Combined use of Paramagnetic and X-ray Absorption spectroscopiesas a tool in the structural analysis of metallo-proteins

VIII° Scuola di Nazionale di Luce di SincrotroneLaboratori Nazionali Frascati,10-21 ottobre 2005

Bubacco Luigi Department of Biology, University of Padova, Italy.

In the biological prospective what are the relevant questionsto be addressed by a structural techniquein studing a the metal site of a protein?

a) Type o f proteins ligands (patterns recognition in the sequence?)

Genome wide search

Several complete genomes are available

Easy case: the ligandsof the metal ions define aregions of the protein beckbone

A HNXXH patter can beidentified in the sequences

Difficult case : the ligandsof the metal ion camefrom different regionsof the protein beckbone

In the biological prospective what are the relevant questionsto be addressed by a structural techniquein studing a the metal site of a protein?

a) Type o f proteins ligands (patterns recognition in the sequence?)

b) Type o f exogenous ligands (exchangeble coordination positionfor catalitically active enzyme)

c) Number of ligands

Pattern ID Ligand Pattern Count

0 his,his,his 54

1 cys,his,his 13

2 his,his,H2O 10

3 his,his,OH 6

4 his,his,mto 5

5 cys,his,met 4

6 cys,cys,cys 3

Pattern ID Ligand Pattern

Count

0 cys,his,his,met 129

1 his,his,his,his 38

2 his,his,his, H2O 32

3 cys,glu,his,his 10

4 c2o,his,his,his 9

5 cys,his,his,his 6

6 his,his,his,tpq 6

7 cuz,his,his, H2O 6http://metallo.scripps.edu/analysis/

In the biological prospective what are the relevant questionsto be addressed by a structural techniquein studing a metal site?

a) Type o f proteins ligands

b) Type o f exogenous ligands (exchangeble coordination position)

c) Number of ligands

d) Bond lenghts (distances)

Statistics for histogram: Cu-His N = 771Min = 1.6000 Max = 2.6000

Avg = 2.0918

Sites with four ligands

Statistics for histogram: Cu-His N = 700 Min = 1.6000 Max = 2.6000Avg = 2.0872

Sites with five ligands

In the biological prospective what are the relevant questionsto be addressed by a structural techniquein studing a metal site?

a) Type o f proteins ligands

b) Type o f exogenous ligands (exchangeble coordination position)

c) Number of ligands

d)Bond lenghts (distances)

f) Coordination geomentry (MXAN)

g) Ligand’s orientation

When can electron paramagnetic resonance be applied ?

a) Presence of a paramgnetic center copper … [Cu(I) Cu(II)]iron…manganese…nickel…..cobalt…

Biologically relevant forms that can not be studied:

Cu(I) d10 diamagneticZn(II)

Cw EPR on copper centers

NN

OO

N

2400 2600 2800 3000 3200 3400

Magnetic field (G)

A||

g||

2,14 2,16 2,18 2,20 2,22 2,24 2,26 2,28 2,30 2,32 2,34 2,36100

120

140

160

180

200

220

240

SODCarbpep

Hclp

saTymonoHcA

ToluicTy

NitroTy

HccmNiRaf

Hcov

hmTyhmTyhmTy

MimoTy

ncTy

AII

10-4 c

m-1

gII

Peisach Blumberg plot for Cu(II) center

Cw EPR on copper centers

NN

OO

N

2600 2800 3000 3200 3400 3600

Magnetic Field (G)

EPR line of a paramagnetic center in a solidinhomogeneously broadened.

Each spin packet can be considered independently from the other having its own Larmor frequency

Evolution of the magnetisation in a pulsed experiment.

Free induction decay and corresponding Fourier transform

Relaxation processes

The z-component of the magnetisation reverts to its equilibrium value Mo with a time constant called:Longitudinal relaxation time, T1 , this relaxation process is giving up energy to the surrounding and it is also called spin-lattice relaxation.

The randomisation of the spin direction on the xy-plane due to the fanning-out

occurs exponentially with a time constant that is called

Transverse relaxation time, T2. The relaxation involves the relative

orientation of the spins , T2 is also called spin-spin relaxation time.

