Spectroscopic Probes of Protein Dynamics: Hemoglobin and ...franzen/public_html/CH736/lecture/Hb_Mb_Lecture.pdf · Structural heterogeneity in myoglobin has been proposed based on

Spectroscopic Probes of Protein Dynamics:

Hemoglobin and Myoglobin

NC State University

N N

NN

O O-O O-

Fe

The iron in heme is the binding site for oxygen and peroxide

Heme is iron protoporphyrin IX.

Functional aspects in Mb

O|||O

N N

NN

O O-O O-

Fe




1. Discrimination againstCO binding.

O|||C

N N

NN

O O-O O-

Fe




1. Discrimination againstCO binding.

2. O2 is the physiologicallyrelevant ligand, but it canoxidize iron (autooxidation).

3+

Myoglobin

PDB: 1A6G

SCOPClass: All α proteinsSuperfamily:

Globin-likeFamily: Globins

Vojetchovsky,Berendzen,Schlicting

The substrate of DHP binding site

Lebioda et al., J.Biol.Chem. (2000) 275, 18712

4-iodophenol

Catalytic activity in a hemoglobin

Tribromophenol

DibromoQuinone

DHP + TPB + H2O2

DHP + DBQ + H2O

Conformational SubstatesProteins can assume a huge number of slightly different structures (conformational substates), which can be represented by nearly isoenergetic minima in an energy landscape with a typical barrier height of 10 kJ/mol.

What is the functional role of these substates?

kBT

Enzymatic catalysis involves lowering of the energy barrier

∆H*

Protein fluctuations are certainly required for catalysis.However, the important question is:How is specificity built into the catalytic mechanism?

∆H*

Non-equilibrium relaxationProtein-substrate, protein-protein and protein-DNA interactions usually do not occur under equilibrium conditions in living systems.

Non-equilibrium relaxations from one state to another occur in many situations in biology:

Ligand binding and transportSignalingElectron TransferEnzymatic CatalysisProtein Folding

MbCO

The peptide backboneis shown as a ribbonthat follows the a-helicalstructure of myoglobin.

The structure shown isat equilibrium.

Conformational substatesare called A states.

Teng, Srajer, MoffatNature Struct. Biol. (1994), 1, 701

Mb:COThe photoproduct.

Iron moves out of the heme planewhen CO is photolyzed.

CO moves to a dockingsite and is parallel to the heme plane.

Conformational substatesare called B states.

Deoxy Mb

The deoxy structurehas no CO ligand.

The protein backbonehas shifted to permita water to enter thedistal pocket.

This form is often referred to an S state.

MbCO

Mb:CO

Deoxy Mb

A comparison ofthree structures showsthe changes in bothproximal and distalhistidines.

k12

k23

Protein Data Bank 1A6N 1AJG 1AJH

Deoxy Mb

Mb:CO

MbCO

These structuresrepresent the three states of a kinetic scheme.

k21

k32

Teng, Srajer, MoffatNature Struct. Biol. (1994), 1, 701

Absorption Spectroscopy:Introduction to

Vibronic Coupling

Porphine orbitals

eg eg

a2u a1u

The four orbital model is used to represent the highest occupied and

lowest unoccupied MOs of porphyrins

eg π∗

a1u πa2u π

The two highest occupiedorbitals (a1u,a2u) are nearly equal in energy. The egorbitals are equal in energy.Transitions occur from:a1u→ eg and a2u → eg.

