ESEEM and Pulsed ENDOR of Metalloenzymes(Multi-frequency, S-D bands) Studies and something else…

Arnold M. RaitsimringAndrei AstashkinJohn EnemarkEric KleinKayunta Johnson-Winters

Debbie Baute and Daniela GoldfarbThe Weizmann Institute of Science, Rehovot

76100, Israel

Oleg G. Poluektov Chemistry Division, Argonne National Laboratory, Argonne, Illinois 60439

Peter Caravan, Harvard Medical School, 149 Thirteenth Street, Suite 2301 Charlestown, Massachusetts 02129

Y. Song, T. J. Meade, Departments of Chemistry; Biochemistry and Molecular and Cell Biology and Radiology, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208,USA

Cys

SO4,PO4AsO4

pterinAxial 17O

Equatorial 17O

(D)

Sulfite oxydase

Cl

ESEEM

Hfi/nqi parameters Structure functions

0 10 20 30 40 50 60 70

Frequency/MHz

50 52 54 56 58 60

Δ=1.65 MHz

6200 6250 6300 6350 6400 6450

Magnetic field /G

FT E

SEEM

ES

E a

mpl

itude

0 10 20 30 40 50

Frequency/MHz

Δ≈3.1MHz

2νH

νH

FT E

SEEM

magnetic field/G

3350 3400 3450 3500 3550

ρ

MoS

OH

S

S

O

Ka and X band FTs of the four-pulse integrated ESEEMs and ESE detected field sweep EPR spectra of R55M in H2O buffer. The field positions at which spectra were acquired are shown by the rectangles. Operational frequencies : 17.44 GHz (left panel) and 9.47 GHz (right panel).

bacterial SO, R55M mutant

B≅ 10 MHz

Ku band

X-band

Red spectrum –D -buffer

Most splittings are ~0.45 MHz, giving a nuclear quadrupole coupling constant of ~1.5 MHz. ForR160Q the splittings and the constant are unusually large (~1.4 and 4.5 MHz, respectively).Experiments were performed in Ka‐band (~29.3 GHz), employing τ‐integrated 4‐pulse ESEEM.Temperature ~20K. (r160q is human SO mutant)

Maximum Quadrupole Splittings of Axial 17O Ligands (near gz)

frequency/MHZ

0 5 10 15 20 25

05

1015

2025

Quadrupole splittings/2

1H “folded” frequency

17O I

1H “folded” frequency

0 5 10 15 20 25

05

1015

2025

frequency/MHZ

gy

HYSCORE projection; fundamental line line; /2~0.55MHz; nqcc= 3.6MHz

Quadrupole splittings

Example of HYSCORE spectrum at gy position B= 10,806 G; Ka-band (left). Right – same spectrum after 450 rotation. Quadrupole splittings are readily measured from projection selected by cursors.

Plant SO, “blocked form

Plant SO

Q ≈ 0.7 MHz

A ≈ 4.4 MHz

νO ≈ 6.07 MHz

HYSCORE of lpH R160Q in H217O.

(axial 17O)νmw = 29.548 GHzBo = 10523 G (gZ) tp = 14, 14, 27, 14 nsτ = 180 nsT = 20 K

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

02

46

810

1214

1618

2022

2426

2830

3234

3638

40

(17,23.5)

gy

0

MH

z

0 5 10 15 20

-20

-15

-10

-5

MHz

A≅17 MHz(-14.5 2.5 )

Ka-band (strong interaction)

W-band (weak interaction)

10 12 14 16 18 20 22 24 26 28 30

nqcc=6.8MHzδ ≈ 1.05 MHz

HYSCORE of lpH R160Q in H2

17O. (equatorial 17O) 29.55 GHz

(-+ quadrant)

MHz

Ka-band experiment,Plant (At) SO

33S

nqcc/2

Ka-band ( 29 GHz) ESEEM spectra of [33S]At-SO , primary echo (a) FT spectra of the field-integrated two-pulse ESEEM of [33S]At-SO (upper trace) and [32S]At-SO (lower trace).(b) HYSCORE spectrum of [33S]At-SO obtained at the low-field EPR turning point (gZ) where the Zeeman and hyperfine interactions approximately cancel each other. The frequency of this inter-doublet transition is: νid e2Qq/2and as e2Qq/h≈36 MHz.The major offdiagonal cross-peaks at (3,11) MHz belong to |1/2↔|−1/2 transitions of 33S.

I≅3.4-3.5 MHz

Cl

Ka-band experiment; 29.56 GHzHYSCORE detection of Cl- in SO.|T⊥| = 0.2 ± 0.05 MHz, aiso = 4 MHz,e2Qq/h = 3 MHz

Tanford, C. and Roxby R. Biochemistry 1972, 2192–2198.Bashford, D. and Karplus, M. J.Phys. Chem. 1991 9556–9561

Cys

SO4,PO4AsO4

pterinAxial 17O

Equatorial 17O

(D)

Sulfite oxydase

Cl

Cys H- C-band

Ka-band

W-band

Ku-band

P- X-Ku bandS- Ka-band

As- W-band?Ka band

Pulsed ENDOR of Gd based MRIagents: Ka, W or D-band?