Two pulses experiment

Electron Spin Echo detected EPR

W band (95 GHz) spectrum of Azurin (van Gastel)

τ τT

Hahn echo

Three pulses experiment

a b e f g

The nuclear modulation

The modulation that arises from the magnetic interactionsof the electron spin with the nuclear spins of the nearby atoms, such anisotropic hyperfine interactions are in the order of magnitude of the nuclear Zeeman energy consequentially the nuclear spin transitions frequencies contributes to the modulation on the ESE

Modulation function

VMOD = 1-k/2+ k/2[cos (ωβ τ) - cos (ωα τ) – 1/2 cos ((ωα + ωβ) τ) - 1/2 cos((ωα - ωβ) τ)]

k = 4 Pa Pf = [(ωI B)/(ωα ωβ)]2

ωα = [(A/2-ωI)2 + ( B/2)2]

ωβ = [(A/2-ωI)2 - ( B/2)2]

A = A|| cos2 θ + A⊥ sin2 θ

B = (A|| + A⊥) sin θ cos θ

A⊥ = Ad⊥ + aiso

A|| = Ad|| + aiso

Electron spin energy diagram for the remote nitrogen of a Cu(II) coordinated imidazole

Ms = ½

Ms = - ½

NQI lines

Broad line

Zeeman Shf NQI

(a)

Zeeman Shf NQI

Doublequantum line

NQI lines

0 1 2 3 4 5Frequency (MHz )

NQI lines Double

quantum line

Nuclear quadrupolar interaction

e2qQ = quadrupole coupling constant

η = qzz

qxx qyy ν ± = ¾ e2qQ (1 ± η/3)

ν o = ½ e2qQ η

Cu(II) N N

gz qz

qxqy

0 1 2 3 4 5Frequency (MHz )

NQI lines Double

quantum lineφ is the angle between the plane

defined by the His’ atoms and theperpendicular equatorial planeof the Cu(II)

Cu(II) N N

φ ≠ 0

Cu(II) N N

φ ≅ 0

Electron spin energy diagram for the remote nitrogen of a Cu(II) coordinated imidazole

Ms = ½

Ms = - ½

NQI lines

Broad line

Zeeman Shf NQI

(a)

Zeeman Shf NQI

Doublequantum line

NQI lines

0 1 2 3 4 5Frequency (MHz )

NQI lines Double

quantum line

0 1 2 3 4 5Frequency (MHz )

NQI lines Double

quantum line

ν d = double quantum transition frequency

ν d = 2 [ (ν i+ aiso/2 )2 + (e2qQ /4)2 (3 + η2)]1/2

aiso = isotropic electron nuclear coupling

ν i = nitrogen frequency

τ = 120 ns

Inte

nsity

[a.u

]

τ = 120 ns

b. Half-met tyrosinase + nitrophenolB=332.5 mT

c. Half-met tyrosinase + mimosineB=337.5 mT

a. Half-met tyrosinase B=336.0 mT

τ = 144 ns τ = 144 nsτ = 144 ns

τ = 168 ns τ = 168 nsτ = 168 ns

τ = 208 ns τ = 192 ns

frequency [MHz]

τ = 208 ns

0 1 2 3 4 5 6

τ = 280 ns

0 1 2 3 4 5 6

τ = 280 ns

0 1 2 3 4 5 6

τ = 280 ns

τ dependance analysis of the ESEEM

spectra of Halfmet Ty

and its complexes with inhibitors

2400 2600 2800 3000 3200 3400

Magnetic field (G)

τ τT

Hahn echo

345 mTIn

tens

ity [a

.u]

344 mT342 mT

b. Half-met tyrosinase + nitrophenol c. Half-met tyrosinase + mimosinea. Half-met tyrosinase

336 mT 337.5 mT332.5 mT

325 mT 327.5 mT322 mT

315 mT 315 mT312 mT

300 mT 300 mT300 mT

0 1 2 3 4 5 6

286 mT

0 1 2 3 4 5 6

285 mT

0 1 2 3 4 5 6

287 mT

frequency [MHz]

Field dependence analysis

of the ESEEM

spectra of Halfmet Ty

and its complexes with inhibitors

2400 2600 2800 3000 3200 3400

Magnetic field (G)

How do we single out the individual contributions of the remote nitrogens for more then one His ligand ?

HYSCORE (hyperfine sublevel correlation spectroscopy)

π/2 π/2 π/2

π

τ τt1 t2 Preparation Evolution

Mixing

Detection

0 2 4 6 8-8

-6

-4

-2

0

2

4

6

8

02

46

8 0

2

4

6

8

frequenc

y (MHz)

frequency (MHz)

HYSCORE spectroscopy on Octopus vulgarisHalf-metHc

0 1 2 3 4 5Frequency (MHz)

NQI lines Double

quantum line

Frequency (MHz)

Freq

uenc

y(M

Hz)

How does the magnitude of the coupling affects the experiment ?

