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Exploring the conformational    flexibility of leghemoglobin: a    framework for examination of      heme protein axial li...
Outline     Introduction          Iron Protoporphyrins          Self-regulation          Leghemoglobin     Methods an...
Iron Protoporphyrin (IX)     Key component of many proteins and enzymes             Electron transfer proteins         ...
Self-regulation     Heme coordination can be altered by intramolecular         ligand switching where rearrangements allo...
Leghemoglobin     Small oxygen carrier found in         the roots of legumes     Heme pocket             Larger        ...
Outline     Introduction     Methods and Results             Mutagenesis             Electronic Absorption           ...
Mutagenesis     A H61Y variant was made and         introduced into the heme         pocket in order to study the        ...
Electronic Absorption        Ligand-bound derivatives were prepared by the         addition of excess ligand to solutions...
Electronic Absorption        Ferrous (reduced) H61Y produced peaks at 429.5 and 557 nm similar to the phenolate rLb (not ...
Electronic Absorption     Reduction of dithionite followed by removal by         anaerobic gel filtration and exposure to...
pH Titrations        Does hydroxide bound heme compete with                               pH 7.0         tyrosine ligatio...
X-ray Absorption (EXAFS)     In both cases, the iron lies in the plane of         the porphyrin ring     rLb           ...
Magnetic Circular Dichroism (MCD)     The H61Y MCD spectrum is         consistent with high spin ferric         heme sign...
Electron Paramagnetic Resonance (EPR)        H61Y:          High spin species with g values at              6.43, 5.56 a...
Reduction Potential     Obtained spectroelectrochemical data         for H61Y at various applied potentials     Nernst p...
Conclusions        They used spectroscopic characterization to demonstrate that this conformational         mobility can ...
Conclusions     EXAFS data clearly indicates that H61Y has a 6-coordinate         heme geometry and a shorter Fe-O bond l...
Future Aims     This decrease in reduction potential raises questions:          The alkaline form of Chlamydomonas hemog...
SupplementalN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204                                     ...
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Pletneva group presentation