M1

The transitions from ground state π orbitalsa1u and a2u to excited state π* orbitals eg

can mix by configuration interaction

eg π∗

a1u πa2u π

Two electronic transitionsare observed. One is verystrong (B or Soret) and the other is weak (Q).The transition moments are:MB = M1 + M2MQ = M1 - M2 ≈ 0

M1 M2

The porphine ring is an aromatic ring that has a fourfold symmetry axis

The ring and metal can beconsidered separately.The ring has been succesfullymodeled using the Goutermanfour orbital model.In globins the iron is Fe(II)and can be either high spin or low spin.MbCO ------ low spinDeoxy Mb - high spin

N N

NN

Four orbital model of metalloporphyrin spectra

|By0⟩ = 1

2 a2uegy + a1uegx

|Qy0⟩ = 1

2 a2uegy – a1uegx

|Bx0⟩ = 1

2 a2uegx + a1uegy

|Qx0⟩ = 1

2 a2uegx – a1uegy

There are four excited state configurations possible inD4h symmetry. These are denoted B (strong) and Q(weak).

The absorption cross sectionThe absorption cross section for a Franck-Condon active transition is proportional to:

Here i and f represent individual vibrationallevels in each electronic manifold. The polarization can be σ = x, y, or z.

<i|eσ|f>2

Ef – Ei – hω – iΓ f

The Herzberg-Teller expansionThe Herzberg-Teller expansion of a particularvibronic level is given by:

This expansion describes how electronic statesn and r can mix by virtue of distortions of themolecular geometry.

|nv⟩ = |n⟩ 0|v⟩ +r| ∂H∂QK

|n u|QK|v

En,v – Er,u|r⟩|u⟩Σ

KΣr

Vibronic matrix elements

By0| ∂H∂Q |Qy

0 = a2u| ∂H∂Q |a2u

– a1u| ∂H∂Q |a1u + egy| ∂H∂Q |egy – egx| ∂H∂Q |egx

Qy0| ∂H∂Q |Qy

0 = a1u| ∂H∂Q |a1u

+ a2u| ∂H∂Q |a2u + egy| ∂H∂Q |egy + egx| ∂H∂Q |egx

Interstate Herzberg-Teller coupling:

Intrastate Jahn-Teller coupling:

Vibronic theory of absorptionThe extinction coefficient is proportional to the square of the transition moment:

FC part

Vibronicpart

∈ = <i|eσ|f>2<0|v>2

Efv – Ei0 – hω – iΓ fΣv +

<r| ∂H∂QK

|f><u|QK|v><i|eσ|r>Ef ,v – Er,u

2

<0|u>2

Efv – Ei0 – hω – iΓ fΣK

Σr

Transition moments in the four orbital model with configuration interaction

The mixing of the B and Q states is representedby the angle α:

rσ0 ≡ G|eσ|Qσ0

Rσ0 ≡ G|eσ|Bσ

0

rσ = G|eσ|Qσ = cos α G|eσ|Qσ0 + sin α G|eσ|Bσ

0

= cos α rσ0 +sin α Rσ0

Rσ = G|eσ|Bσ = cos α G|eσ|Bσ0 + sin α G|eσ|Qσ

0

= cos α Rσ0 +sin α rσ0

The B and Q bands for B1g and B2gsymmetry vibronic matrix elements

Franck-Condonpart

Herzberg-Teller part

∈ = Rσ0 2 sin2 α

EQ0 – hω 2 + ΓQ2

+cos2 α

EB0 – hω 2 + ΓB2

+ Rσ0 2b g

2

sin α sin 2αhωK

+cos α cos 2αEQ

0 – EB0 + hω

EQ1 – hω 2 + ΓQ2

+

cos α sin 2αhωK

+sin α cos 2αEQ

0 – EB0 – hω

EB1 – hω 2 + ΓB2

Vibronic lineshapes as a function of angle α for B1g vibronic modes

Vibronic lineshapes for B1g modes as a function of vibronic strength (α =1o)

Vibronic lineshapes for B1g modes as a function of vibronic strength (α =15o)

The B and Q bands for A2gsymmetry vibronic matrix elements

Franck-Condonpart

Herzberg-Teller part

∈ = Rσ0 2 sin2 α

EQ0 – hω 2 + ΓQ2

+cos2 α

EB0 – hω 2 + ΓB2

+ Rσ0 2a2g

2

cos2 α

EQ0 – EB

0 + hω2

EQ1 – hω 2 + ΓQ2

+

sin2 α

EQ0 – EB

0 – hω2

EB1 – hω 2 + ΓB2

Vibronic lineshapes for A2g modes as a function of vibronic strength (α =15o)

