Interaction and Associated Conformational Changes in the Modulation of the Redox Properties in Flavoproteins

THE ROLE OF THE N(5) INTERACTION AND ASSOCIATED CONFORMATIONAL CHANGES IN THE MODULATION OF THE REDOX PROPERTIES IN FLAVOPROTEINS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Mumtaz Kasim, M.Sc. * * * * * The Ohio State University 2002 Dissertation Committee: Dr. Richard P. Swenson, Advisor Approved by Dr. Gary Means ________________________ Dr. Caroline Breitenberger Advisor Dr. Mark Foster Department of Biochemistry ABSTRACT Many biochemical processes exploit the remarkable versatility of flavoenzymes and their flavin cofactors by modulating the numerous interactions of the cofactor with the apoflavoprotein. The interaction between the protein and N(5) of the flavin cofactor has been of particular interest for this very reason. In the Clostridium beijerinckii flavodoxin, the four residue reverse turn –Met56-Gly-Asp-Glu59- provides the majority of the critical interactions with the flavin mononucleotide cofactor (FMN) that contribute to the binding and differential stabilization of its three redox states. This turn undergoes a conversion from a mix of cis/trans peptide configurations that approximates a type II turn in the oxidized state to a type II′ turn upon reduction. This change results in the formation of a new hydrogen bond between the N(5)H of the reduced flavin and the carbonyl group of Gly57 of the central peptide bond of the turn, an interaction that contributes to the modulation of the oxidation-reduction potentials of the cofactor. Systematic replacement of the second and third residues of the turn (Gly57 and Asp58) with –Gly-Gly-, -Gly- Ala-, -Ala-Gly- and –Ala-Ala- dipeptidyl sequences resulted in an altered stability of the FMN semiquinone that was directly correlated to the conformational energetics of the turn. In addition, sequential elimination of all side chain interactions in various combinations through an alanine-scanning mutagenesis approach proved the overriding ii importance of the main chain interactions with the N(5)H of the FMN and the associated conformational change in this loop to be the primary determinant of the thermodynamic stabilization of the FMN semiquinone. In contrast, in the structurally homologous FMN-binding domain of cytochrome P450 reductase from Bacillus megaterium, a main chain hydrogen bond to N(5) is present in the oxidized state. 15N-NMR studies indicate that in this case, a conformational change occurs in the flavin at the N(5) position. Sequence specificity of the type I′ turn adopted by the residues –Tyr536-Asn-Gly-His539- was tested by replacement of the central residues of the turn (Asn537 and Gly538) with –Gly-Gly-, -Gly-Ala-, -Ala-Gly- and –Ala-Ala- dipeptidyl sequences. These mutations established the critical role of the position of the glycine residue in maintaining turn stability. We conclude that the N(5) interaction and the associated conformational change, as well as sequence specificity of the turn involved in flavin binding, play a critical role in determining the redox properties of flavoproteins. iii To my dad, who always believed in me, and to my husband, who taught me to believe in myself. iv ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Richard P. Swenson, for his guidance, support and patience. I would like to also thank my family who always had faith in me and who continue to understand my long absence away from home. I would like to thank past and present members of the Swenson laboratory, especially, Dr Lawrence Druhan, Dr. Fu-Chung Chang, Dr. Yucheng Feng, Dr. Luke Bradley, James Wu, Kun-Yun Yang, Tracey Murray and Michelle Nauerth, for valuable help and insight. I would like to thank the people in Buckeye Tang Soo Do, whose support and friendship helped me through the years. I would like to thank my friends: Joe Davis, Paul Erwin and especially Shane Mellor, for being there when I needed them and for making these past few years more fun than I thought was possible. Finally, I would like to thank my many friends in Graduate School, in particular: Milan Jovanovic, Craig McElroy, Srisunder Subramanium, Ryan Pereira and Manoj Nair for providing a very interesting work environment. v VITA April 6, 1972……………………………… Born – Bombay, India 1993……………………………………….. B. Sc. Life Sciences/Biochemistry, St. Xavier's College, University of Bombay 1995………………………………….……. M. Sc. Biochemistry, University of Bombay 1996 – present…………………………….. Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Kasim, M. and Swenson, R. P. “Alanine-Scanning of the 50’s Loop in the Clostridium beijerinckii Flavodoxin: Evaluation of Additivity and the Importance of Interactions Provided by the Main Chain in the Modulation of the Oxidation- Reduction Potentials.” Biochemistry, 40, 13548–13555, (2001). 