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J. Biol. Chem., Vol. 281, Issue 7, 4318-4325, February 17, 2006

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pH-dependent Equilibrium between Long Lived Near-UV Intermediates of Photoactive Yellow Protein^*

From the Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

The long lived intermediate (signaling state) of photoactive yellow protein (PYP_M), which is formed in the photocycle, was characterized at various pHs. PYP_M at neutral pH was in equilibrium between two spectroscopically distinct states. Absorption maxima of the acidic form (PYP_M^acid) and alkaline form (PYP_M^alkali) were located at 367 and 356 nm, respectively. Equilibrium was represented by the Henderson-Hasselbalch equation, in which apparent pK_a was 6.4. Content of - and/or -structure of PYP_M^acid was significantly greater than PYP_M^alkali as demonstrated by the molar ellipticity at 222 nm. In addition, changes in amide I and II modes of -structure in the difference Fourier transform infrared spectra for formation of PYP_M^acid was smaller than that of PYP_M^alkali. The vibrational mode at 1747 cm^-1 of protonated Glu-46 was found as a small band for PYP_M^acid but not for PYP_M^alkali, suggesting that Glu-46 remains partially protonated in PYP_M^acid, whereas it is fully deprotonated in PYP_M^alkali. Small angle x-ray scattering measurements demonstrated that the radius of gyration of PYP_M^acid was 15.7 Å, whereas for PYP_M^alkali it was 16.2 Å. These results indicate that PYP_M^acid assumes a more ordered and compact structure than PYP_M^alkali. Binding of citrate shifts this equilibrium toward PYP_M^alkali. UV-visible absorption spectra and difference infrared spectra of the long lived intermediate formed from E46Q mutant was consistent with those of PYP_M^acid, indicating that the mutation shifts this equilibrium toward PYP_M^acid. Alterations in the nature of PYP_M by pH, citrate, and mutation of Glu-46 are consistently explained by the shift of the equilibrium between PYP_M^acid and PYP_M^alkali.

Received for publication, June 13, 2005 , and in revised form, December 19, 2005.

^* This work was supported by grants-in-aid for Scientific Research (C), for Scientific Research on Priority Areas "Molecular Nano Dynamics" from Ministry of Education, Culture, Sports, Science and Technology and a grant from a Foundation for Nara Institute of Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Spring-8/Japan Synchrotron Radiation Research Institute (JASRI), Sayo, Hyogo 679-5198, Japan.

² To whom correspondence should be addressed. Tel.: 81-743-72-6101; Fax: 81-743-72-6109; E-mail: imamoto{at}ms.naist.jp.

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				J. Hendriks and K. J. Hellingwerf pH Dependence of the Photoactive Yellow Protein Photocycle Recovery Reaction Reveals a New Late Photocycle Intermediate with a Deprotonated Chromophore J. Biol. Chem., February 20, 2009; 284(8): 5277 - 5288. [Abstract] [Full Text] [PDF]