Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1986 The er actions of nitric oxide and nitrite with hemerythrin Judith Mary Nocek Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Inorganic Chemistry Commons Recommended Citation Nocek, Judith Mary, "The er actions of nitric oxide and nitrite with hemerythrin " (1986). Retrospective Theses and Dissertations. 8026. https://lib.dr.iastate.edu/rtd/8026 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. For more information, please contact [email protected]. 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Other University Microfilms International The reactions of nitric oxide and nitrite with hemerythrin by Judith Mary Nocek A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department: Chemistry Major: Inorganic Chemistry Approved: Signature was redacted for privacy. In Charge of Majdr^WoVk Signature was redacted for privacy. Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1986 il TABLE OF CONTENTS Page LIST OF SYMBOLS AND ABBREVIATIONS xv I. INTRODUCTION 1 A. Statement of the Problem 1 B. Review of Previous Studies on Hemerythrin 6 C. Aqueous Chemistry of NO and NO^"" 77 II. EXPERIMENTAL WORK 98 A. Instrumentation 98 B. Preparation of Reagents and of Hemerythrin and Myoglobin in Their Various Oxidation Levels 104 C. Preparation of Nitric Oxide and Nitrite Derivatives of Hemerythrin 112 D. Isotopic Enrichment of Samples 121 E. Reactivity Studies with the Nitric Oxide and Nitrite Derivatives of Hemerythrin 123 III. RESULTS AND DISCUSSION 131 A. Oxidation of Hemerythrin by Nitrite 131 B. Assay of Aqueous Solutions of Nitric Oxide 155 C. Preparation and Characterization of NO Adducts of (Semi-met)o and (Semi-met)r 165 0. Preparation and Characterization of NO Adducts of DeoxyHr and DeoxyF'Hr 185 E. Attempts to Prepare a Nitric Oxide Adduct of Methemerythrin 236 F. Reactions of DeoxyNO 244 G. Reactions of DeoxyF"NO 275 ii i Page IV. SUMMARY AND CONCLUSIONS 279 A. Preparation, Characterization, and Reactivity of the Nitric Oxide and Nitrite Derivatives of Hemerythrin 279 B. Comparisons to the Physiologically Occurring Reactions: Oxygenation and Auto-Oxidation 284 C. Comparisons to the Reactions of Nitrite and Nitric Oxide with Hemoglobin 288 0. Implications About Nitrite Metabolism 295 V. REFERENCES 300 VI. ACKNOWLEDGEMENTS 311 VII. APPENDIX A 312 VIII. APPENDIX B 315 iv LIST OF FIGURES Page Figure 1.1. Respiratory diagrams for hemerythrin from Themi s te zostericola (A) and Siphonosoma ingens (B) (20) 7 Figure 1.2. Primary structure of hemerythrin from Phascolopsis gouldii (43) 11 Figure 1.3. Molecular model of the tertiary structure for hemerythrin (46) 13 Figure 1.4. Schematic representation of the quaternary struc­ ture for hemerythrin from Phascolopsis gouldii (55) 15 Figure 1.5. Quaternary structure of hemerythrin from Phascolopsis gouldii (41) 16 Figure 1.6. Active site structures at 2.0 A resolution for metHr (A) and metN^- (B) from Themiste dyscritum (45) 18 Figure 1.7. Structures of synthetic model compounds for hemerythrin 22 Figure 1.8. Stereodiagrams showing the vicinity of the ac­ tive site and the locations of HO 149 (A) and Tyr 109 (B) (65) ^ 23 Figure 1.9. Proposed mechanism for the conversion of deoxyHr to oxyHr (3) 27 Figure 1.10. Optical spectra for derivatives of hemerythrin 29 Figure 1.11. Optical spectra of metHr in 50 nW phosphate at pH 6.5 (A) and in 50 mM Tris-sulfate at pH 7.5 (B) 31 Figure,1.12. Order of the energies of the valence orbitals of the Fe^^-OgZ" center of oxyhemerythrin (72) 33 Figure 1.13. Order of the energies of the magnetic states re­ sulting from antiferromagnetic coupling of high- spin Fe2+ and high-spin Fe3+ ions (A) and from an isolated high-spin Fe^"*" ion with 0 > 0 (B) 38 Figure 1.14. EPR spectra for derivatives of hemerythrin 39 V Page 1.15. Resonance Raman spectra obtained with = 514.5 nm for oxyheirerythrin prepared with 58 atom % iSQ-enriched 0^ gas (82) 41 1.16. Isotopic substitution effects on the resonance Raman spectrum of oj^hemerythrin (80,81,83) 42 1.17. Resonance Raman enhancement profiles for oxyheme rythrin (80) 43 1.18. IH-NMR spectra for derivatives of metHr at 30°C (89) 54 1.19. IH-NMR spectra for semi-metN," (A), deoxyN ~ (B), and deoxyHr (C) (89) 55 1.20. Comparison of the ordering of the energies of the magnetic states for two ferrous sites with 0 » J (A) and with P « J (B) 57 1.21. Summary of the oxidation levels attainable for hemerythrin 60 1.22. EPR spectra for the conversion of (semi-met)o to (semi-met)^ 67 1.23. EPR spectrum of NO in phosphate buffer at pH 6.5 78 1.24. Molecular orbital diagrams for the binding of NO to iron in a linear geometry (A) or a bent geometry (B) 89 1.25. Structures of binuclear Iron nitrosyl compounds 94 1.26. Bidentate coordination modes for the nitrite ligand 97 2.1. EPR spectrum for an S = 1/2 center having rhombic symmetry 100 3.1. Spectral time course for the oxidation of deoxyHr with excess nitrite 132 3.2. Semi-log plot of the absorbance changes at 380 nm as a function of time for the oxidation of 0.0682 mM deoxyHr with 6.91 nW NaNO, in 50 tiW phosphate (pH 7.05) 133 vi Page Figure 3,3. Comparison of the absorbante changes at 380 nm calculated from an NLLSQ fit with the experi­ mental absorbance changes 134 Figure 3,4. pH dependence of the second-order rate constant for the initial fast phase of the oxidation of deoxyHr by excess nitrite 137 Figure 3.5. Time course for the oxidation of deoxyHr by ex­ cess nitrite followed by EPR spectroscopy 139 Figure 3.6. Spectral time course of the oxidation of deoxyHr by excess nitrite in the presence of NaN^ 143 Figure 3.7. Optical spectra before (A) and after (B) the addi­ tion of excess NaN, at 3 1/2 hours after mixing deoxyHr with nitrite 146 Figure 3.8. EPR spectra obtained after addition of excess NaNOg to (semi-met)o (A) and (semi-met)p (B) 147 Figure 3.9, Proposed structure for semi-metNOg" 151 Figure 3.10. Time course for the 1:1 reaction of deoxyHr with NaNOg followed by EPR spectroscopy 153 Figure 3.11. Time course for the reaction of oxyHr with ex­ cess NaNOg followed by EPR spectroscopy 157 Figure 3.12. Time course for the intracellular oxidation by nitrite of O^-depleted erythrocytes from Phascolopsis goudlii 158 Figure 3.13. EPR spectra of assay mixtures containing Fe2+(EDTA) and either gaseous NO (A), a 2-fold excess of NaNOg (B), or a saturated aqueous solution of NO (C) 160 Figure 3.14.