H2O Counting

Gd aq8 water ligands

-4 -3 -2 -1 0 1 2 3νRF- ν17O/MHz

17O Mims ENDOR spectra of Gdaq in H2O17/ methnol solution collected in 3 mw bands (black-Ka -band, red-W-band and green- D band ) ;± ½ electron manifolds. For Gdaq cfi parameter, D, ~300G; for D/B 0.01 cfi effects can be neglected

16 18 20 22 24-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006no

rmal

ized

EN

DO

R in

tens

ity

RF frequency/MHZ

W-band experiment

Gdaq/8

-0.020

-0.015

-0.010

-0.005

0.000

0.005

RF frequency/ MHz24 25 26 27 28 29 30

D-band experiment

Gdaq/4

HSA boundGdaq/4

RF frequency/MHz

W-band experiment

17 18 19 20 21 22 23

0.992

0.994

0.996

0.998

1.000

1.002

17O W-band experiment, background test

RF frequency/MHz

Pulsed Dipolar SpectroscopyUsing Gd-based labels Ka-W band

Scheme 1. Modification of oligonucleotides withGd(III) chelates via click chemistry.

9000 9500 10000 10500 11000 11500 12000

0

Field/gauss

Ka-band

-500 0 500 1000 1500 2000 2500 3000

-0.08

-0.07

-0.06

-0.05

-0.04

-0.03

-0.02

-0.01

0.00

0.01

lnV

t

Initial 4-pulse DEER kinetics,various average concentrations

1:2:3

obs

pump

/ns

Blue, green and red curves: calculated kinetics using Gaussians in which δ=14Å andxo varied as 55,60 and 65Å.

0 500 1000 1500 2000 2500 3000

0.980

0.982

0.984

0.986

0.988

0.990

0.992

0.994

0.996

0.998

1.000

λo≅0.016

Via

0 500 1000 1500 2000 2500 3000

0.980

0.982

0.984

0.986

0.988

0.990

0.992

0.994

0.996

0.998

1.000

λo≅0.016

Via

0 500 1000 1500 2000 2500 3000

0.980

0.982

0.984

0.986

0.988

0.990

0.992

0.994

0.996

0.998

1.000

t/ns

λo≅0.016

Via

20 40 60 1000 80

)/)xxexp{()r(f 220 δ−∝

x/Å20 40 60 1000 8020 40 60 1000 80

)/)xxexp{()r(f 220 δ−∝

tt1.0021.0021.002

-500 0 500 1000 1500 2000 2500 3000

-0.08

-0.07

-0.06

-0.05

-0.04

-0.03

-0.02

-0.01

0.00

0.01

t/ns

lnV,

ln V

int

-500 0 500 1000 1500 2000 2500 3000

0.020

0.015

0.010

0.005

0.00

-lnVi

a

-500 0 500 1000 1500 2000 2500 3000

-0.08

-0.07

-0.06

-0.05

-0.04

-0.03

-0.02

-0.01

0.00

0.01

t/ns

lnV,

ln V

int

-500 0 500 1000 1500 2000 2500 3000

-0.08

-0.07

-0.06

-0.05

-0.04

-0.03

-0.02

-0.01

0.00

0.01

t/ns

lnV,

ln V

int

-500 0 500 1000 1500 2000 2500 3000-500 0 500 1000 1500 2000 2500 3000t/ns

ACKNOWLEDGMENTThis research was supported by the Binational Science Foundation

(USA-Israel, BSF#2006179) and NIH 1R01 EB005866-01

)12(~22

2)()()(2

+++=+= II

IIII mQBmmmν

νννν βασ

ESEEM- combination lines

νI νβνα

aiso+Tzz

2Q

I=5/2- 5linesI=1- 2 lines

2νI +B2/ 2νI

2νI

quadrupole sublines4QIν

B)Im(Q)T(aIνα,βν ZZiso 4

212~

21

++++±−=

For S=1/2

IνB

)Im(Q)T(aIνα,βν ZZiso 4

212~

21

++++±−=

For S=1/2)12(42

3~ 2

−= II

qQeQ

So, for fixed orientation the “fundamental” spectrum without quadrupole interaction is 2 lines and quadrupole interaction splits each line in 2I sub-lines

νI νβνα

aiso+Tzz

2Q

I=5/2- 5linesI=1- 2 lines

HOH3.1 Å

SS

OMо

Scys

O

(V)

2–X SO3

cSO R138(hSO R160)

wild type cSO active site R138Q cSO active site

cSO Q138(hSO Q160)

Wild Type and R138Q Active Site Structures from Chicken SO

Oax

Oeq Oeq

Oax

MPTMPT

Kisker, C., et al., Cell 1997, 91, 973  Karakas, E., et al., J. Biol. Chem. 2005, 39, 33506