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

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P141. Effect of Weak Interaction on the Electronic Structure and
Electrochemical Properties of Pseudoazurin Met16X Mutants
Y. Obara a , R. F. Abdelhamid a , K. Fujita b , D. E. Brown b , D. M. Dooley b , H. Tanaka c ,
I. Kitagawa c , M. Okada c , and T. Kohzuma a
a
Institute of Applied Beam Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan
e-mail: 06nd603s@hcs.ibaraki.ac.jp
b
Department of Chemistry, Montana State University, Bozeman, Montana 59717, U. S. A.
c
Hitachi Ltd , Omika-cho 7-1-1, Hitachi, Ibaraki 319-1292, Japan
Pseudoazurin (PAz) is a blue copper protein, which functions as an electron carrier in several denitrifying
bacteria. Very recently, we have reported the spectroscopic and electrochemical properties of Met16Phe,
Met16Tyr, and Met16Trp mutants to elucidate the effects of the π-π interaction at the active site of blue copper
protein [1, 2]. The introduction of aromatic amino acid in the vicinity of the blue copper active site shows
a drastic spectroscopic changing and significantly higher redox potential as compared to the wild-type protein.
Two different types of PAz mutants, Met16Lys and Met16Glu were constructed and studied to further explore
the effects of weak interactions involving electrostatic effects on the structure and function of the blue copper
active site.
Wild-type PAz exhibits three intense absorption bands at 454 (ε = 1700 M -1 cm -1 ) and 594 (ε = 3700 M -1 cm -1 ) nm
due to the SCys→Cu(II) ligand to metal charge transfer (LMCT) and a d-d transition at 753 nm (ε = 1700 M -1 cm -
1 ). These assignments are based on the electronic structure analysis by Solomon and co-workers [3]. Perturbation
of the ratios of the LMCT bands (A~460/A~600) by the substitution at Met16 reflects the structural changes around
the active site [4]. The ratio of the absorption at 460 and 600 nm, A~460/A~600 of Met16Lys and Met16Glu
pseudoazurin were estimated to be 0.54 and 0.64, respectively. These values are larger than that of wild-type
PAz, and the larger A~460/A~600 values of Met16Lys and Met16Glu suggest the larger population of rhombic
structure. X-band EPR spectra of the Met16Lys and Met16Glu variants showed almost identical spectra to the
spectrum of Met16Val variant, which shows rhombic EPR spectral pattern. The redox potentials of Met16Lys
and Met16Glu mutants were evaluated to be 306 and 245 mV vs NHE (pH 7.0), respectively. The redox
potentials of the Met16Lys and Met16Glu are reasonably explained by the electrostatic effect of the side chain
groups of Met16Lys and Met16Glu variants.
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.
References:
[1] R. F. Abdelhamid, Y. Obara, Y. Uchida, T. Kohzuma, D. M. Dooley, D. E. Brown, H. Hori, J. Biol. Inorg.
Chem, 12, 165 (2007).
[2] R. F. Abdelhamid, Y. Obara, T. Kohzuma, J. Inorg. Biochem, 102, 1373 (2008).
[3] E. I. Solomon and M. D. Lowery, Science, 259, 1575 (1993).
[4] A. A. Gewirth and E. I. Solomon, J. Am. Chem. Soc., 110, 3811 (1988).
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