long term and with my own experimental contributions
An intriguing question which is still open for cytochrome p450cam what the principal oxygenating
species is there? Putatively it was assigned to oxyferril π-cation radical (classical compound I)
in analogy with chloroperoxidase, however there is no definitive experimental evidence for that yet.
To resolve the problem an original approach was proposed and implemented by C. Algad and D. Hilvert, LOC/ETH,
a mutant of p450cam with the cysteine ligand substituted by selenocysteine was synthesized. Hybrid QM/MM
calculations by S. Cohen et. al., 2006 of the active species indicate that Se-compound I will be formed
faster and consumed more slower comparing with wild type enzyme i.e. could be detected more easily.
In addition, 77Se enrichment is affordable and the presence of the radical on selenocysteine
can be detected via hyperfine interaction. Currently we conduct detailed EPR investigation of the resting
and substrate bound forms of mutant in frozen solution (C. Algad and D. Hilvert, LOC/ETH) and single crystals (I. Schlichting, F. Schwarz,
R.L. Shoeman, K. von König, MPI-Heidelberg, F. Lendzian, TU-Berlin). With I. Garcia Rubio and A. Schweiger†, LPC/ETH.
The Fe(III) ions in high-spin state show highly anisotropic EPR spectra. As a consequence the determination of
ligand hyperfine couplings in orientationally disordered systems containing high-spin Fe is a demanding task
for spectroscopists. For example nitrogen ENDOR spectra of metmyoglobin, well resolved at gzz position
(B0⊥heme plain), are structure less for in-plain orientations. In HYSCORE single-quantum 14N correlation
peaks become ridges and disappear, double-quantum ridges are more resistive. The goal here is to develop a strategy to obtain
highly resolved hfi map for the high-spin complexes using a set of advanced pulse EPR techniques and
multiple mw frequencies (in collaboration with M. Fittipaldi and S. van Doorslaer, University of Antwerp).
The paramagnetic properties of iron in heme are strongly affected by axial ligands. When the ligands are "
weak" (Fe is still in high-spin state) their assignment is a complicated task. However detailed analysis of
g-factors helps to make a conclusion about iron coordination as it was done for docking site of heme in the
CcmE protein shown on the left (in collaboration with M. Braun, L. Thöny-Meier, Microbiology/ETH).
With I. Garcia Rubio and A. Schweiger†, LPC/ETH.
Multiple-photon
processes are known in magnetic resonance. Examples are a) modulation of the static
magnetic field during microwave field irradiation; b)
tetrachromatic excitation (J. Hyde in cw EPR and S.Vega in pulse NMR).
Following to classical work of Shirley, who has implemented Floquet theory
to solve a eigenvalue problem of the periodic spin Hamiltonian, these
cases can be reformulated in a different language: case a) involves a
microwave σ photon and multiple radiofrequenczπ photons , σ±nxπ
; in case b) n microwave photons are involved, nxσ
, where n is an odd number. The case b) is well treated theoretically and many details
where verified experimentally. The case a) is less studied in sense of pulse magnetic
resonance, but routinely used in cw EPR practically since its discovery.
In this direction we found that an electron spin echo on two-photon transition,
σ±π (mw±rf), can easily be excited; longitudinal oscillating field can be used to manipulate mw transitions zeroing transition probability at certain field amplitude (new kind electromagnetically induced transparency). With Moritz Kälin, Matvey Fedin, Arthur Schweiger, LPC/ETH.