Ligand recombination is a sum of single exponential processes at room temperature

Difference spectrum from nanosecond transient absorption spectroscopy.

kescape

kbimolecular

His - FeP::CO

His-FeP + CO

His-FeP-CO

kgeminatehν

S(t) = Φgee– (k gem + k esc)t + Φbie– k bit

The heme iron center moves out of the heme plane and the porphyrin macrocycle

domes upon deligation of CO

CO is photolyzed Fe displacement

PlanarHeme

DomedHeme

The ligation of CO changes the spin state of the heme iron

dz2

dx2-y2

dxz,dyz

dxy

dz2

dx2-y2

dyz

dxz

dxy

Low spin Fe(II) High spin Fe(II)

S = 0 S = 2

Absorption spectra for Absorption spectra for MbCO MbCO and and deoxy deoxy MbMb

Soret Band Q Band

Infrared Spectroscopy

Structural heterogeneity in myoglobin has been proposed based on the observation

of multiple CO stretching bands• The bands are observed at 1966 cm-1,

1945 cm -1, and 1927 cm -1.• The heterogeneity has been attributed to

conformational substates.• Rebinding to each substate is also

observed to be non-exponential.• Are there many relevant tiers of

substates?

The origin of the A states is the hydrogen bonding conformations to CO

L29

H64F43

V68

Distal

Proximal

Protein Data Bank 2MGK

Infrared spectra of myoglobin

L29

H64F43

V68

Distal

ProximalQuillin, Phillips et al.JMB 1993, 234, 140-155

Wild type Mb IR spectra show multiple bands

Mutants at the V68 position also show multiple bands

L29

H64F43

V68Distal

Proximal

The H64V mutant shows a single IR band

L29

H64F43

V68Distal

Proximal

DFT calculation of νCO frequencies

Multiple hydrogen bonding interactions

Single hydrogen bonding interaction


No hydrogen bonding interaction


DFT calculation in an applied electric field+ + + + + + + + + + + +

− − − − − − − − − − − − − − − −

F

Franzen JACS (2002) 124, 13271


Park and BoxerJPC 1999, 103, 9013

Stark tuning rate is 2.4 cm-1/(MV/cm).This is value predictedfrom correlations shownon right.

Franzen JACS (2002) 124, 13271

Cryogenic Spectroscopy

At less than 10K photolyzed Mb*CO recombines by nuclear tunneling

k = Ae– HBA/RT

Current hypothesisThe iron coordinate controls the barrier to CO rebinding and explains the distribution of energy barriers.

Austin et al. Biochemistry 1975, 14, 5355

Experimental observation:

Mb*CO at t1

Steinbach et al. Biochemistry1991, 30, 3988

Iron relaxation hypothesis rests on the assignment of charge transfer band III

Experimental observation:

Mb*CO at t2



Experimental observation: Kinetic holeburning

Mb*CO t2 - t1



Assignment of band IIIThe interpretation of the data depends critically on the assignment of band III.

Band III has been assigned based on single crystal absorption data.

However, there have been problems with the interpretation from the very earliest experiments.

Interpretation of kinetic hole burning

Conformational substatesare correlated with theband III maximumand theenthalpy of rebindingandthe rate constant distribution.


ν (cm-1)

H (kJ/mol)

log(t/s)-6 -5 -4 -3 -2

Ultrafast Spectroscopy

Ligand dynamics, protein dynamics, and fast recombination can be

monitored by ultrafast spectroscopy

Mode lockedTi:sapphire Laser

ZAP!MbCOSample

Time resolution from100 fs to nanoseconds.

