Role of Neighboring FMN Side Chains in the Modulation of Flavin Reduction Potentials and in the Ener

1
Role of Neighboring FMN Side Chains in the
Modulation of Flavin Reduction Potentials and in
the Energetics of the FMNApoprotein Interaction
in Anabaena Flavodoxin.

• Carrie George
• BMB III
• December 6, 2004

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Ferredoxin in Photosynthesis
3
Where does Flavodoxin come in?

• During low iron conditions flavodoxin replaces
  ferredoxin.
• Flavodoxin contains FMN instead of iron
• FMN is an isoalloxazine ring capable of holding
  an unpaired electron on one of its Nitrogens

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Oxidation States of FMN
FMNH
Semiquinone Intermediate
1e-
1e-
Hydroquinone
Quinone
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Flavodoxins

• Aka Flds
• FMN resides in a 20 Å pocket inside the
  flavoprotein
• 2 classes of flavodoxins
• Short chain 150 aa
• Long chain 170 aa

http//strucbio.biologie.uni-konstanz.de/kay/pdfs
/flavodoxin.pdf
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FMN Reduction Potentials

• E -?G/F (F is faradays constant)
• So if -?G favors products, E favor products
• FMN has 2 reduction potentials
• Oxidized Semiquione Eox/sq
• Semiquinone Reduced Esq/rd
• FMN binding to protein causes a shift in its
  reduction potentials
• Eox/sq gets more
• Esq/rd gets more
• Sq intermediate is stabilized

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Contributions to ?E

• FMNApoflavodoxin Interactions
• FMN has contacts with residues 10-15 (P binding
  loop), 56-62 (ß3/a3 loop), and 90-99 (ß4/a4 loop)

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4

• Hydrogen Bonding
• Asp90 NH --- N1 and O2
• Gln99 NH --- O2
• Asp97 CO --- N3
• Gly60 NH --- O4
• Thr56 CO --- N1
• Ile59 NH --- N5

3
2
5
1
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Contributions to ?E

• Electrostatic Interactions
• Asn58
• Ile59
• Gly60
• Tyr94
• Asn97
• Aromatic Stacking
• Tyr94 face-face w/ isoalloxazine ring
• Trp57 interacts with the phosphate

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Objective of the Experiment

• Explore the effects of specific H bond
  interactions and the electrostatic environment on
  FMN reduction potentials
• Methods used
• Site-directed mutagenesis create FMNFld
  mutants
• UV-vis spec compare spectral properties of
  mutant vs. WT (has the environment changed?)
• Fluorescence Determine dissociation constants
• Circular Diochroism effect on overall protein
  structure
• Photoreduction formation of Sq form of FMN
• EPR compare amounts of Sq stabilized
• ESEEM effect of residues on electron density
  distribution

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Anabaena Fld Mutants

• 5 mutants were made to modify polarity and H
  bonding network
• Thr56Gly loss of H bond
• Thr56Ser displacement of H bond
• Asn58Cys more negatively charged
• Asn58Lys add charge
• Asn97Lys add charge
• 3 mutants were made to change the electrostatic
  environment on the surface of the protein
• Glu20Lys - neg. to pos. charge
• Asp65Lys - neg. to pos. charge
• Asp96Asn - neutralization

Negatively Charged Surface residues in red
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Expression Levels

• Expression levels of all mutants were similar to
  wild type
• No major structural changes
• CD data show no changes in secondary structure of
  WT and mutants
• Thr56Gly lost a portion of its FMN during
  purification
• Weak FMNprotein interactions in this mutant

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Photoreduction

• Reduction of oxidized Fld was initiated by
  reaction with a highly reductive dRfH
  (5-deazoriboflavin) radical.
• Reactions were made anaerobic by evacuation and
  flushing with O2 free Ar
• Reaction mixtures were irradiated with 150W
  light.
• Absorption spectra taken after successive
  irradiation steps to measure concentrations of
  each redox state

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Determination of Concentration and Reduction
Potentials

• Measure potential of solution using a sat.
  calomel reference electrode and record absorbance
  spectra during successive irradiations
• Determine sq from absorbance at 580nm ox
  Fldtotal sq during early photoreduction
  rd Fldtotal sq during late
  photoreduction
• Determine midpoint potentials from the Nernst
  Equation E Em (0.059/n)log(ox/rd)