Coordination mode for histidine ligandsMay be hard to get by X-ray absorption

Cuε

δ

b

ac Cu

δε

c

ba

Effects on the NQI parameters upon chemical substitutionon the imidazole ring

Cu

δ

ε

V.DUCROS et al., TYPE-2 CU-DEPLETED LACCASE FROM Coprinus Cinereous (PDB 1A65)

The hydrogen bond to the non coordinatingNitrogen of the ligand His

The hydrogen bond and NQI parameters

One more resason why the hydrogen bond is so relevant?

Cu(I) → Cu(II)… that is protein function

Pattern ID Ligand Pattern Count

0 his,his,his 54

1 cys,his,his 13

2 his,his,H2O 10

3 his,his,OH 6

4 his,his,mto 5

5 cys,his,met 4

6 cys,cys,cys 3

Pattern ID Ligand Pattern

Count

0 cys,his,his,met 129

1 his,his,his,his 38

2 his,his,his, H2O 32

3 cys,glu,his,his 10

4 c2o,his,his,his 9

5 cys,his,his,his 6

6 his,his,his,tpq 6

7 cuz,his,his, H2O 6http://metallo.scripps.edu/analysis/

Complexation of Copper(II) with Carbonate ligand in Aqueous Solution:A CW and Pulsed EPR Study

P.M. Schosseler, B. Wehrli, and A. SchweigerInorg. Chem. 1997, 36, 4490-4499.

CuOH2

OH2

2 OHH O

2OCO H 2

2HO CO pH 5.5

2OH

OH2

CuO

OC=OO=C

O

OpH 8.0

Detection by HYSCORE of a directly coordinated water moleculein copper proteins active sites

half-met tyrosinase

1 0 1 2 1 4 1 6 1 8 2 01 0

1 1

1 2

1 3

1 4

1 5

1 6

1 7

1 8

1 9

2 0

a

frequ

ency

ν2

f r e q u e n c y ν 1

1 0 1 2 1 4 1 6 1 8 2 0

b

H2O D2O

• Cu(II) coordinated H2O molecule with a dipolar coupling constant

of about 5 MHz

Structural model for the active site of Halfmet Ty

His324

His364

binuclear metal center

Arthropods oxy hemocyanin (Hazes et al.)

binuclear metal center

Mollusc oxy hemocyanin (Cuff et al.)

binuclear metal center

plant met phenol oxidase (Krebs et al.)

CuB

Copper site B 183 187 214 Oda AHNPIHY14YTSYDPLFFLHHSNVERLFTIWQ Odb THNAIHA14YTSFDPLFWLHHSQVDRLWAVWQ Odc AHNHIHA14TTTFDPIFILHHSNVDRIWAIWQ Odd LHNTIHS14FAAYDPIFFLHHSNIDRIWATWQ Ode AHNAIHS14YAAYDPIFYLHHSNVDRLWVIWQ Hpd LHNALHS14YTAFDPVFFLHHANTDRLWAIWQ Odf VHNSIHY14YSSFDPIFYVHHSNVDRLWAIWQ Hpg SHNAIHS14YTAYDPLFLLHHSNVDROWAIWQ Odg GHNAIHS14YTSYDPLFYLHHSNTDRIWSVWQ Soh GHNAIHS14YTSYDPLFYLHHSNTDRIWSVWQ Ysg LHNRVHV 9MSPNDPLFWLHHAYVDRLWAEWQ YNc VHNEIHD 9VSAFDPLFWLHHVNVDRLWSIWQ YHs MHNALHI 9GSANDPIFLLHHAFVDSIFEQWL YMm MHNALHI10GSANDPIFLLHHAFVDSIFDQWL Ece LHNWGHV21TSLRDPIFYRYHRFIDNIFQKYA Ecd LHNWGHV20TSLRDPIFYRYHRFIDNIFQKYA Lp2 LHNWGHV21TSLRDPIFYDWHRFIDNIFHEYK Pia LHNTAHV21TATKDPSFFRLHKYMDNIFKKHT

deletion

The perpendicular orientation of the aromatic planes ofthe HISs is conserved in all type 3 sites studied.

