ИСТИНА |
Войти в систему Регистрация |
|
ИСТИНА ИНХС РАН |
||
The electron acceptors of Photosystem I (PS I) have very low reduction potentials, because one of the PS I main functions is the ability to effectively reduce low-potential NADP to NADPH. The most common low-potential artificial electron acceptor is methylviologen (MV), which interacts with the terminal PS I iron-sulfur cluster FA/FB. The ability of MV to interact with the quinones in the A1-site of the PS I is currently not well understood, while the molecular mechanisms of electron transfer on the acceptor site of PS I remain to be clarified. Here we address this issue by incorporating the high potential quinone, 2,3-dichloro-1,4-naphthoquinone (Cl2NQ) into the A1 binding sites of PS I to prevent forward electron transfer to the iron-sulfur clusters. To incorporate Cl2NQ we used PS I from the menB variant of Synechocystis sp. PCC 6803. Inactivation of the menB gene, which codes for a naphthoate synthase, inhibits the biosynthesis of phylloquinone and in its absence, plastoquinone-9 (PQ) binds to the A1A and A1B sites. Because PQ binds relatively weakly, it can be substituted to a wide variety of naphthoquinones by incubation in solution. The kinetics of the charge recombination in PS I complexes containing either PQ (PQ-PS I) or Cl2NQ (Cl2NQ-PS I) in the A1-site was studied by transient absorption spectrometry at 700 and 820 nm. In the absence of MV the kinetics of P700+ reduction in PQ-PS I was mainly due to the back-reaction from the terminal FA/FB iron-sulfur centers. Addition of low concentration of MV resulted in a significant deceleration of recombination kinetics cased by the electron flow from FB- to MV, thus preventing the back-reaction. Further increase of the MV concentration did not essentially affect the P700+ reduction kinetics. In case of Cl2NQ-PS I samples, the flash-induced kinetics of P700+ re-reduction was significantly faster, then in PQ-PS I samples, because the reduction potential of Cl2NQ was more positive than that of FA/FB and the electron transfer to the iron-sulfur clusters did not take place. Thus the back-reaction was due to recombination from the Cl2NQ-. The addition of low concentration of MV did not affect the kinetics of P700+ reduction. The increase of MV concentration resulted in slowing down of the recombination. The essential decrease of the effect of MV was due to the structural and thermodynamic constraints preventing its interaction with Cl2NQ in the A1-site. However, at high concentration of MV the electron flow either directly from Cl2NQ or via the iron-sulfur clusters FX, FA and FB to MV was observed in a fraction of PS I. The continuous-wave EPR experiments data provided evidence for this assumption. Analysis of P700+ decay kinetics exhibits decrease of the reduction rate and significant growth of P700+ signal amplitude in the presence of high concentration of MV.