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Pletneva group presentation

  1. 1. Exploring the conformational flexibility of leghemoglobin: a framework for examination of heme protein axial ligationNeesha Patel et al., Archives of Biochemistry and Biophysics 418 (2003) 197-204
  2. 2. Outline  Introduction  Iron Protoporphyrins  Self-regulation  Leghemoglobin  Methods and Results  Conclusion  Future AimsN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 2
  3. 3. Iron Protoporphyrin (IX)  Key component of many proteins and enzymes  Electron transfer proteins  Peroxidases  Monooxygenases  Oxygen carriers - heme  What are the mechanisms by which the protein structure controls the specific chemical reactivity of the heme group?  Axial ligands – most influential  Understand the relationships that exist between different classes of heme proteinsN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 3
  4. 4. Self-regulation  Heme coordination can be altered by intramolecular ligand switching where rearrangements allow for the replacement of axial ligands  pH dependent alkaline transition of ferricytochrome c: methionine is replaced by 2 lysines  C-heme reduction: axial ligation of His-His switches to His-Met  Self-regulation provides the framework for the investigation of heme structure-function relationships.N. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 4
  5. 5. Leghemoglobin  Small oxygen carrier found in the roots of legumes  Heme pocket  Larger  More accessible  More flexible  Distal  Flexibility allows for the binding of a mobile switching ligand at a low temperature.N. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 5
  6. 6. Outline  Introduction  Methods and Results  Mutagenesis  Electronic Absorption  X-ray absorption (EXAFS)  Magnetic circular dichroism (MCD)  Electron Paramagnetic Resonance (EPR)  Reduction Potential  Conclusion  Future AimsN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 6
  7. 7. Mutagenesis  A H61Y variant was made and introduced into the heme pocket in order to study the intrinsic conformational flexibility of the heme.  Purification:  Recombinant soybean leghemoglobin a (rLb)  H61Y variantN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 7
  8. 8. Electronic Absorption  Ligand-bound derivatives were prepared by the addition of excess ligand to solutions of ferric and ferrous rLb  Phenolate-bound rLb was also prepared  Most notable difference:  H61Y: green  rLb: red  The ferric H61Y mutant is distinct from the high and low spin species from the rLb.  Prominent band at 600 nmN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 8
  9. 9. Electronic Absorption  Ferrous (reduced) H61Y produced peaks at 429.5 and 557 nm similar to the phenolate rLb (not shown).  Loss of Tyrosine ligand  5 coordinate heme structure  Addition of cyanide to the ferric H61Y gave similar peaks to the cyanide bound rLb  Ferric H61Y also bound other strong-field ligands such as nicotinate and azide with similar peaks to that of rLb.  Although these compete for the 6th coordinate position, the binding for H61Y was weaker  Kd H61Y = 19,000 ± 1000 μM  Kd rLb = 4.8 ± 0.2 μM  Weak field ligands don’t significantly alter the spectrum (not shown).N. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 9
  10. 10. Electronic Absorption  Reduction of dithionite followed by removal by anaerobic gel filtration and exposure to O2 is known to form stable oxy-derivatives in rLb.  Similar attempts in H61Y were unsuccessful  Likely due to rapid auto-oxidation of the ferrous-oxy derivativeN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 10
  11. 11. pH Titrations  Does hydroxide bound heme compete with pH 7.0 tyrosine ligation at alkaline conditions?  The spectrum of the H61Y variant did not change with increasing pH, but did with decreasing pH (green to red).  The intensity of the 602 nm band decreases and pH 3.7 shifts to a longer wavelength  There’s a decrease in intensity of the soret band at 401 nm and a band shift to 370 nm at an acidic pH pH 8.5  Consistent with the protonation of the proximal histidine  Similar pKa reports: rLb pKa of 4.4 and a H61Y pKa of 4.6 pH 3.8  Same protonation process  Final species in both rLb and H61Y is likely a non-covalent, 4-coordinate, protein-heme complexN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 11
  12. 12. X-ray Absorption (EXAFS)  In both cases, the iron lies in the plane of the porphyrin ring  rLb  Short bond Fe-OH2: 190 ± 5 pm  Coordination of water  H61Y  Short bond Fe-O: 185 ± 5 pm  Significantly lower Debye-Waller factor  Atom was part of a heavier unit than water, indicating a ligation of Tyr61  Stronger ligation to heme  The Fe-Nε bond length in H61Y is identical within experimental error to the rLb value, strongly implicating a 6- coordinate His-Tyr hemeN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 12
  13. 13. Magnetic Circular Dichroism (MCD)  The H61Y MCD spectrum is consistent with high spin ferric heme signals.  The soret band at 413 nm confirms the EXAFS assignment as 6-coordinate.  High spin ferric heme systems contain 2 ligand-metal charge transfer bands, CT1 and CT2 that change with axial ligation  rLb His-H2O: 635 and 1100 nm (not shown)  H61Y His-Tyr: 621 and 830 nm  Identical to whale myoglobin which is known to have His-Tyr ligationN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 13
  14. 14. Electron Paramagnetic Resonance (EPR)  H61Y:  High spin species with g values at 6.43, 5.56 and 1.95  Small proportion of low-spin heme at 2.67, 2.17 and 1.79  Low spin His-Tyr species arising from freezing induced conformational changes.  The ferric heme rearranges to a low spin species at low temperature  This low spin derivative is a bis-histidine species generated by ligation of His61 to the heme  rLb: 6.01, 2.74, 2.27 and 2.00 (previously published)  Rhombic splitting of the g=6 is common among high spin Fe-Tyr species such as whale myoglobin and catalase.N. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 14
  15. 15. Reduction Potential  Obtained spectroelectrochemical data for H61Y at various applied potentials  Nernst plot of the absorbance data at 405 nm to the Nernst equation  Reduction Potential: -127 ± 5 mV  Decrease of 148 mV from the value obtained from rLb.  Low reduction potential accounts for the failure to isolate an oxy-derivative of H61YN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 15
  16. 16. Conclusions  They used spectroscopic characterization to demonstrate that this conformational mobility can be used as a platform for alteration of active site architecture.  The electronic absorption spectrum of the ferric H61Y is similar to that of rLb  Evidence of tyrosine ligation at room temperature  The soret band at ~600 nm is characteristic of other 5 coordinate tyr-ligated heme proteins including the H93Y mutant in horse heart and humans.  The spectra obtained upon reduction of H61Y is consistent with the loss of the tyrosine ligand.  Strong field ligands compete for the 6th coordinate position.  The decrease in intensity of the soret band at 401 nm and a shift to 370 nm at an acidic pH for rLb and H61Y are consistent with the protonation of the proximal histidine  Same protonation process  Non-cavelent protein-heme 4 coordinate complexN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 16
  17. 17. Conclusions  EXAFS data clearly indicates that H61Y has a 6-coordinate heme geometry and a shorter Fe-O bond length and stronger ligation to heme.  Consistent with an Fe-Tyr coordination geometry.  MCD and EPR experiments provided further evidence for the 6-coordinate assignment and tyrosine ligation.  From the presence of high spin ferric heme  H61Y wasn’t able to form a stable, physiological oxygen- bound heme complex due to the 150 mV decrease in reduction potential when compared to rLbN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 17
  18. 18. Future Aims  This decrease in reduction potential raises questions:  The alkaline form of Chlamydomonas hemoglobin is able to form functional O2 bound complexes despite using 6- coordinate His-Tyr ligation  They need to expore the features that define the differences between the different hemes, but their results provided a solid framework for further investigation.N. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 18
  19. 19. SupplementalN. Patel et al. Archives of Biochemistry and Biophysics 418 (2003)197-204 19

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