Nd:YLF Laser

Regenerativeamplifier

BBO ContinuumDelay line

Frequency doubled 100 fs pulses 1000 Hz

Ultrafast near-infrared spectroscopy shows that a protein relaxation

follows photolysis

Jackson, Lim, Anfinrud Chem. Phys. 1994, 180, 131-140

Band III at 10 ps, 10 ns,100 ns, and 10 µs

Non-exponential band IIIdecay in buffer solution η = 1cp

Mb:CO

Ultrafast mid-infrared shows CO trapping in the distal pocket

C O

O C

Two CO orientationsin infrared bands

B1 and B2

Spectral bands arerotamers of CO in Mb

Nature structural BiologyLim, Jackson, Anfinrud, 1997, 4, 209

What is the structure origin of the viscosity dependent protein relaxation?

The heme iron out-of-plane motion could change heme spectra

- band III a1u,a2u → dπ charge transfer band and therefore its frequency maximum is thought to depend on the heme iron position.

- rebinding enthalpy H is thought to depend on the heme iron position and there is a connection between H and the position of band III (kinetic hole burning).

The iron hypothesis“The red shift of the Soret band and band III by the same amount ∆ν = 140 cm-1 , suggests that changes in a single coordinate affect both the Soret band and band III. We expect that the primary coordinate associated with the heme relaxation from Mb* to Mb involves heme doming, characterized by the iron out-of-plane displacement”

Srajer and Champion Biochemistry (1991), 30, 7390

“The similarity of the deoxyheme (Soret) spectral changes to those observed for hemoglobin suggests that they correspond to a displacement of the iron relative to the heme plane that is coupled to a protein conformational change on the proximal side of the heme.”

Ansari, Hofrichter, Eaton et al. Science (1992), 256, 1976

The iron hypothesis“As the effective coordinate involved in the time-dependent barrier (responsible for non-exponential NO rebinding kinetics), we focus on the Fe-heme distance. It is, in turn, modulated by the protein relaxation. The latter is the major factor that regulates the distribution of geminate rebinding rates over a picosecond time scale at room temperature.”

Petrich, Karplus, and Martin Biochemistry (1991), 30, 3986

Iron

Dockingsite

What structural feature is responsible for the relaxation?

An alternative view of relaxationProtein relaxation involves CO dockingnot the iron out-of-plane motion.1. Iron out-of-plane motion occurs in < 2 ps. 2. NO recombination is biexponential. The two components are due to the rotamers of NO.

3. The Soret band and band III shift couple to distal mutations, and are not coupled to proximal mutations.

4. Band III is a vibronically coupled charge transfer band! Band III is coupled exclusively to non-totally symmetric modes.

5. MCD spectra are consistent with vibronic coupling model.6. Temperature dependent νFe-L mode is consistent withanharmonic coupling and not conformational substates indeoxy Mb.

Ultrafast NO recombination kinetics show an acceleration in rate with

increasing ηMaximum entropy method1. Two populations2. Tends towards a single

population at high η

Picosecond kinetics of photolyzed MbNO

Shreve, Franzen, and Dyer JPC 1999, 103,7969

FTIR data show that there are two populations of NO

Miller, Chance et al. Biochemistry 1997, 40, 12199

Photoproduct statescan interconverteven at 10 K

The lower wavenumberband corresponds to alower barrier

B1 B2

Protein relaxation can be monitored by Soret band and Band III

frequency shifts

Franzen and Boxer JBC 1997, 272, 9655

Band III shift Soret band shift

Comparison oftime-dependentfrequency shifts

290 K250 K

250 K

250 K

Observed in 75%glycerol/buffer

η = 30 cP at 290 Kη = 300 cP at 250 K

Time-resolved absorption spectroscopy shows protein relaxation following photolysis does

not depend on the proximal ligand

CO rebinding progressis monitored by ∆A.

∆A

Protein relaxation monitoredby ∆ν of the absorption band.

MbCO in 75% glycerol/buffer solution.

