Complete thermal-unfolding profiles of oxidized and reduced cytochromes c

Journal of the American Chemical Society

Volumn 126, Issue 45, 2004, Pages 14684-14685

  Uchiyama, Susumu ^a   Ohshima, Atsushi ^a   Nakamura, Shota ^a   Hasegawa, Jun ^b   Terui, Norifumi ^c   Takayama, Shin Ichi Joseph ^c   Yamamoto, Yasuhiko ^c   Sambongi, Yoshihiro ^d   Kobayashi, Yuji ^a  

^a OSAKA UNIVERSITY   (Japan)

^b DAIICHI SANKYO CO   (Japan)

^c UNIVERSITY OF TSUKUBA   (Japan)

^d HIROSHIMA UNIVERSITY   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL ENZYME; CYTOCHROME C;

AMINO ACID SEQUENCE; ARTICLE; CIRCULAR DICHROISM; CYCLIC POTENTIOMETRY; ELECTRON TRANSPORT; ENZYME ACTIVE SITE; ENZYME ACTIVITY; ENZYME ANALYSIS; ENZYME METABOLISM; ENZYME STABILITY; ENZYME STRUCTURE; NONHUMAN; OXIDATION REDUCTION REACTION; PARAMETER; PROTEIN FOLDING; PSEUDOMONAS AERUGINOSA; REDUCTION; SEQUENCE ANALYSIS; STRUCTURE ANALYSIS; TECHNIQUE; TEMPERATURE DEPENDENCE; THERMODYNAMICS; THERMOPHILIC BACTERIUM;

CIRCULAR DICHROISM; CYTOCHROMES C; HEAT; OXIDATION-REDUCTION; PROTEIN FOLDING; THERMODYNAMICS;

EID: 8844241450     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja046667t     Document Type: Article

References (28)

