Denver 2008

The

2+ Mn -bicarbonate

complex in a frozen solution revisited by pulsed W-band ENDOR. Alexey Potapov and Daniella Goldfarb.

Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel.

Pulse sequences.

Introduction

1 Dasgupta, J.; Tyryshkin, A.; Kozlov, Y.; Klimov, V.; Dismukes, G. J. Phys. Chem. B 2006, 110, 5099—5111.

Hˆ = ge βe B0Sˆz + SˆDSˆ + π/2

π/2

MW

echo

π/2

π

Quadrupole interaction Zero-field splitting (ZFS) Zeeman interaction Electron Zeeman interaction Isotropic and anisotropic hyperfine interaction

π

MW

echo

Davies ENDOR

π

RF MW

Mims ENDOR

π

RF

π/2

π

W-band

FID FID-detected Davies ENDOR

π

RF

tmix

π/2

MW

echo

π

RF

(Sˆ(Aisoi + Ti )Iˆi − gn βn B0 Iˆi + Iˆi Pi Iˆi ) i

π/2

Mims Variable Mixing Time (VMT) ENDOR used for determining the absolute sign of the hyperfine coupling.

X-band

The central transition ms = |-1/2> <-> |-1/2> is resolved

The transition is broad and often not resolved

The intensities of transitions other than ms = |-1/2> <-> |-1/2> are smaller compared to central one.

The intensities of the other transitions are often comparable to central transition

The approximation of

The Electron spin is quantized along the effective axis determined by the ZFS and the external magnetic field.

g e β e B0 >> D is valid. The Electron spin is quantized along the direction of external magnetic field For 13C and 23Na the approximation of

The nuclear spin is quantized along the effective axis determined by the anisotropic hyperfine (and quadrupole) interaction and the external magnetic field.

g n β n B0 >> T|| , P||

All pulse ENDOR sequences suffer from a suppression hole at the nuclear Larmor frequency

is valid. The nuclear spin is quantized along the direction of external magnetic field. Shapes of the suppression holes RED – Mims ENDOR (t = delay between two first pulses) BLUE – echo-detected Davies ENDOR (t = length of the first inversion pulse) BLACK – FID-detected Davies ENDOR (t = length of the first inversion pulse) A is the hyperfine coupling.

ENDOR-effect

The replacement of Mg2+ cofactor by Mn2+ in many enzymes does not alter their catalytic activity, therefore Mn2+ often serves as an EPR probe to characterize the active site of the protein. Structural information can be extracted using advanced EPR techniques, probing subtle hyperfine interactions of Mn2+ with surrounding nuclei. One of the frequently found ligands of Mn2+ in proteins are carboxylate side chains. The Mn2+-bicarbonate complex serves as the simplest model system for the interactions of metal ion with such side chains. Such a complex has been already studied by means of Xband (9.5 GHz) pulsed EPR techniques1 proposing that two carbonates are coordinated to the Mn2+: one in a bidentate mode and the other in a monodentate mode. X-band measurements has several short comings when high spin systems are concerned and these can lead to ambiguous structure In this work we present pulse W-band ENDOR measurements of Mn2+ 13C enriched bicarbonate complexes aiming at the determination of the coordination shell of the Mn2+. The spectra reveal two types of 13C nuclei and the determined hyperfine couplings are interpreted in terms of Mn-13C distances, showing that there are two monodentate bound carbonate ligands in the complex. This conclusion was further corroborated by DFT (density functional theory) calculations. In addition, distant and proximal 23Na ions were resolved in the ENDOR spectra, indicating the presence of complexes with carbonate ligands bridging Mn2+ and Na+ cations.

X-band vs. W-band

The difference in Larmor of 23Na and 13C is small and their ENDOR spectra overlap. Data analysis requires subtraction of 13Clabelled and unlabelled bicarbonate.

1

The difference in Larmor frequencies is enough to prevent the overlap of ENDOR spectra

0

-10

-8

-6

-4

-2

0

2

A*t

νL (13C)

6

8

10

Field-swept echo-detected EPR spectrum of Mn2+-bicarbonate complex

Results 13C-ENDOR

4

3.20

3.25

+

+

3.40

3.45

3.50

3.55

3.60

DFT calculations.

+* * +

Mims (τ=350 ns)

3.35

Magnetic field, T

23Na-ENDOR

νL (23Na)

3.30

a

b

c

d

NaHCO3 100 mM

Davies (tinv=300 ns) Na2CO3 500 mM

FID-detected Davies (tinv=300 ns)

NaHCO3 100 mM

Mims Variable Mixing Time tm=0.5 ms

34

35

36

37

38

39

NaHCO3 500 mM

ν RF , MHz -2

Simulations (in RED): two slightly inequivalent nuclei with Aiso1=1.2 MHz T1=0.7 MHz Aiso2=1.0 MHz T2=0.6 MHz, or a distribution of hyperfine values.

-1

0

νRF-ν

ENDOR spectra recorded under significantly difference experimental conditions producing different suppression holes provide unique simulation parameters.

1 23

Na

Two types of 23 Na nuclei are identified from variations of lines intensities.

1H-ENDOR

-0.92

echo intensity

Davies 1H-ENDOR spectra RED-Hexa-aqua complex H2O/methanol BLUE-H2O/methnol-d4 BLACK-Bicarbonate in H2O/methanol

-0.96 -0.97 -0.98

Lower intensity of lines with small hyperfines indicates the displacement of methanol molecules

-0.99 -1.00 -1.01

-8

-6

-4

-2

0

2

νRF-νH, MHz

4

6

distance, Å

a. one monodentate carboxylate

1.15 , [-0.76 -0.59 1.35]

3.07

b. two monodentate carboxylates I

I 1.00 , [-0.70 -0.56 1.26] 1.73 , [-0.66 -0.58 1.23]

3.17 3.12

c. two monodentate carboxylatesII

0.91 , [-0.70 -0.55 1.25] 1.69 , [-0.66 -0.58 1.24]

3.15 3.12

d.monodentate+bidentate 1.02 , [-0.69 -0.55 1.25] carboxylate 4.60 , [-1.13 -0.88 2.01]

3.07 2.65

Conclusions.

-0.94 -0.95

calculated Aiso, [Txx, Tyy, Tzz ] MHz

The geometry optimization has been performed using Gaussian 03 program, The resulting geometry was used as an input for Orca program, which was used for hyperfine couplings calculations

The proton spectrum carries information on the solvent molecules.

-0.93

complex

2

8

Bicarbonate displaces also the water molecules

13C

W-band Davies and Mims ENDOR measurements, corroborated with DFT calculations, showed that in a water/methanol solution Mn2+ is coordinated to two bicarbonates/carbonate ligands in a monodendate mode. Moreover, the carbonate was found, at least in part of the complexes, to coordinate also Na+ and it acts as a bridging ligand. In addition, this study demonstrates the resolving power of W-band ENDOR and its simplicity as compared to X-band for the characterization of the coordination shell of Mn2+. Moreover, it shows that for small hyperfine couplings the suppression effect of pulse the ENDOR measurements has to be taken into account and several measurements under different conditions have to be carried out to obtain a unique analysis of the spectra. This research has been supported by Israel Science Foundation (ISF). D. G. holds the Erich Klieger Professorial Chair in Chemical Physics.