Mossbauer Spectroscopy, or nuclear gamma resonance spectroscopy, is one of the more highly sensitive techniques in spectroscopy. The technique examines transitions that occur from the nuclear ground state and a low energy excited state.1 The technique can be applied to many isotopic nuclei, but the most common is 57Fe. Applications of 57Fe Mossbauer Spectroscopy range from phase transformation studies in materials to probing catalytic properties of metals found in many proteins and enzymes. The following review will examine Mossbauer Spectroscopy and its application in finding the intermediate oxidation state of the single iron atom during the enzymatic cycle of phenylalanine hydroxylase (PheH). PheH is a monooxygenase that contains a single iron, which is responsible for catalyzing the hydroxylation of phenylalanine and in result producing tyrosine as shown in Figure 1.2
FIgure 1. The three step reaction of phenylalanine hydroxylase starts at a high spin Fe(II) and then goes to low spin Fe(IV) with the addition of O2. After hydroxylation of phenylalanine Fe(IV) reverts back to high spin Fe(II).
Mossbauer Spectroscopy
Rudolf L. Mossbauer was a graduate student at Heidelberg in 1957 when he started investigating resonant absorption of gamma rays. Previously, intranuclear properties were not considered relevant to chemical bonding simply because of the large energy difference, so essentially atoms were treated as if they were unbound. But, Mossbauer found that chemical bonds did affect the absorption of gamma rays.3 The Mossbauer Effect conjoined nuclear emission of gamma rays and nuclear absorption by the use of conservation of momentum and the Doppler Effect. By applying these classical concepts, the detection of quantum effects such as isomer shifts, quadrupole splittings, and hyperfine splittings possible, and they can be related to the properties and the environment of the Mossbauer nuclei.
Theory
Nuclear resonant absorption can be described as emission of a photon from an atomic species and absorption by the same species. One factor causes the separation of the emission and absorption lines that essentially limits nuclear resonant absorption, and that factor is recoil of the nucleus created from the emission of a gamma ray. Rudolf Mossbauer described it in terms of trying to throw a rock out of a boat in the summer compared to doing then on a frozen lake in the winter.4 The boat acting as the nuclei and the thrown rock is acting as the emission. In the summer, the boat absorbs some kinetic energy in the opposite direction of launch, consequently taking away from the kinetic energy put on the rock. When the rock is thrown in the winter, the boat does not absorb the energy as before, but instead the frozen lake absorbs and limits the energy loss. Nuclei recoil energies, or kinetic energy lost to the lake after throwing the rock, are characterized by the phonon energy of the lattice. When the lattice is loosely bound, the recoil energy is large, as is in the same case of the boat in water during the summer time. When the lattice of the nuclei is tightly bound, recoil energy is minimal, which is the case when a rock is thrown from a boat during the winter with a frozen lake. The process of observing different recoil energies that exist in a solid were sought out while Mossbauer was in graduate school and consequently lead to the Mossbauer Effect, which landed him a co Nobel Prize in Nuclear Physics in 1961.
Mossbauer Effect
When Mossbauer was studying gamma ray scattering, he noticed an increase in scattering of 191Ir at low temperatures, which was counter to classical predictions of the time. He hypothesized three different types of recoil energies for a lattice of atoms.3 The first type of recoil energy is large enough so that the atom will eject itself from the lattice, which ranges from 15 to 30 eV. The second type of recoil energy is too small for the atom to be ejected but still larger than the phonon energy of the lattice, so the atom will remain in the lattice but distribute its recoil energy throughout the lattice as an integer number of photons. The last type of recoil energy is lower than the phonon energy and as a result gives an increase of scattering at low temperatures. This is known as the Mossbauer Effect. Mossbauer interpreted this Figure 2. is the recoil energy. The shaded area is overlap of emission and absorption frequencies that are not affected by recoil that are necessary for nuclear resonance.