Cu

Second shell structural featuresCys-His bond

TYRO_HUMAN/111-456 FAHEAPAFLPWHRLFLLRWEQEIQKLTGTYR2_HUMAN/117-462 FSHQGPAFVTWHRYHLLCLERDLQRLIGTYR1_HUMAN/121-471 FSHEGPAFLTWHRYHLLRLEKDMQEMLQTYRO_NEUCR/1-373 CTHSSILFI…TWHRPYLALYEQALYASVQHCYA_OCTDO/388-696 CLHGMPVFPHWHRVYLLHFEDSMRRHGHCYB_HELPO/1-274 CVHGMPTFPSWHRLYVEQVEEALLDHGPPO_VITVI/122-440 QVHASWLFLPFH RYYLYFNERILAKLIDPPO_VICFA/112-431 QVHGSWLFFPFH RWYLYFYERILGSLINPPO_MALDO/108-425 Q I HNSWLFFPFH RYYLYFFEKILGKLINPPOD_LYCES/102-430 QVHNSWLFFPF H RWYLYFYESNAGKLIDPPOB_SOLTU/107-428 QVHFSWLFFPF H RWYLYFYERILGSLINPPOB_LYCES/106-435 QVHNSWLFFPF H RWYLYFYERILGKLIDPPOA_LYCES/106-434 QVHNSWLFFPF H RWYLYFYERILGSLIDPPO_SPIOL/120-455 EVHASWLFPSF H RWYLYFYERILGKLIN

http://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=TYRO_HUMANhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=TYR2_HUMANhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=TYR1_HUMANhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=TYRO_NEUCRhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=HCYA_OCTDOhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=HCYB_HELPOhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=PPO_VITVIhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=PPO_VICFAhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=PPO_MALDOhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=PPOD_LYCEShttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=PPOB_SOLTUhttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=PPOB_LYCEShttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=PPOA_LYCEShttp://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl?name=PPO_SPIOL

NN

O O

N

cw Pulsed techniques

a) Half met Ty

b) Half met Ty, D2O exchanged

c) Sample a plus p-Nitrophenol

d) Sample a plus L-mimosine

10 12 14 16 18 2010

11

12

13

14

15

16

17

18

19

20

frequency ν1 [MHz]

afre

quen

cy ν

2 [M

Hz]

10 12 14 16 18 20

c

10 12 14 16 18 20

b

10 12 14 16 18 2010

11

12

13

14

15

16

17

18

19

20

d

Structural model for the active site of half-met Ty

His324

His364

Paramagnetic NMR on [Cu(II) Cu(II)] met Type 3 sites

a b c

d

[Cu(II) Cu(II)]S =1/2 S =1/2

S =1

• The fast relaxation rate of the paramagnetic center allows for the selection of the contributions to the NMR signal.

• First observed for binuclear copper complexes

• Experiments at room temperature in solution

When can this technique be applied ?

a) Presence of a fast relaxing paramgnetic center (Cu(II), Co(II), Fe(III))

b) The protein size poses a limit around 30-40 KD

c) Protein solubility, since high concentrations are requiered

binuclear metal center

plant met phenol oxidase (Krebs et al.)

[Cu(I) Cu(I)]deoxy

[Cu(II) Cu(II)]met

Paramagnetic NMR

H2O2

[Cu(II) O22- Cu(II)]oxy

±O2

[Cu(I) Cu(II)]half-met

Cw and pulsed 1D and 2D EPR

Reversible oxygenbinding Hcs

Tyrosinase

Paramagnetic NMR

Cuε

δ

• at room temperature a paramagneticexited state S = 1 is present

• histidines have Nε co-ordinationto the metal ion

Paramagnetic NMRBinuclear copper proteins

Cuε

δ

H2O

D2O

• at room temperature a paramagneticexited state S = 1 is present for themet-Tyrosinase

• All six histidine residues are bound to the metal ions

Coordination mode for histidine ligands

Cuε

δ

b

ac Cu

δε

c

ba

Schematic representation of the T1 relaxation rates and NOE connectivities expectedfor the ring proton NMR signals of a histidine co-ordinated to a paramagnetic Cu(II) ion.