1. (a) Moore, G. R.; Pettigrew G. W. Cytochromes c: Evolutionary, Structural, and Physicochemical Aspects; Springer-Verlag: Berlin, 1990.
2. (b) Battistuzzi, G.; Borsari, M.; Sola, M.; Francia, F. Biochemistry 1997, 36, 16247-16258.
3. (c) Terui, N.; Tachiiri, N.; Matsuo, H.; Hasegawa, J.; Uchiyama, S.; Kobayashi, Y.; Igarashi, Y.; Sambongi, Y.; Yamamoto, Y. J. Am. Chem. Soc. 2003, 125, 13650-13651.
4. One hundred twenty structures of cyt c including their wild type and variants are registered in the Protein Data Bank.
5. (a) Fisher, M. T. Biochemistry 1991, 30, 10012-10018.
6. (b) Bixler, J.; Bakker, G.; McLendon, G. J. Am. Chem. Soc. 1992, 114, 6938-6939.
7. (c) Pascher, T.; Gray, G. B. Science 1996, 271, 1558-1560.
8. (a) Lett, C. M.; Berghuis, A. M.; Frey, H. E.; Lepocl, J. R.; Guillemette, J. G. J. Biol. Chem. 1996, 271, 29088-29093.
9. (b) Uchiyama, S.; Hasegawa, J.; Tanimoto, Y.; Moriguchi, H.; Mizutani, M.; Igarashi, Y.; Sambongi, Y.; Kobayashi, Y. Protein Eng. 2002, 15, 455-461.
10. The airtight pressure-proof cell compartment with quartz windows was designed to tolerate 10 atm where the boiling temperature of water is higher than 180 °C. The pressure in the compartment was kept by nitrogen gas, which was connected to a gas cylinder via a pressure-reducing valve. The temperature of the cell was controlled and monitored by an electric heater and a thermistor mounted in the cell compartment on the bottom of the compartment. Ordinary CD cells, with any path lengths, were able to be set in the compartment. They were free from distortion because there was no pressure difference between the outside and inside of the cell. The distortion of the window of the compartment was small so that the baseline correction was easily done by the subtraction of a blank.
11. To verify reliability of the pressure cell compartment, we measured three CD spectra (200-250 nm) of PA at 25 °C: in the pressure-proof cell compartment at 10 atm, the same cell compartment at 1 atm, and the ordinary cell compartment at 1 atm. There was no difference in the spectra among them. Next, we tested thermal unfolding of PA in the ordinary cell compartment at 1 atm and in the pressure-proof cell compartment at 10 atm below 100 °C. The thermal unfolding profiles showed no difference between them. Together, these results prove that high pressure and the cell shape do not affect CD measurement.
12. 2. The method established here worked well, PA and HT being kept reduced in the temperature ranges to complete thermal unfolding.
13. The oxidized and reduced cyts c (final 10 μM) in 50 mM phosphate buffer (pH 7.0) and the same buffer containing 0.5 mM DTT, respectively, were subjected to the CD and visible spectra measurements. The temperature-dependent CD ellipticity change at 200-250 nm and the visible absorption change at 500-600 nm were followed in cuvettes of 1-mm and 1-cm path lengths, respectively. The spectra were recorded from 40 to 160 °C with temperature intervals of 2-20 °C. The CD ellipticity change at 222 nm (for both proteins) and absorption changes at 551 nm (PA) and at 552 nm (HT) were followed versus temperature.
14. Sanbongi, Y.; Ishii, M.; Igarashi, Y.; Kodama, T. J. Bacteriol. 1989, 171, 65-69.
15. (a) Hasegawa, J.; Yoshida, T.; Yamazaki, T.; Sambongi, Y.; Yu, Y.; Igarashi, Y.; Kodama, T.; Yamazaki, K.; Kyougoku, Y.; Kobayashi, Y. Biochemistry 1998, 37, 9641-9649.
16. (b) Hasegawa, J.; Shimahara, H.; Mizutani, M.; Uchiyama, S.; Arai, H.; Ishii, M.; Kobayashi, Y.; Ferguson, S. J.; Sambongi, Y.; Igarashi, Y. J. Biol. Chem. 1999, 274, 37533-37537.
17. (c) Hasegawa, J.; Uchiyama, S.; Tanimoto, Y.; Mizutani, M.; Kobayashi, Y.; Sambongi, Y.; Igarashi, Y. J. Biol. Chem. 2000, 275, 37824-37826.
18. (d) Sambongi, Y.; Uchiyama, S.; Kobayashi, Y.; Igarashi, Y.; Hasegawa, J. Eur. J. Biochem. 2002, 269, 3355-3361.
19. (e) Yamamoto, Y.; Terui, N.; Tachiiri, N.; Minakawa, K.; Matsuo, H.; Kameda, T.; Hasegawa, J.; Sambongi, Y.; Uchiyama, S.; Kobayashi, Y.; Igarashi, Y. J. Am. Chem. Soc. 2002, 124, 11574-11575.
20. ps, previously derived values under an oxidized condition at pH 3.64b were used. Detailed procedures were provided as Supporting Information. The thermodynamic parameters were determined by averaging those obtained at more than three different independent measurements.
21. (a) We assumed that the estimated ΔG values of the unfolding at 10 atm and at 25 °C are the same as that at standard condition (1 atm and 25 °C) based on the published study. Hawley, S. A. Biochemistry 1971, 10, 2436-2442.
22. (b) The redox potential of cyt c is scarcely affected by the pressure from 1 to 200 atm. Smith, E. G. J. Am. Chem. Soc. 1995, 117, 6717-6719.
23. (a) Hilgen-Willis, S.; Bowden, E. F.; Pielak G. J. J. Inorg. Biochem. 1993, 51, 649-653.
24. (b) Winkler, J. R.; Wittung-Stafshede, P.; Leckner, J.; Malmstrom, B. G.; Gray H. B. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4246-4249.
25. 0 is the redox potential.
26. (a) Fern, T.; Poscia, A.; Ascoli, F.; Santucci, R. Biochim. Biophys. Acta 1996, 1298, 102-108.
27. (b) Komar-Panicucci, S.; Weis, D.; Bakker, G.; Qiao, T.; Sherman, F.; McLendon, G. Biochemistry 1994, 33, 10556-10560.
28. (c) Bhuyan, A. K.; Udgaonkar, J. B. J. Mol. Biol. 2001, 312, 1135-1160.