Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1986 Oxidation and reduction reactions of hemerythrin and [mu]-sulfidohemerythrin: mechanism of reduction and implications for electron transfer Linda Lorraine Pearce Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Inorganic Chemistry Commons Recommended Citation Pearce, Linda Lorraine, "Oxidation and reduction reactions of hemerythrin and [mu]-sulfidohemerythrin: mechanism of reduction and implications for electron transfer " (1986). Retrospective Theses and Dissertations. 8109. https://lib.dr.iastate.edu/rtd/8109 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. 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Zeeb Road, Ann Arbor, Michigan 48106 8627144 Pearce, Linda Lorraine OXIDATION AND REDUCTION REACTIONS OF HEMERYTHRIN AND MU- SULFIDOHEMERYTHRIN: MECHANISM OF REDUCTION AND IMPLICATIONS FOR ELECTRON TRANSFER Iowa State University PH.D. 1986 University Microfilms i n tsrn Slti O n âl SOO N. Zeet Road, Ann Artwr, Ml 48106 PLEASE NOTE: In ail cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages 2. Colored illustrations, paper or print 3. Photographs with dark background 4. Illustrations are poor copy 5. Pages with black marks, not original copy 6. Print shows through as there is text on both sides of page 7. indistinct, broken or small print on several pages J 8. Print exceeds margin requirements 9. Tightly bound copy with print lost in spine 10. Computer printout pages with indistinct print 11. Page(s) lacking when material received, and not available from school or author. 12. Page(s) seem to be mis^ng in numbering only as text follows. 13. Two pages numbered . Text follows. 14. Curiing and wrinkled pages 15. Dissertation contains pages with print at a slant, filmed as received 16. Other University Microfilms International Oxidation and reduction reactions of hemerythrin and y-sulfidohemerythrin: Mechanism of reduction and implications for electron transfer by Linda Lorraine Pearce A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Chemistry Major: Inorganic Signature was redacted for privacy. Charge of Major Wor Signature was redacted for privacy. Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1986 ii TABLE OF CONTENTS Page ABBREVIATIONS XV GENERAL INTRODUCTION 1 SECTION I. THE REDUCTION OF METHEMERÏTHRIN TO DEOXYHEMERYTHRIN 16 STATEMENT OF THE PROBLEM 16 PREVIOUS WORK 16 EXPERIMENTAL 22 RESULTS 30 DISCUSSION 61 REFERENCES CITED 75 SECTION II. COMPARISONS OF THE OXIDATION AND REDUCTION KINETICS OF HEMERYTHRIN AND U-S^" HEMERYTHRIN 77 INTRODUCTION 77 STATEMENT OF THE PROBLEM 32 EXPERIMENTAL 84 RESULTS AND DISCUSSION 90 CONCLUSION 133 REFERENCES CITED 137 SECTION III. THE OXIDATION OF DEOXYHEMERYTHRIN TO THE SEMI-MET OXIDATION LEVEL BY CHROMATE 140 INTRODUCTION 140 EXPERIMENTAL 141 RESULTS AND DISCUSSION 144 iii REFERENCES CITED 161 GENERAL CONCLUSIONS 162 LITERATURE CITED FOR GENERAL INTRODUCTION AND GENERAL CONCLUSION 168 APPENDIX A: CHROMOUS SCRUBBING TOWERS 170 APPENDIX B; CALIBRATION OF EPR CRYOSTAT TEMPERATURE 171 iv LIST OF FIGURES Page Figure 1. The active site structure of metHr (left) and 3 metazidoHr (right) at 2.0 Â resolution Figure 2. Quaternary and tertiary structures of 4 octameric Hr Figure 3. UV-visible absorption spectra of metHr at pH 7 6.0 (a), pH 8.0 (b) and metN^" ( ) produced after addition of NaN^ to (a) or (b) Figure 4. (Semi-met)Q and (semi-met)gHr EPR spectra at 11 4 K, pH 