The discovery of a new type electron transfer center - binuclear copper complex CuA in cytochrome C oxidase has been rather surprising, considering the abundant mononuclear type 1 copper site which is prevalent in blue oxidases. Nice to mention that mixed valence nature of the CuA which has in oxidazed state one unpaired electron per two coppers Cu(1.5)Cu(1.5) was discovered by EPR. The complex has characteristic hyperfine pattern consisting 7 lines with splitting half of value typical for thiolate coordinated Cu (f.e. in azurin). Claire Slutter (who came to Weizmann from CalTech) made weak ligand mutations in CuA fragment in order to check how the ligands modulate electronic properties (and finally ET rate) of the complex. With Claire Slutter and Daniella Goldfarb
Ascorbate oxidase and laccase are blue copper oxidases which contain four copper binding sites per functional unit (see figure on the left); a type 1 site involved in electron uptake from the substrate, a type 2 site, and an EPR silent type 3 site which consists of a pair of anti-ferromagnetically coupled Cu(II) ions. The latter three Cu(II) binding sites are proximal and constitute a trinuclear center which is the catalytic dioxygen reduction site. To reveal the details of the dioxygen reduction at the trinuclear center we studied binding of an exogenous ligand - azide, trying to observe it via electron-nuclear double resonance. It was found that binding of azide to the protein leads to appearance of a new EPR signal at g<2 accompanied by decrease in intensity of the type 2 signals. The g < 2 signals in both proteins are assigned to an S = 1 dipolar coupled Cu(II) pair whereby the azide binding disrupts the anti-ferromagnetic coupling of the type 3 Cu(II) pair. Due to fast spin-lattice relaxation and a weakness of observed signals attributed to the azide bound trinuclear complex we were not able to observe clearly azide 14N signals in ENDOR despite our efforts. Only due to energy of Daniella Goldfarb a huge amount of experimental data was converted in a reasonable model. With Daniella Goldfarb.
The idea here was to
estimate "a glass disorder" hopping that exists some medium range
order (nanoscale) in the glassy materials. Non-Kramers ions, Tb3+ and Ho3+, were used
to probe the ligand field distribution. For rare earth non-Kramers
ions a degeneracy of energy levels is completely eliminated by local electric fields
but the resulting splitting of a non-Kramers doublet (could be two closely
spaced singlet levels as well) can be in a range reachable by EPR spectrometers. Partially expected, but any way surprisingly, very well resolved
spectra, look on the picture on the left, were observed at 9.4 GHz and
35.4 GHz. Probably, in between, the spectra where as good as at used
frequencies, but we had only X- and Ka-band pulse spectrometers. The latter
was designed specially for this project in 1991.
It features 2W pulse IMPATT source (pi/2-pi pulse sequence was 50-100
ns for g=2 with overcoupled rectangular cavity); operate in absolutely stable way at temperatures down to 1.5 K; two orientations of the microwave magnetic field respect
to static one are possible. Coming back to the glasses, we have observed the holmium
and terbium spectra with well resolved hyperfine structure in more than
ten different glasses. Attempts to estimate a dispersion of the zero-field splitting for selected,
via observing frequencies, paramagnetic centers gave a strange values
like 1% respect to these frequencies. Note that only one parameter of
the related spin Hamiltonians was used for the simulations - zero field
splitting. It was my PhD work. With S. Orlinskii and R. Rakhmatullin, MRS/KSU.
My diploma work was done in the MRS lab of KSU in the group guided by Andrej Alekseevich Antipin who was a
great experimentalist from whom I have learned how to do scientific work and how to deal with microwaves. Here
we have studied heat propagation through solids at helium temperature using a combination of EPR and heat pulse
techniques. The heat phonons were injected to the crystal LiYF4 doped with Er3+ using the
thin metallic film on its surface heated by short current pulse. Passing the crystal the phonons induce
transitions between Stark levels of Er3+
(as is shown on the left) creating non-equlibrium population of the excited states. EPR on first two
excited state was used to monitor phonon dynamics and their interaction with electron spins. This was a PhD
work of Yu. Rosentzwaig. With Yu. Rosentzvaig, R. Rakhmatullin, A. A. Antipin, MRS/KSU
This was my first scientific and experimental work (semester projects, 3rd and 4th year at the University).
The question was to observe a light echo in ruby in a way that the echo is radiated in the direction opposite
to the first excitation pulse. The work was done in Zavoisky Kazan Physical Institute of Russian Academy of
Science (KFTI) with V. Lobkov and E. Shtyrkov