phenomenon by using energy and momentum calculations and showed that although the average emission process has recoil energy that shifts the emission energy out of the absorption energy range, a small fraction of atoms are not affected by recoil and absorption is possible. Figure 2. shows the distribution of emission and absorption, and the overlap of the two curves give the small fraction of recoilless atoms that are needed for resonance.5 To excite the less probable recoilless frequencies, a Doppler shift is required. The Doppler shift is obtained by moving the source or the absorber in the opposite direction of the recoil of emission. For example, 57Fe has a transition energy of 14.4 keV from nuclear excited spin state to ground state and has recoil energies that limit nuclear resonance by 10-2 eV, represented as in figure 2. Recoil-free energies, represented as the shaded area in figure 2., differ by 10-8 eV, which are detectable by shifting the Mossbauer source a few mm/s towards the absorber. By this way of Doppler tuning, the difference in chemical environment between the emitter and absorber can be observed. The resulting spectrum that arises from the differences in chemical environment is an inverted Lorentzian shape because no detection occurs from the emitting source when photons are in resonance with the absorber, but when resonance is not occurring there is detection of photons, which becomes the baseline of the spectrum. The spectrum produced varies by small changes in velocity that are small Doppler shifts needed for the recognition of the recoilless energies that are essential for nuclear resonance between two opposing Mossbauer nuclei. The occurrence of nuclear resonance enables the characterization of quantum effects of nuclear transitions produced by differences in chemical environment of the emitting and absorbing nuclei. The quantum effects are characterized by isomer shifts, quadrupole splittings, and hyperfine splittings. Because of the aim of this review, the focus will be on isomer shifts and quadrupole splittings.
Isomer Shift
The more common effects seen in Mossbauer transitions are isomer shifts. The isomer shift depends largely on the electronic charge density interacting with the change in nuclear charge density between the emitter and absorber in the Mossbauer system. The energy of the interaction between the nuclear and electronic charge is given , where is the nuclear charge density at a 3-dimensional point 'r' and V(r) is the external potential (orbital s-electrons) at the same point 'r', and dτ is the volume that it is integrated over.6 By Taylor series expansion of , the first three terms are
The first term contributes to the overall potential to the system. The second term is the dipolar interaction but it goes to zero because of the symmetry of the charge of the nucleus. The third term in Equation 3, describes the electric field gradient in the form of a 3x3 tensor, which can be written in terms of its principle values where i=x, y, z or i=1, 2, 3. Continuing with the examination of the third term, a few mathematical tricks yield information about the monopolar interaction and quadrupolar interaction (Eq. 4), which have different effects on spectra in Mossbauer Spectroscopy.
-v
v=velocity
0
+v
δ(v)=Isomer shift
Fig. 3 A spectrum of an isomer shift that is a function of velocity.
The monopolar interaction describes the isomer shift, while the quadrupole interaction gives a quadrupole splitting, which depends on the electric field gradient and will be discussed later. Focusing on the monopolar interaction, the spherically symmetric electronic potential is where is the electronic charge density at the nucleus. Treating the nucleus as a homogeneously charged sphere, the monopolar interaction energy between the nuclear charge density (ρ(r)n) and the electron charge density () goes as the integral . Integration over the radii of the emitting and absorbing nuclei gives the isomer shift, and the isomer shift can be measured with Mossbauer Spectroscopy from the difference of applied Doppler velocities between absorption lines of the sample and a source, which is shown in figure 3. Different compounds have different isomer shifts due to the differences in the electron densities of s-orbitals about the nucleus, which can cause the resonant velocity to be negative or positive. The isomer shift is commonly studied when compounds have few impurities or external chemical interactions, but for this review, the quadrupolar interaction dominates and is due to ligand binding to a single Fe atom inside the Phenylalanine hydroxylase.
Quadrupole Splitting
As previously indicated, the resonant absorption of 57Fe is a transition from nuclear spin state of ½ to nuclear spin state of 3/2. For a quadrupole splitting to occur, the nuclear spin (I) has to be greater than one, so a quadrupole splitting can only occur at the excited spin state I=3/2.6 The monopolar interaction comes from a spherically symmetric distribution of charge due to the interaction of s-orbital electrons, but the quadrupole interaction, , arises from a non-symmetric distribution of charge. This interaction will only be present for the interaction of p, d, and f-orbital electrons with the nucleus because of their lack of spherical orbital symetry. These orbitals produce an electric field gradient (EFG) that can be written as a tensor in terms of its largest principle value and an assymetry paramer η= (Eq. 5).