6.3 (50 mM MES) Figure 5. UV-visible absorption spectra of (semi-met)^Hr 12 (solid line), and semi-met azideHr (dashed line), the latter obtained after addition of azide to (semi-met)j^Hr in 50 mM MES, pH 6.3, 0.15 M NagSO^ Figure 6. Oxy (solid line) and deoxyHr (dashed line), 14 UV-visible absorption spectra, 50 mM MES, 0.15 M Na2S0^, pH 6.3 Figure I-l. UV-visible absorption spectra of the second 20 stage product of reduction of T. zostericola metHr (3) V Figure 1-2. Non-linear least squares fits of the 32 absorbance at 380 ran versus time data for the reduction of 0.1 mM P. qouldii metHr by a 10-fold molar excess of NagSgO^ at pH 7.0 (50 mM HEPES), 0.15 M NagSO^ Figure 1-3. UV-visible absorption spectra of the products 37 of reduction of 0.1 mM P. qouldii metHr at pH 7.0 Figure 1-4. UV-visible absorption spectra of the products 39 of reduction of 0.1 mM P. qouldii metHr at pH 6.3 Figure 1-5. UV-visible spectra of second stage product in 40 the reduction of P. qouldii metHr at pH 6.3 before and after exposure to Og Figure 1-6. UV-visible absorption spectra of the products 43 of reduction of 0.1 mM P. qouldii metHr at pH 8.2 Figure 1-7. UV-visible absorption spectra of the second 44 stage product in the reduction of P. qouldii metHr at pH 3.2 before and after exposure to O2 Figure 1-8. Semi-met EPR signals obtained during reduction 45 of 0.1 mM P. qouldii metHr with a 20-fold molar excess of NagSgO^ at pH 7.0 (50 mM HEPES) and 20 "C vi Figure 1-9. Semi-met EPR signals obtained during reduction 46 of 0.1 mM P. qouldii metHr with a 20-fold molar excess of NagSgO^ at pH 6.3 [50 mM MES) and 20 *C Figure I-IO. Semi-met EPR signals obtained during the 47 reduction of 0.1 mM P. qouldii metHr with a 20-fold molar excess of dithionite at pH 8.2 (50 mM EPPS) and 25 *C Figure I-lla. EPR spectra during reduction of 0.1 mM T. 49 zostericola metHr with a 20-fold molar excess of dithionite Figure I-llb. EPR spectra during reduction of 0.1 mM T. 50 zostericola metHr with a 20-fold molar excess of dithionite after addition of 50 mM N-~ Figure 1-12. Semi-met EPR signals obtained after reduction 51 of 1.0 mM P. qouldii metHr with one equivalent of Cr^+Caq) at pH 7.0 (50 mM HEPES), 0.15 M «*2804 Figure 1-13. Semi-met EPR signals obtained after reduction 53 of 1.0 mM P. qouldii metHr with one equivalent of [cr(15-aneN,)(H20)2]^* at pH 7.0 (50 mM HEPES), 0.15 M NagSO^ Figure 1-14, ^^Fe Mossbauer spectrum of the second stage 54 product during the reduction of T. zostericola Hr using 1.5 equivalents of [cr(15-aneN^)(H20)2]^*, obtained at 4.2 K in a magnetic field of 2.2 kgauss vii Figure 1-15. MSssbauer spectrum of the second stage 56 product during the reduction of P. qouldii Hr using excess dithionite at pH 6.3 (50 mM MES), 0.15 M NagSO^, obtained at 100 K Figure 1-16. MSssbauer spectrum of the second stage 57 product during the reduction of P. qouldii Hr using two equivalents of [crCl5-aneN^)CH20)2^ pH 8.2 (50 mM EPPS), 0.15 M NagSO^, obtained at 100 K Figure 1-17. Power saturation of the EPR signal obtained 59 at 6.4 K for semi-metazidoHr Figure 1-18. Temperature dependences of the half- 60 saturation powers for semi-metazidoHr, squares; (semi-met)^Hr, circles; p-S "semi-metHr, triangles Figure II-l. UV-visible absorption spectra of u-S ~metHr 79 and u-S "semi-metHr in anaerobic Tris/acetate pK 8.0 Figure II-2. Active site of metHr (left) and proposed 81 active site of p-S "metHr (right) Figure II-3.