2. Kasim, M. and Swenson, R. P. “Conformational Energetics of a Reverse Turn in the Clostridium beijerinckii Flavodoxin is Directly Coupled to the Modulation of its Oxidation-Reduction Potentials.” Biochemistry, 39, 15322-15332, (2000). 3. Swenson, R. P., Kasim, M., Bradley, L. and Druhan, L. “Role of Conformational Dynamics and Associated Electrostatic and Hydrogen Bonding Interactions in the Regulation of Redox Potentials in the Clostridium beijerinckii Flavodoxin.” In Flavins and Flavoproteins 1999 (Ghisla, S., Kroneck, P., Macheroux, P., and Sund, H., Eds) Agency for Scientific Publishing, Berlin, 183-186, (2000). vi FIELDS OF STUDY Major Field: Biochemistry vii TABLE OF CONTENTS PAGE Abstract……………………………………………………………………….. ii Dedication…………………………………………………………………….. iv Acknowledgments…………………………………………………………….. v Vita……………………………………………………………………………. vi List of Tables…………………………………………………………………. x List of Figures………………………………………………………………… xii List of Abbreviations…………………………………………………………. xvi Chapters: 1. Introduction…………………………………………………………... 1 2. Materials and Methods………………………………………………. 34 3. Conformational Energetics of a Reverse Turn in the Clostridium beijerinckii Flavodoxin is Directly Coupled to the Modulation of its Oxidation- Reduction Potentials………………………..…………… 42 Introduction………………………………………………………….. 42 Materials and Methods………………………………………………. 49 Results……………………………………………………………….. 49 viii Discussion…………………………………………………………… 71 4. Alanine-Scanning of the 50’s Loop in the Clostridium beijerinckii Flavodoxin: Evaluation of Additivity and the Importance of Interactions Provided by the Main Chain in the Modulation of the Oxidation-Reduction Potentials……………………………………… 84 Introduction………………………………………………………….. 84 Materials and Methods………………………………………………. 91 Results……………………………………………………………….. 91 Discussion…………………………………………………………… 101 5. Cloning and Characterization of the FMN-Binding Domain of Cytochrome P450 Reductase From Bacillus Megaterium…………… 113 Introduction………………………………………………………….. 113 Materials and Methods………………………………………………. 123 Results……………………………………………………………….. 123 Discussion…………………………………………………………… 153 6. The FMN-Binding Domain of P450BM-3: Investigation into the Possible Mechanisms of Redox Tuning……………………………... 159 Introduction………………………………………………………….. 159 Materials and Methods………………………………………………. 163 Results……………………………………………………………….. 164 Discussion…………………………………………………………… 184 7. General Conclusions and Future Directions…………………………. 190 List of references……………………………………………………………… 196 ix LIST OF TABLES TABLE PAGE 1. Turns in the flavodoxin from Clostridium beijerinckii………………… 15 2. Idealized dihedral angles of hydrogen bonded β turns………………… 17 3. Sequence and midpoint potential comparisons of several flavodoxins... 18 4. Oxidation-reduction midpoint potentials for the Clostridium beijerinckii flavodoxin…………………………………………………. 20 5. Energies of β turn formation for the Type II and Type II′ turns……….. 23 6. Relative free-energy changes for refolding from the Type II to the Type II′ turn……………………………………………………………. 24 7. Energies of β turn formation for the Type I′ turn……….…………….. 31 8. Relative free-energy changes for refolding from the extended to the Type I′ turn conformation……...………………………………………. 32 9. Oxidation-reduction midpoint potentials, FMN dissociation constants and Gibbs free energy changes of wild-type and mutant C. beijerinckii flavodoxins…………………………………………………………….. 57 10. 15N Chemical shifts for free and bound FMN in the oxidized state, pH 7.0, 300°K for C. beijerinckii………………………………………... 67 11. 1H-15N HSQC temperature coefficients for the Clostridium beijerinckii wild-type and mutant flavodoxins in the oxidized state….. 70 x 12. Oxidation-reduction midpoint potentials, FMN dissociation constants and Gibbs free energy of FMN binding for wild-type and mutant C. beijerinckii flavodoxins……………………………….……………….. 97 13. 15N Chemical shifts for free and bound FMN in the oxidized state, pH 7.0, 300°K for BM-3…………………………………………………… 145 14. 15N Chemical shifts for free and bound FMN in the reduced state, pH 7.0, 300°K for BM-3…………………………………………………… 149 15. Coupling constants for N(3)H and N(5)H in the oxidized and fully reduced states of BM-3…………………………………………….…. 150 16. 15N Chemical shifts for free and bound FMN in the oxidized state, pH 7.0, 300°K for –537Ala-Ala-…………………………………………… 172 xi LIST OF FIGURES FIGURE PAGE 1. The structures of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)……………………………………………. 2 2. The three oxidation states of the isoalloxazine ring…………………… 4 3. The UV-visible spectra of the flavin cofactor in different redox states... 5 4. Structure of the Clostridium beijerinckii flavodoxin…………………... 12 5. Comparison of the

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