In the spin state, the nucleus has a nuclear quadrupole moment (Q). The interaction of the nuclear quadrupole moment and the EFG gives the quadrupole coupling and can be expressed as a Hamilitonian, . Its energies can be expanded to give Eq. 6.7
The quadrupole energy is expressed by nuclear spin state, nuclear spin operators and the nuclear constant terms, , the spin operator about the z axis and , the raising and lowering operators that correlate with the spin operators and . For simplicity, for a sytem that has η=0 and Vxx= Vyy, the system has rotational symmetry about the z-axis. This in part gives a quadrupole splitting between some nuclear spin states. Using the quantum notation state with I being the nuclear spin quantum number and m the orientation in relation to the EFG, the quadrupole energy for these states are = and = and is illustrated in an energy diagram and spectrum. (Figure 4.)
-v
v=velocity
0
+v
Eq=
I=3/2
I=1/2
m=±3/2
m=±1/2
Mossbauer transitions
Figure 4. The Mossbauer spectrum of a quadrupole interaction and energy diagram showing allowed Mossbauer transitions and the quadrupole splitting (Eq) of nuclear spin I=3/2. is the isomer shift.
Phenylalanine Hydroxylase
Phenylalanine hydroxylase, PheH, is a single, non-heme, iron monooxygenase that converts phenylalanine to tyrosine.8 PheH belongs to a family of tetrahydrobiopterin dependent aromatic amino acid hydroxylases that include tryptophan and tyrosine hydroxylase. PheH in mammals is found in the liver where its main role is to catalyze the first, and rate limiting, step for the conversion of excess pheynylalanine to tyrosine from the everyday diet.2 Mutations in PheH can cause an excess of phenylalanine in the blood system and result in phenylketonuria, whose symptoms include slowed brain development. All of the hydroxylases require the cofactor tetrahydrobiopterin, which functions as an electron donor, for catalytic activity.9 The PheH discussed here comes from the bacteria Chromobacterium violaceum (Cv), which closely resembles the fold and coordinating active site of the human PheH, whose crystal structure is shown in Figure Figure 5. Human PheH showing bidentate ligand glutamate (Glu), binding of two histidines (His), tetrahydropterin (BH4) cosubstrate and a nonreactivve thienyl alanine.
5.10 The crystal structure of the human PheH shows the iron bound by two histidines and a glutamate accompanied by thienyl alanine, which acts as a nonreactive substrate analogue.10 CvPheH has two to three water molecules that are bound to the active site.11 The binding of the amino acid substrate along with the interaction of the tetrahydropterin causes the elimination of two water molecules and converts glutamate from a monodentate to a bidentate ligand. After these reactions occur, a site for molecular oxygen to bind onto the iron atom is generated. When the molecular oxygen binds, the oxidation state of iron has been suggested to change from Fe(II) to Fe(IV) and after hydroxylation to revert back to Fe(II). Utilizing Mossbauer Spectroscopy and freeze quench methods, measurements at different proposed times of the overall reaction could give insight on the change in oxidation states of the single iron.12
Finding the Intermediate Oxidation State of Fe in Phenylalanine Hydroxylase
Panay et al.2 used Mossbauer Spectroscopy at 4.2 K to examine the state of the iron during the hydroxylation reaction in the Chromobacterium violaceum PheH. Freeze quench samples were made with a 1:1 ratio anaerobic mixture of CvPheH-Fe(II)-Phe-6-MePH4 complex with an O2 saturated buffer, so only one turnover was possible. Freeze quench along with Mossbauer Spectroscopy was administered at 0, 20, 100, and 400ms (Figure. 6).
As shown in figure 6., two, broad, distinct peaks with one peak close to 0 mm/s and the other close to 3 mm/s are present in the Mossbauer spectrum of a sample freeze quenched at 0 ms time. Simulation gave two overlapping splittings of Fe(II) with isomer splittings at 1.20 mm/s and 1.30 mm/s and two quadrupole splittings of 2.00 mm/s and 3.50 mm/s. This spectrum agrees with the first step in the three step reaction of PheH, which has a high spin Fe(II). Taurine:RKG dioxygenase (TauD) is a dioxygenase with a similar active site containing a mononuclear Fe having similar Mossbauer spectra. Evidence on Fe(II)→Fe(IV)→Fe(II) conversion during its catalytic cycle has been provided by interpretation of Mossbauer spectra; the Mossbauer spectra is supported by simulation, density functional theory, and X-ray diffraction.