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Reduction Potentials
Ox/Sq Glu20Lys, Thr56Gly, Thr56Ser
Ox/Sq WT, Asp65Lys, Asp96Asn
Sq/Rd Glu20Lys, Thr56Gly, Thr56Ser
Sq/Rd WT, Asp65Lys, Asp96Asn
Slope stays about the same, but both midpoint
potentials change for the mutants, esp. Asn58Lys
and Thr56Gly ?ESq/Rd -11 to 63 mV ?EOx/Sq
-23 to 19 mV Potentials tend to converge with
each other for the mutants
Ox/Sq Asn58Cys, Asn58Lys, Asn97Lys
Sq/Rd Asn58Cys, Asn58Lys, Asn97Lys
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Reduction Potentials

• Less Sq is stabilized in the mutants as reduction
  potentials converge

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Absorption spectra of oxidized forms WT vs.
mutants

• Extinction coefficients and maxima at 460nm is
  smaller for mutants and the shoulder at 480nm is
  lost in 2 mutants environment around the flavin
  has been modified

WT bold line Thr56Gly thin line
Thr56Ser broken thin line Asn58Cys dotted
thin line Asn58Lys broken bold line Asn97Lys
dotted bold line
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Absorption spectra of semiquinone Fld WT vs.
mutant

• Isosbestic point for mutants is different
• Less sq stabilized in mutants, especially
  Thr56Gly and Asn58Lys

WT
Thr56Gly
Sq 580nm
Ox 480nm
Asn58Lys
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UV-vis properties
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EPR

• EPR (Electron Paramagnetic Resonance) Method
  for detecting species with free radicals, similar
  to NMR

• Apply continuous wave of magnetic field
• Two possible electron spin orientations create
  two distinct energy levels
• Resonance occurs when ?Ms1
• Pass microwave radiation through resonating
  molecules and determine concentration from the
  absorbance

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EPR

• ?E h? gßB
• where
• ?E is the energy difference between the two spin
  states
• h is Planck constant
• v is the microwave frequency
• g is the Zeeman splitting factor
• ß is the Bohr magneton
• B is the applied magnetic field.
• Both WT and mutants gave same value for the
  Zeeman splitting factor g2.005 (data not shown)

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EPR and ESEEM

• ESEEM (Electron Spin-Echo Envelope Modulation)
  Variation of EPR using pulsed waves of magnetic
  field
• An echo is created that cause nuclear spins to
  occur in nearby atoms.
• Measure of echo vs. time gives information about
  electron distribution in a molecule.
• Mutants show similar ESEEM data to WT (not shown)
  
• EPR and ESEEM data show that the change in
  individual residues has no effect on the electron
  density distribution of FMN.

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Effect of pH on Reduction potentials

• Slopes
• EOx/Sq
• WT -51 ?
• Thr56Gly -61 ?
• Asn58Lys -65
• ESq/Rd
• WT -51 below pH 7, 0 above
• Thr56Gly -33 ?
• Asn58Lys -89 ?

?
Sq becomes more stable as pH increases moreso for
mutants than WT
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Fluorescence Data

• Binding of FMN to protein quenches the
  fluorescence by FMN.
• WT Ox Fld has only fluoresces 5 as much as free
  FMN
• All mutants are the same except
• Asn58Lys 9
• Thr56Gly 29
• FMN in these mutants has more exposure to the
  external environment

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Fluorescence DataDetermination of Dissociation
Constants

• Begin with pure Ox FMN read fluorescence
• Add protein and allow to reach equil. read
  again
• Repeat successively
• Determine Kd from this equation
• F Ffinal Fd(dCF - CA Kd dCF)
• (CA Kd dCF)2 4CAdCF1/2/2

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Determining binding affinity profiles from Kds

• Reduction potentials are related to binding
  affinities by this thermodynamic cycle.
• Now that we have a relationship between the Es
  and Kd for Ox FMN we can calculate ?GOx.

?GSq and ?GRd can be calculated from ?GSq ?GOx
F(EOx/Sq EOx/Sqfree) ?GRd ?GOx F(EOx/Sq
ESq/Rd EOx/Sqfree ESq/Rdfree)
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Binding Affinity Profiles

• A
• ? WT
• ? Thr56Gly
• ? Thr56Ser
• ? Asn58Cys
• Asn97Lys

B ? Glu20Lys Asp96Asn ? Asn58Lys ? Asp65Lys
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What do these data tell us?