Franzen and Boxer JBC 1997, 272, 9655

Near-infrared bands of heme in deoxy myoglobin

MagneticCircularDichroism

Absorption

CircularDichroism

Single crystalpolarizedabsorptionspectra

c axis

a axis

a,c axis

Frequency (cm-1)

Band III

IIII

IVII

Eaton et al. JACS 1978, 100, 4991

Near-infrared charge transfer bands of the heme in deoxy myoglobin

Eaton et al. JACS 1978, 100, 4991

Raman Spectroscopy

A resonance Raman spectrum is obtained by laser light scattering

Laser Spectrograph

Detector

Sample

Inelastic light scattering produces a frequency shift. There is exchange of energy between the vibrations of the molecule and the incident photon.

Lens

Resonance Raman spectrum for Resonance Raman spectrum for excitation of hemeexcitation of heme SoretSoret bandband

Soret BandB Band Excitation Laser Q Band

Raman spectrum

Soret (B) band Resonance Raman spectra of MbCO and Deoxy Mb

ν8

B band Resonance Raman spectra of MbCO and Deoxy Mb

The cooperative R - T switch relies on iron displacement to communicate

between α and β subunits

Hemoglobin is composed of two α and two βsubunits whose structures resemble myoglobin.

Eaton et al. Nature Struct. Biol. 1999, 6, 351

Ultrafast resonance Raman spectroscopy shows that heme doming occurs in ≈1 ps

Equilibrium HbCO

Difference spectra obtainedby subtraction of the redspectrum from spectra obtained at the timedelays shown.

Franzen et al. Nature Struct. Biol. (1994) 1, 230

The frequency of the iron-histidine vibration shows strain in T state

The comparison of photolyzedHbCO in the R state andthe equilibrium T state.Hb*CO at 10 ns Fe-His = 230 cm-1

Deoxy HbFe-His = 216 cm -1The lower frequency indicatesweaker bonding interactionand coupling to bending modes.

λexc = 435 nmFe-His

Deoxy HbT-state

Hb*CO10 nsR-state

The motion of the F-helix tugs on the proximal histidine and introduces strain

The frequency lowering in the T state arisesfrom weaker Fe-His ligation and from anharmonic coupling introduced by thebent conformation of the proximal histidine.

FeN

NH

FeN

NH

R state T state

Time-resolved resonance Raman can follow the R - T structure change

Strain is introduced instages as intersubunitcontacts are made.Based on the x-ray datait was proposed that theiron displacement fromthe heme plane is a triggerfor the conformational changes.

Hb*CO

10 ns

100 ns400 ns1 µs

8 µs

15 µs40 µs60 µs

120 µs

Deoxy Hb

Time evolution

200 210 220 230 240Raman Shift (cm-1)

Scott and Friedman JACS 1984, 106, 5877

Many Peroxidases belong to the Cytochrome c Peroxidase family

PDB: 1A2FCytochrome c Peroxidase (CCP)Class: All α proteinsSuperfamily:

Heme peroxidasesFamily: CCP-like Goodin and McCree

Scripps Institute

PDB: 2ATJHorseradish Peroxidase (HRP)Class: All α proteinsSuperfamily:

Heme peroxidasesFamily: CCP-like Hendrickson et al.

Biochemistry (1998)37, 8054

Dehaloperoxidase is a peroxidase that belongs to the globin family

PDB: 1A6GMyoglobin (Mb)Class: All α proteinsSuperfamily:


Vojetchovsky,Berendzen,Schlichting

PDB: 1EW6Dehaloperoxidase (DHP)Class: All α proteinsSuperfamily:


Lebioda et al. J.Biol.Chem. 27518712 (2000)

MbDHP

Comparison of DHP and Mb Structures比起DHP和Mb 结构

Resonance Raman spectroscopy of iron-histidine stretching mode of

deoxy dehaloperoxidase

The frequency of the Fe-His mode is intermediate betweenthat of myoglobin (HHMb) and horseradish peroxidase (HRP).

