Studies of heme-proteins by high-field multi-frequency ESR
with high sensitivity
Masayuki Hagiwaraa, Haruhiko Yashiroa,b, Masaki Horitanic, and Hiroshi HoridaKYOKUGEN (Center for Quantum Science and Technology under Extreme Conditions),
Osaka University, bPRESTO, Japan Science and Technology, cBiometal Science Laboratory,
RIKEN Spring-8 Center, and dGraduate School of Engineering Science, Osaka University

To elucidate the electronic structures of transition metal ions in a transition metal complex
is of great importance in chemistry and biology. Electron spin resonance (ESR)
spectroscopy has long been used extensively to investigate structural and electronic
environments of the paramagnetic metal centers. For many years, the conventional low
frequencies (< 35 GHz) ESR apparatus have been used for metal complexes with odd numberof unpaired electrons, i.e. half-integer spin systems (Kramers system), whereas paramagnetictransition metal complexes with an integer spin (non-Kramers system) were hardly
investigated by this method. Thus, the paramagnetic ions with an integer spin and a large
zero-field splitting (ZFS) have been regarded as ESR-inactive or ESR-silent species underconventional ESR techniques. The necessity for observing the ESR spectra in theseparamagnetic species is to extend the frequency and magnetic field range of ESR. In recentyears, high-field multi-frequency ESR (HFMF ESR) apparatus have been developed at someplaces including KYOKUGEN in Osaka University to investigate a variety of spin systems inthe transition metal complexes [1].
Iron ions are ubiquitous in biological systems, namely metalloproteins. Among ironcontaining metalloproteins, we have a particular class called hemoproteins, which haveiron-porphyrin, i.e. heme, as the prosthetic group in the active center. The electronic statesof the ferric (Fe3+) hemoproteins with a half-integer spin have been investigated in detail byconventional ESR techniques. Several years ago, the ferric high-spin myoglobin (Mb) wasmeasured with our HFMF ESR apparatus and the ZFS parameters were determined precisely[2]. On the other hand, the electronic structures of the ferrous (Fe2+) high-spin hemoproteins
with an integer spin have not been definitely analyzed. In spite of many efforts to detect theESR absorption of deoxy Mb and deoxy hemoglobin (Hb) by means of HFMF ESR
techniques, no meaningful results have yet been reported. It should be noted that theparamagnetic ions are buried in the biological macromolecule and thus the concentration of
the paramagnetic ions is very dilute and hard to detect the ESR signals as compared with thecondensed chemical substances.
The main aim of our study is to detect the ESR signals of hemeproteins with an integer spinsuch as the deoxy Mb and the deoxy Hb, and to analyze their electronic structures that

correlate with their biological functions, but it must be meaningful to establish analyticalmethods for an S = 2 integer spin system by using Mn(III)-containing protein molecules,
namely, Mn(III)-protoporphyrin reconstituted Mb, Mn(III)Mb. The aquomet Mb used in thisstudy was extracted from native sperm whale muscle and the Mn(III)Mb was prepared byreconstitution of apo Mb with the manganese (III) protoporphyrin IX. For HFMF ESR
measurements, we used a frozen solution of the Mn(III)Mb in potassium phosphate at pH 7.0.
The HFMF ESR measurements were performed with a millimeter wave vector network
analyzer and a 16 T superconducting magnet. In order to obtain a high sensitive ESR spectrafrom biological substances with dilute paramagnetic ions, we have developed home madeTE011 single-mode cylindrical cavities for 33, 50, 70, 90, and 130 GHz with high stablematching mechanism and the sensitivity of 1010 spins/G at T = 1.5 K [3].
Figure 1 shows the HFMF ESR spectra of the Mn(III)Mb in a frozen solution at 10 K forseveral frequencies between 33.3 GHz and 122.1 GHz and the magnetic fields up to 14 T [3].
To analyze the EPR spectra, we adopted the standard spin Hamiltonian for S = 2 spin statecomprised of the Zeeman and the ZFS terms given by,222
H = BB· ~g ·S + D (Sz – 2) + E (Sx – Sy )(1),
where B is the electron Bohr magneton, B the external magnetic field, ~g the g tensor ofMn3+ spins, S the S = 2 spin operator, D and
E the axial and rhombic ZFS parameters,
respectively. The observed MFEPR spectra(blue lines) were analyzed with the spectral
simulation software, and the red lines in Fig.
1 are the simulation results. The agreement
between the experimental and the simulated
spectra is satisfactory. Assuming isotropic
g-values, gx = gy = gz = 2.00, the ZFS
parameters were determined by thisFig. 1. HFMF ESR spectra of the Mn(III)Mb
comparison to be D = -3.79 0.03 cm-1 andE = 0.08 0.01 cm-1. In the presentation, these ZFS parameters will be compared with thosein Mn(III) porphyrin complexes and other Mn(III) complexes.
Finally, if we have the time, we will briefly talk about ESR studies on the deoxy Hb (Fe2+:S = 2), which is now in progress.

[1] J. Krzystek, A. Ozarowski, J. Telser, Coord. Chem. Rev. 250 (2006) 2308.
[2] Y. Miyajima, H. Yashiro, T. Kashiwagi, M. Hagiwara, H. Hori, J. Phys. Soc. Jpn. 73(2004) 280.
[3] M. Horitani, H. Yashiro, M. Hagiwara, and H. Hori, J. Inog. Biochem. 102 (2008) 781.