• In general If electrostatic properties within 14
  ? away are modified, changes occur in both EOx/Sq
  and ESq/Rd.
• Except Thr56Gly only ESq/Rd is changed
• Small changes occur when the electrostatic
  environment 20 ? away is modified.

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What do these data tell us?

• More specifically
• How the protein environment modulates ESq/Rd.
• How it modulates EOx/Sq.
• How pH effects both potentials
• Role of specific residues on stability of
  proteinFMN complex.

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Protein Environment Affects the ESq/Rd

• N1 is usually not protonated in the hydroquinone
  so is less stable than the other forms because of
  the negatively charged environment.
• So, removing negative charge or adding positive
  charge to this location stabilizes the
  hydroquinone (depending on location relative to
  the ring)
• Asn58Lys (4 ? away) ?E 32 mV
• Asn97Lys (5 ? away) ?E 18 mV
• Asp96Asn (10 ? away) ?E 13 mV
• Asp65Lys (13 ? away) ?E 16 mV
• Glu20Lys (20 ? away) ?E 9 mV
• Theoretical estimation -4 mV per negatively
  charged residue shift in ESq/Rd

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Protein Environment Affects the ESq/Rd

• Solvent accessibility lowers the potential
  (stabilizing the Sq state)
• Thr56Gly (increases the internal cavity) ?E
  63 mV

20 ? cavity
44 ? and 16 ? cavities
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Protein Environment Affects the EOx/Sq

• Ox to Sq transition of WT Fld from other species
  (i.e. A. nidulans)
• Sequence conservation between A. nidulans and
  Anabaena suggest it will do the same.
• Previous study Ile59Lys makes the potential more
  negative (less stabilization of Sq)

• Asn58Lys ?E -23 mV
• Increase in conformational energy of Asp58-Ile59
• And/or breakage of hydrophobic interactions
  between FMN and Asp 58

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pH Affects the Reduction Potentials

• Change in slopes of ESq/Rd andEOx/Sq vs. pH
  suggest protonation of residue(s) in the FMN
  environment (not of FMN itself) modulates the
  reduction potential.
• Thr56Gly and Asp58Lys result in a shift of the
  pKa of the protein from 6 to gt8.
• Structural changes caused by these mutations
  possible affect
• Decrease in ESq/Rd vs. pH slope
• Increase in ESq/Rd
• Increase of pKa
• Because solvent may enter the cavities.

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Thr56Gly Has a Large Effect the ApoFldFMN
interaction

• Highest increase in Kd (184-fold!)
• Why???
• Thr56 Interactions with FMN
• H bond with two O atoms
• Hydrophobic interactions with 7 atoms
• H bond to Asp100 and Ala101 near FMN
• Replace with Gly (very large increase in Kd)
• Lose H bond and most other interactions
• Make FMN more solvent accessible
• Replace with Ser (very small increase in Kd)
• Keep H bond and lose a few interactions
• Little change in solvent accessibility

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Asn97Lys Has a Smaller Effect on the ApoFldFMN
interaction

• 3.2-fold increase in Kd
• Why???
• Asp97 interactions
• H bond with N3 of FMN, Tyr94, Gln99, and 2 with
  Asp100
• Hydrophobic interactions with 2 atoms of FMN
• Replace with Lys
• Break one H bond with Asp100 and lose hydrophobic
  interactions with FMN

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Conclusion

• Redox properties of FMN are modified by solvent
  accessibility to isoalloxazine ring, and
  hydrophobic and electrostatic properties of
  residues in the environment.
• Specific examples have been presented

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References

• Nogués, et al. 2004. Role of Neighboring Side
  Chains in the Modulation of Flavin Reduction
  Potentials and in the Energetics of the
  FMNApoprotein Interaction in Anabaena
  Flavodoxin. Biochemistry 43 15111-21.
• Hoover et al. 1999. Comparisons of Wild-type and
  Mutant Flavodoxins from Anacystis nidulans.
  Structural Determinants of the Redox Potentials.
  J. Mol. Bio. 294 725-43.
• Berg, Tymoczko, and Stryer. 2002. Biochemistry
  5th ed. Ch 19 The light reactions of
  photosynthesis. 538-39
• Electron Paramagnetic Resonance What is EPR?
  2000. http//www.chem.queensu.ca/eprnmr/EPR_summar
  y.htm