Franzen et al., JACS (1998), 120, 4658-4661

Model for proximal hydrogen bonding

Peroxidase Catalytic TriadAsp-His-Fe

Franzen JACS 2001,123, 12578

Asp

HisFe

This is the “push”In peroxidase mechanism

Model for proximal hydrogen bonding

Dehaloperoxidase Catalytic TriadC=O-His-Fe?

Franzen JACS 2001,123, 12578

Backbone C=O

HisFe

Hydrogen bond strengthis intermediate betweenMb and HRP/CcP.

General expression for Raman scattering transition polarizability

The transition polarizability involves an electricfield interaction for the incident wave σ and thescattered wave ρ:

αρσ if=

i|eσ|n n|eρ| fEn – E f – hω – iΓn

Σn

Raman scattering transition polarizability for vibronic bands

Substituting in the H-T expansion we have for σ:

αρσ 01

′ =i0|eσ|r0

r0| ∂H /∂Q |n1

Er0 – En11|Q|0 n1|eρ|i1

En1 – Ei0 – hω – iΓnΣv

αρσ 01

′′ =i0|eσ|n0 0|Q|1

r1| ∂H /∂Q |n0

Er1 – En0r1|eρ|i1

En0 – Ei0 – hω – iΓnΣv

and for ρ:

Vibronic matrix elements for B1g and B2g vibronic modes

αxybg = – bgRσ02

sin2 α sin 2αhωΚ

+sin α cos α cos 2α

EB0 – EQ0 + hωΚ

EQ0 – hω 2 + ΓQ2

–

cos2 α sin 2αhωΚ

+sin α cos α sin 2α

EB0 – EQ0 – hωΚ

EB0 – hω 2 + ΓB2

– bgRσ0 2

sin α cos α sin 2α

EB0 – EQ0 – hωΚ

–sin2 α sin 2α

hωΚ

EQ1 – hω 2 + ΓQ2

+

cos2 α sin 2αhωΚ

–sin α cos α sin 2α

EB0 – EQ0 + hωΚ

EB1 – hω2

+ ΓB2

Vibronic Raman excitation profile as a function of angle α for B1g modes

Predicted Jahn-Tellerenhancement

Vibronic Raman excitation profile for the Q band for B1g modes

Vibronic Raman excitation profile as a function of vibronic strength of B1g

modes for α = 1o

Vibronic Raman excitation profile as a function of vibronic strength of B1g

modes for α = 15o

Vibronic matrix element for A2gvibronic modes

αxya2g = – a2gRσ

0 2cos α sin α

1/ EB0 – EQ0 + hωΚ

EQ0 – hω – iΓQ+

1/ EB0 – EQ0 – hωΚ

EB0 – hω – iΓB–

1/ EB0 – EQ0 – hωΚ

EQ1 – hω – iΓQ–

1/ EB0 – EQ0 + hωΚ

EB1 – hω – iΓB

The A2g modes can be easily assigned from the depolarization ratio. These modes are prediected to have ρ = ∞. Experimentally, large ρ >> 0.75are observed.

Vibronic Raman excitation profile as a function of vibronic strength of A2g

modes for α = 15o

Raman spectra for the Soret, Q, and III

Franzen et al. JACS (2002) 124, 7146

Q band Resonance Raman spectra

REPs of B1g modes

ν11

ν10

ν19

ν20 ν21

REPs of A2g modes

Band III resonance Raman spectra


REP of high frequency band III modes

ν10

ν13

ν11

ν19ν28


B1g modes

REP of high frequency band III modes


Significance of Mb substatesConformational substates must include functional tests forspecific functional states.

The biological role of myoglobin is consistent with relaxationand conformational states of the distal pocket.

There are specific conformational states that are important1. The docking site (transient)2. The distal histidine interaction (closed form)3. The open form

Inhomogeneous broadening is a background that is always present.

