorganische chemie. Ik ben daarom in hoge mate dankbaar voor de supervisie. Fred wil ik bovendien bedanken voor de aangename alledaagse begeleiding en de praktische, theoretische en tekstuele bijdragen die een onmisbare rol hebben gespeeld. Chronologisch vervolgend, wil ik mijn dank richten tot Marelle Boersma en Anita van Berkel. De eerste prille maanden in 1987/1988 op lab 5 heb ik veel steun gehad op praktisch gebied. Een voor jullie misschien onverwacht dank-je-wel voor de secure administratie van de experimenten in het verleden. Dagenlang heb ik doorgebracht met het lezen en vergelijken van resultaten. De vruchten van de secure boekhouding zijn op indirecte wijze terug te vinden in hoofdstuk 3 tot en met 5: de door Marelle als '? hydrogenase' gelabelde ingevroren monsters hebben geleid tot de ontdekking van het marellase (momenteel prismaan eiwit geheten). Met de verhuizing naar lab 1/2 en de start van Ronnie Wolbert begon de oogst fase van het promotie onderzoek. Jouw punctuele aanvang van experimenten in de ochtend combineerde uitstekend met mijn meer naar de tweede helft van de dag verschoven ritme. Ondanks het onaangename parfum van Desulfovibrio, ontsnappingen van 300 liter water en het grote aantal gels en blots was jij altijd in een goede stemming. Samen met Fred Hagen, Marc Verhagen, Dirk Heering en de afstudeervakstudenten vormde lab 1/2 een hecht team. Deze verbondenheid uitte zich door de gemeenschappelijke hoge affiniteit voor ons koffiezet apparaatje, de assay-koelkast, Granucci en Sa Lolla.Mar c Verhagen wil ik bedanken voor zijn hulp bij de afhandeling van allerlei zaken gedurende de laatste maanden. Door hulp met het opwekken van antilichamen en N-terminale aminozuur volgorde bepaling hebben Anita Kaan en Walter van Dongen het onderzoek naar het prismaan eiwit versneld. Willy van den Berg en Jack Stokkermans hebben door de zeer prettige samenwerking en de opheldering van de primaire structuur van de prismaan eiwitten uit Desulfovibrio vulgaris (Hildenborough) en D. desulfuricans (ATCC 2777'4) ervoor gezorgd dat het prismaan eiwit onderzoek in een stroomversnelling is geraakt. De samenwerking met Hans Wassink en Huub Haaker resulteerde in een hoofdstuk over het molybdeen-ijzer eiwit. Bedankt Hans voor de isolatie van het enzym en het leren van een aantal technieken. Huub, de vele discussies over nitrogenase en gerelateerde onderwerpen heb ik zeer op prijs gesteld. Rafael Israels, Martina Duyvis, Joop van Helvoort, Carine Stevens, Wim Hilbrandts, Leonard Mallee, Sander Arendsen en Gerrit Portier hebben door hun afstudeervak te doen in ons lab in belangrijke mate bijgedragen tot de sfeer, gezelligheid en wetenschappelijke productie. Bedankt voor de energie die jullie hebben gestoken in het onderzoek. Door de inspirerende samenwerking met Mike Jetten en Christof Holliger van de vakgroep Microbiologic kon ik kennismaken met koolmonoxide dehydrogenase en F-430. Peter Mutsaers, Leo de Folter, Auke van Heuzen, Dhr. Walinga, Tom Bongers, Jo Haas, Marja Snoek, Willem van Berkel, Adri Westphal en Simon Albracht hebben mij vertrouwd gemaakt met diverse technieken. Martin Bouwmans, Bery Sachteleben en Laura Ausma zorgden voor allerlei administratieve zaken. Thomas Link has contributed by involving me in the study of the soluble domain of the Rieske protein. The efforts of Dick Dunham and Dick Sands have strenghtened the importance of Chapter 5. Many thanks for your valuable contributions. I am grateful to Barry Smith and Bob Eady who allowed me to spend a part of my postdoctoral time on the dutch graduation. De praktische en/of morele steun van alle reeds met naam genoemde personen, Axel Berg, Rene Wientjens, Chris Oldfield, Jacques Benen, Phil Bastiaens, Ewart Pap en alle overige (ex)leden van de vakgroep Biochemie heb ik als zeer prettig ervaren. Naast de aangename uren op het Lab zorgden huisgenoten, de leden van de twee 'eetclubs', de werkgroep fossielen Wageningen en andere vrienden ervoor dat de terugkeer naar de geboortestad Wageningen aangenamer was dan ik ooit had kunnen vermoeden. Tenslotte wil ik mijn ouders en broer Leo bedanken, die door het fijne familieleven in de weekenden/ vakanties en door hun gezonde nieuwsgierigheid naar mijn werk een zeer motiverende invloed hebben gehad. Er resteert een persoon die ik vanuit deze positie niet kan bedanken: door zijn vroegtijdige overlijden is Jan Redeker niet meer in ons midden. Jan, ik benj e erkentelijk voor jouw bijdrage tot hoofdstuk 2, jouw herinnering leeft voort in mijn gedachte. The research was carried out at the Department of Biochemistry, Agricultural University, Wageningen, The Netherlands. The investigations were supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO). CONTENTS Page Chapter 1: Introduction 1 Chapter 2: Redox properties of the iron-sulfur clusters 33 in activated Fe-hydrogenase from Desulfovibrio vulgaris (Hildenborough) Chapter 3: Novel Electron Paramagnetic Resonance Signals 45 from an Fe/S Protein containing Six Iron Atoms Chapter 4: Purification and biochemical characterization of 55 a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough) Chapter 5: Multi-frequency EPR and high-resolution Mossbauer 65 spectroscopy of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough): characterization of a supercluster and superspin model protein Chapter 6: The third subunit
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