ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P95. UV Resonance Raman Spectroscopic Studies of Azurin I and Azurin II
from Alcaligenes Xylosoxidans NCIM
S. Kimura, R.F. Abdelhamid, T. Kohzuma
Applyed Beam Science, Ibaraki University, 310-8512, Mito, Japan
e-mail: 07nd603y@mcs.ibaraki.ac.jp
Azurin is a blue copper protein, which functions as an electron donor to nitrite reductase. A denitrifying bacteria,
Alcaligenes xylosoxidans NCIMB 11015 has two different types of azurins, azurin I (AzI) and azurin II (AzII)
[1]. Resonance Raman spectroscopic technique is strong methods to obtain the structure of a certain
chromophore having specific electronic absorption in the visible region. Oxidized azurin has a intense blue color
due to the SCys→Cu(II) ligand to metal charge transfer (LMCT), but the reduced form of azurin does not have
any electronic absorption in the visible region. UV resonance Raman (UVRR) spectroscopc technique is a
powerful technique to elucidate the structure and dynamics of protein molecules, which does not have electronic
absorption in the visible region. Most of all protein molecules has electronic absorption band in the UV region
due to the electronic absorption of aromatic amino acids. UVRR spectra of AzI and AzII were measured to
elucidate the protein structural differences between the oxidized and reduced forms under the various conditions.
UVRR of AzI and AzII were measured by the excitation at 244 nm. UVRR of AzI and AzII were readily to be
assigned according to the previous report [2]. Raman bands at 1628cm -1 (Y8a, ring stretch mode), 1207cm -1
(Y7a, ring-C stretch mode), and 1173cm -1 (Y9a, CH in-plane bending mode) are contributed from tyrosine
residues. Raman bands at 1360 cm -1 and 1340 cm -1 are assigned to W7 Fermi doublet for N1C8 stretching mode
of tryptophane indole. The intensity ratio of the W7 Fermi doublet (I1360/I1340) is known to reflect the
environment of tryptophane indole [2, 3]. The intensity ratio of the W7 Raman bands of AzI and AzII were
calculated to be 1.0 and 0.8, respectively. The environment of tryptophane residue in AzI and AzII are estimated
to be hydrophobic and hydrophilic, respectively, from the intensity ratio of the W7 Fermi doublet Raman bands
of AzI and AzII. Detailed UVRR spectroscpic studies of those oxidized and reduced azurins from Alcaligenes
xylosoxidans NCIMB 11015 under the various pH conditons will be disscussed.
Acknowledgement: A part of this work is supported by Research Promotion Bureau, Ministry of Education,
Culture, Sports, Science and Technology (MEXT), Japan to TK, a Grant-in-Aid for Scientific Research from
JSPS (No. 18550147), Japan to TK, and the Project of Development of Basic Technologies for Advanced
Production Methods Using Microorganism Functions by the New Energy and Industrial Technology
Development Organization (NEDO) to TK.
_____________________________________________________________________
214