Deoxy Mb MbCO

The 150 cm-1 B1g mode is ν18 in MbCOand a mixed ν18, γ16 mode in deoxy Mb

The side view reveals significant out-of-plane character for ν18 in deoxy Mb

Deoxy Mb MbCO

Magnetic Circular Dichroism

The Perimeter Model

The porphine ring has D4h symmetry.The aromatic ring has 18 electrons.The p system approximates circular electron path.

N N

NN

m = 01

2

3

4

5

-1-2

-3

-4

-5

Φ = 12πeimφ

∆m=1∆m=9

MCD spectra

Franzen, JPC Accepted

∆ε ν = A1 –∂ f ν∂ν + B0 + C0

k BTf ν

∆εm ν

ε ν= A1µBD0

–∂ f ν∂νf

∆Lz = 2A1

D0

MbCO MCD spectra follow the PM

The spectra are A-term MCD as shown by the derivatives of the absorption spectrum (red). The (Q MCD) = 9 x (B MCD).

B Q


Deoxy MCD spectra are anomalous

C-term 4 time larger than MbCO!

B Q

A-term but with vibronic structure

MCD spectra: Vibronic couplingin the Perimeter Model


MCD spectra: Vibronic couplingin the Perimeter Model

Metal Porphyrin Vibronic Distortions

Conclusions for band IIIAlthough band III behaves as a ring-to-metalcharge transfer band it is vibronically coupledto the Soret band. It is not the iron-histidinemode that is coupled to the transition, but ratherin-plane non-totally symmetric modes. 1. The polarization arises from vibronic coupling

to in-plane non-totally symmetric modes.

2. The small charge displacement results fromstrong mixing of III with the Soret band.

3. The temperature dependence arises from thermalpopulation of the B1g mode ν18 at 150 cm-1.

Anharmonic Coupling

Temperature dependence of the H93G(Im) axial mode

Franzen, Fritsch, Brewer JPC Accepted

80 K

293 K

Temperature dependence of the H93G(2-Me Im) axial mode

Temperature dependence of the H93G(H2O) axial mode

Differences in anharmonicity

for axial ligands

Experimental approachshows that axial ligands differ.

Horse heart is closest toH93G(4-Me Im).

H93G(2-Me Im) is the most anharmonic.

DFT calculated mode-mode anharmonic

coupling

Comparison with experiment

Frequency shift of axial-ligand mode calculated for displacement of iron doming mode.

Only H93G(1-Me Im) excluded because of Fermi resonance.

Im4-Me Im

2-Me Im

4-Br Im

2,4-diMe Im

Iron-ligand stretching and doming modes

νFe-L νFe-doming

4-Me Im

1-Me Im

143 cm-1 77 cm-1

161 cm-1 81 cm-1

Iron-ligand stretching and doming modes

νFe-L νFe-doming

Im

4-Br Im

70 cm-1

87 cm-1

143 cm-1

170 cm-1

Significance of substatesFunctional test is needed.

In spectroscopy solvation states give rise toinhomogeneous broadening.

Propose the same distinction for kinetics.Non-exponential kinetics in myoglobin is dueto an inhomogeneous distribution.

The inhomogeneous linewidth depends on the environment and hence is not the samefor all proteins.

Myoglobin function depends on specific distal dynamics

Prevent autooxidation: docking site

Discriminate against CO binding: histidine 64

Trigger for O2 release: low pH form?

These functional criteria are all consistent withthe alternative view. They all involve dynamicson the distal side.

A picture of the open and closed states of myoglobin

H64pH 7 νCO = 1943 cm-1

pH 5 νCO = 1967 cm-1

Protein Data Bank 1VXC, 2MGKYang and Phillips JMB 1996, 256, 762

1920 1950 1980 1920 1950 1980

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Spectroscopic Probes of Protein Dynamics: Hemoglobin and ...franzen/public_html/CH736/lecture/Hb_Mb_Lecture.pdf · Structural heterogeneity in myoglobin has been proposed based on