Hydrogen Bonding to P700: Site-Directed Mutagenesis of Threonine A739 of Photosystem I in Chlamydomonas reinhardtii
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- 作者：Heike Witt ; Eberhard Schlodder ; Christian Teutloff ; Jens Niklas ; Enrica Bordignon ; Donatella Carbonera ; Simon Kohler ; Andreas Labahn ; and Wolfgang Lubitz
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：July 9, 2002
- 年：2002
- 卷：41
- 期：27
- 页码：8557 - 8569
- 全文大小：192K
- 年卷期：v.41,no.27(July 9, 2002)
- ISSN：1520-4995
文摘
The primary electron donor P700 of photosystem I is a dimer comprised of chlorophyll a (PB)and chlorophyll a' (PA). PA is involved in a hydrogen bond network with several surrounding amino acidresidues and a nearby water molecule. To investigate the influence of hydrogen bond interactions on theproperties of P700, the threonine at position A739, which donates a putative hydrogen bond to the 131-keto group of PA, was replaced with valine, histidine, and tyrosine in Chlamydomonas reinhardtii usingsite-directed mutagenesis. Growth of the mutants was not impaired. (i) The (P700+
- P700) FTIRdifference spectra of the mutants lack a negative band at 1634 cm-1 observed in the wild-type spectrumand instead exhibit a new negative band between 1658 and 1672 cm-1 depending on the mutation. Thisband can therefore be assigned to the 131-keto group of PA which is upshifted to higher frequencies uponremoval of the hydrogen bond. (ii) The main bleaching band in the Qy region of the (P700+ - P700) and(3P700 - P700) absorption difference spectra is blue shifted for the mutants by ~6 nm compared to thatof the wild type. A blue shift is also observed for the main bleaching in the Soret region. (iii) The (P700+- P700) CD difference spectrum of the wild type reveals two bands at 694 nm (positive CD) and 680 nm(negative CD) of approximately equal area. For each mutant, these two components are blue-shifted tothe same extent. The results strongly suggest that a blue shift of the Qy absorption band of PA is responsiblefor a blue shift of the exciton bands. (iv) Redox titrations yielded a decrease in the midpoint potential forthe oxidation of P700 by 32 mV for the exchange of Thr against Val. (v) ENDOR spectroscopy showsthat the hfc of the methyl protons at position 12 of the spin-carrying Chl PB is decreased due to theremoval of the hydrogen bond to PA. This indicates a redistribution of spin density in P700+ comparedto that in the wild type. This gives evidence for an electronic coupling between the two halves of thedimer in the wild type and mutants.
- P700) FTIRdifference spectra of the mutants lack a negative band at 1634 cm-1 observed in the wild-type spectrumand instead exhibit a new negative band between 1658 and 1672 cm-1 depending on the mutation. Thisband can therefore be assigned to the 131-keto group of PA which is upshifted to higher frequencies uponremoval of the hydrogen bond. (ii) The main bleaching band in the Qy region of the (P700+ - P700) and(3P700 - P700) absorption difference spectra is blue shifted for the mutants by ~6 nm compared to thatof the wild type. A blue shift is also observed for the main bleaching in the Soret region. (iii) The (P700+- P700) CD difference spectrum of the wild type reveals two bands at 694 nm (positive CD) and 680 nm(negative CD) of approximately equal area. For each mutant, these two components are blue-shifted tothe same extent. The results strongly suggest that a blue shift of the Qy absorption band of PA is responsiblefor a blue shift of the exciton bands. (iv) Redox titrations yielded a decrease in the midpoint potential forthe oxidation of P700 by 32 mV for the exchange of Thr against Val. (v) ENDOR spectroscopy showsthat the hfc of the methyl protons at position 12 of the spin-carrying Chl PB is decreased due to theremoval of the hydrogen bond to PA. This indicates a redistribution of spin density in P700+ comparedto that in the wild type. This gives evidence for an electronic coupling between the two halves of thedimer in the wild type and mutants.