Figure 6. Mossbaeur spectra taken at different freeze quench times during the PheH 3 step reaction Fe(II)→Fe(IV)→Fe(II).
In figure 6 at 20 ms time of freeze quench, a new peak appears around 1.00 mm/s, and the two Fe(II) peaks remain. The smooth simulation line gives an isomer shift of 0.28 mm/s and a quadrupole splitting of 1.26, with the second peak sitting under the line from Fe(II) at 0 mm/s. The new peak and simulation suggest that a population of Fe(IV) has formed because Fe(IV) have smaller quadrupole splittings than high-spin complexes such as Fe(II).1 In the TauD system, simulation, DFT calculations, and XRD were used as evidence of a Fe(IV) as an intermediate with an isomer shift of close to 0.30 mm/s and quadrupole splittings of 0.80 mm/s and 1.30 mm/s, which are almost identical to the results for PheH.
In figure 6 at 100 ms time of freeze quench, the spectrum is relatively the same as the 20 ms freeze quench spectrum, except a small loss in intensity occurs from the Fe(IV) peak at 1 mm/s. The small loss is due to the hydroxylation of phenylalanine and the return to the Fe(II) state in the third step of the PheH reaction.
Figure 7. Time formation of Fe(IV) showing increase at 20 ms and decrease of formation from 100 ms to 400 ms.
In figure 6 at 400 ms time of freeze quench, the Fe(IV) peak at 1 mm/s is almost completely gone. This is indicative of the Fe(IV) state returning to Fe(II) state, which follows the final step for the production of tyrosine in PheH.
The spectral results are informative, but is the iron actually working as a catalyst to produce tyrosine? Figure 7 shows the time formation of Fe(IV) that correlate with the same times of the appearance and disappearance of Fe(IV) in the Mossbauer spectra.2 With the evidence of the time formation of Fe(IV), the hydroxylation of phenylalanine to yield tyrosine was also compared with kinetic studies, which would give evidence of iron catalysis. The amount of tyrosine produced from the reaction was measured using high-performance liquid chromatography (HPLC) and tyrosine intrinsic fluorescence.
Figure 8. Left) The production of tyrosine at different steps of the PheH reaction and fit with a curve calculated with the three rate values on the right. Right) Three step reaction of PheH starts with Fe(II), phenylalanine and hydroxypterin which then reacts with O2 to form Fe(IV). Fe(IV)=O intermediate reacts with phenylalanine to yield tyrosine and in result Fe(IV)→Fe(II).
The three step reaction of PheH is described on the right side of Figure 8, and rate constants are: k1=19 mM-1s-1, k2=42 s-1, and k3=6 s-1.2 The left side of Figure 8 shows a surge in production of tyrosine in the first 200 ms that is followed by a slow linear increase and is fit by given rate constants. These rates predict an initial production of Fe(IV) followed by disappearance in ~100 ms, which is compatible with the Mossbauer freeze quench measurements.
Conclusion
Mossbauer Spectroscopy is a highly sensitive technique with applications that range from studying materials to iron containing enzymes. Freeze quench sample preparation is a good tool for examining protein activity at short reaction times. Isomer shifts and quadrupole splittings of different Fe oxidation states at different times in the reaction gave insight on the catalytic role Fe plays in PheH.
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9. Fitzpatrick, P. F., Mechanism of Aromatic Amino Acid Hydroxylation†. Biochemistry 2003, 42 (48), 14083-14091.
10. Andreas Andersen, O.; Flatmark, T.; Hough, E., Crystal Structure of the Ternary Complex of the Catalytic Domain of Human Phenylalanine Hydroxylase with Tetrahydrobiopterin and 3-(2-Thienyl)-l-alanine, and its Implications for the Mechanism of Catalysis and Substrate Activation. J. Mol. Biol. 2002, 320 (5), 1095-1108.
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