A) Streptomyces antibioticus met Tyrosinase

B) S. antibioticus met Tyrosinase plus chloride

C) S. antibioticus met Tyrosinase plus Kojic acid

Cu2+ Cu2+

NN N

NN

OH

O

OO

OH

N

Interaction between the Type-3 Copper Protein Tyrosinase and the Substrate Analogue p-Nitrophenol Studied by NMR

X-Ray spectroscopyAbsorption k-edge spectroscopy-coordination geometry- coordination number/bound length - metal metal distance

EXAFS - Extended X-ray Absorption Fine Structure

- coordination number/bound length- metal metal distance

8950 9000 9050 9100 9150 9200 9250

Energy (eV)

Cu K-edge XANES spectra of the met-Ty of Streptomyces antibioticus, met-Ty complex with Kojic acid (B) and met-Ty-Chloride complex (C).

Mxan simulation of met tyrosinase k-edge data

9000 9040 9080 9120 91600,000

0,004

0,008

0,012

0,016

Energy (eV)

X-R

ay A

bsor

banc

e

fitting met-ty

5 Å

N

N

N

N

Cu NN

N

N

N

N

Cu

N

N

O

O

N

N

N

N

Cu NN

N

N

N

N

Cu

N

N

O

O

N

N

N

N

Cu NN

N

N

N

N

Cu

N

N Cl

R

O

Bubacco et al., Figure 6

Plus Cl-

Plus Kojic acid

Met Ty

Cu K-edge XANES spectra based structural modelsof the met-Ty of Streptomyces antibioticus, met-Ty complex with Kojic acid and met-Ty-Chloride complex

Key information on the coordination number

Metal–metal distance as a key structural feature

Krebs et al.

…… Reaction mechanism

Paramagnetic NMR

Pulsed EPR

X-ray absorption

CuA of Cytochrome oxidase

C.OSTERMEIER,et al., STRUCTURE AT 2.7 A RESOLUTION OF THE Paracoccus Denitrificans CYTOCHROME C OXIDASE

WEFT-NOESY spectra (600-MHz) of SdII-CuA in H2O recorded in a 6 mM protein sample at pH 5.6 and 288 K showing the connectivities between the histidine imidazole ring protons..

Pattern ID Ligand Pattern Count

0 his,his,his 54

1 cys,his,his 13

2 his,his,H2O 10

3 his,his,OH 6

4 his,his,mto 5

5 cys,his,met 4

6 cys,cys,cys 3

Pattern ID Ligand Pattern

Count

0 cys,his,his,met 129

1 his,his,his,his 38

2 his,his,his, H2O 32

3 cys,glu,his,his 10

4 c2o,his,his,his 9

5 cys,his,his,his 6

6 his,his,his,tpq 6

7 cuz,his,his, H2O 6http://metallo.scripps.edu/analysis/

δ= contact shift

δ = Ao (sin2 θ + a cos θ + b)

NMR of paramagnetic proteinsIvano Bertini1 and Claudio Luchinat

[(A/h

) -c]

/b

φ (o)

3b3u 3b2u

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

0 90 180 270 360 The electronic configuration is a keyaspect in electron transferfuncion of CuA in cytochrome oxidase

Molecular biology as a tool to test assigments and consequentiallystructural and functional models

H117

H46

Azurin• Contribution of two His• one His has a 50% stronger coupling

AZURIN PDB file 1AZUSOURCE Pseudomona Aeruginosa

Azurin

H117

H46 Genetic engineering has allowed for the sequence assignment of the two histidines.(Canters et al)

H117G mutant

AZURIN PDB file 1AZUSOURCE Pseudomona Aeruginosa

Second shell structural featuresN190 mutant

Second shell structural featuresH- bonds

SaWTTY SaTyT51A

Km 8.1mM 6.32mM

Kcat 5.4 *104 min-1 7.54*103 min-1

Inhibitor SaWTTY SaTyT51A

Kojic acid 6.7mM 4.8mM

Mimosine 89.8mM 66.8mM

p-toluic acid 228mM 780mM

Benzoic acid 533mM 333mM

Fluoride 12mM 11mM

Chloride 140mM 112mM

In the biological prospective what are the relevant questionsthat can be answered by structural techniquesin studing a metal site?

EXFAS XANES pulsedEPR pNMR

EXFAS XANES pulsedEPR

EXFAS XANES pulsedEPR pNMR

EXFAS XANES

XANES pulsedEPR pNMR

pulsedEPR pNMR

a) Type o f proteins ligands

b) Type o f exogenous ligands

c) Number of ligands

d)Bond lenghts

f) Coordination geomentry

g) Ligand’s orientation/cord. mode

Biochem. J. (2003) 369, Crystal structure of nitrous oxide reductase from Paracoccus denitrificansat 1.6 Å resolution Tuomas HALTIA et al.

binuclear metal center