ESEEM and Pulsed ENDOR of Metalloenzymes Arnold M ......Arnold M. Raitsimring John Enemark Eric...
Transcript of ESEEM and Pulsed ENDOR of Metalloenzymes Arnold M ......Arnold M. Raitsimring John Enemark Eric...
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