Assignment of a kinetic component to electron transfer between iron-sulfur clusters F(X) and F(A/B) of Photosystem I.

We studied the kinetics of reoxidation of the phylloquinones in Chlamydomonas reinhardtii Photosystem I using site-directed mutations in the PhQ(A)-binding site and of the residues serving as the axial ligand to ec3(A) and ec3(B) chlorophylls. In wild type PS I, these kinetics are biphasic, and mutations in the binding region of PhQ(A) induced a specific slowing down of the slow component. This slowing allowed detection of a previously unobserved 180-ns phase having spectral characteristics that differ from electron transfer between phylloquinones and F(X). The new kinetic phase thus reflects a different reaction that we ascribe to oxidation of F(X)(-) by the F(A/B) FeS clusters. These absorption changes partly account for the differences between the spectra associated with the two kinetic components assigned to phylloquinone reoxidation. In the mutant in which the axial ligand to ec3(A) (PsaA-Met688) was targeted, about 25% of charge separations ended in P(700)(+)A(0)(-) charge recombination; no such recombination was detected in the B-side symmetric mutant. Despite significant changes in the amplitude of the components ascribed to phylloquinone reoxidation in the two mutants, the overall nanosecond absorption changes were similar to the wild type. This suggests that these absorption changes are similar for the two different phylloquinones and that part of the differences between the decay-associated spectra of the two components reflect a contribution from different electron acceptors, i.e. from an inter-FeS cluster electron transfer.

Bidirectional electron transfer in photosystem I: determination of two distances between P700+ and A1- in spin-correlated radical pairs.

The spin-correlated radical pair [P(700)(+)A(1)(-)] gives rise to a characteristic "out-of-phase" electron spin-echo signal. The electron spin-echo envelope modulation (ESEEM) of these signals has been studied in thylakoids prepared from the wild-type strain of Chlamydomonas reinhardtii and in two site-directed mutants, in which the methionine residue which acts as the axial ligand to the chlorin electron acceptor A(0) has been substituted with a histidine either on the PsaA (PsaA-M684H) or the PsaB (PsaB-M664H) reaction center subunits. The analysis of the time domain ESEEM provides information about the spin-spin interaction in the [P(700)(+)A(1)(-)] radical pair, and the values of the dipolar (D) and the exchange (J) interaction can be extracted. From the distance dependence of the dipolar coupling term, the distance between the unpaired electron spin density clouds of the primary donor P(700)(+) and the phyllosemiquinone A(1)(-) can be determined. The [P(700)(+)A(1)(-)] ESEEM spectrum obtained in wild-type thylakoids can be reconstructed using a linear combination of the spectra measured in the PsaA and PsaB A(0) mutants, demonstrating that electron transfer resulting in charge separation is occurring on both the PsaA and PsaB branches. The [P(700)(+)A(1B)(-)] distance in the point dipole approximation in the PsaA-M684H mutant is 24.27 +/- 0.02 A, and the [P(700)(+)A(1A)(-)] distance in the PsaB-M664H mutant is 25.43 +/- 0.01 A. An intermediate value of 25.01 +/- 0.02 A is obtained in the wild-type membranes which exhibit both spin-polarized pairs.

Bidirectional electron transfer in photosystem I: electron transfer on the PsaA side is not essential for phototrophic growth in Chlamydomonas.

We have used pulsed electron paramagnetic resonance (EPR) measurements of the electron spin polarised (ESP) signals arising from the geminate radical pair P700(z.rad;+)/A(1)(z.rad;-) to detect electron transfer on both the PsaA and PsaB branches of redox cofactors in the photosystem I (PSI) reaction centre of Chlamydomonas reinhardtii. We have also used electron nuclear double resonance (ENDOR) spectroscopy to monitor the electronic structure of the bound phyllosemiquinones on both the PsaA and PsaB polypeptides. Both these spectroscopic assays have been used to analyse the effects of site-directed mutations to the axial ligands of the primary chlorophyll electron acceptor(s) A(0) and the conserved tryptophan in the PsaB phylloquinone (A(1)) binding pocket. Substitution of histidine for the axial ligand methionine on the PsaA branch (PsaA-M684H) blocks electron transfer to the PsaA-branch phylloquinone, and blocks photoaccumulation of the PsaA-branch phyllosemiquinone. However, this does not prevent photoautotrophic growth, indicating that electron transfer via the PsaB branch must take place and is alone sufficient to support growth. The corresponding substitution on the PsaB branch (PsaB-M664H) blocks kinetic electron transfer to the PsaB phylloquinone at 100 K, but does not block the photoaccumulation of the phyllosemiquinone. This transformant is unable to grow photoautotrophically although PsaA-branch electron transfer to and from the phyllosemiquinone is functional, indicating that the B branch of electron transfer may be essential for photoautotrophic growth. Mutation of the conserved tryptophan PsaB-W673 to leucine affects the electronic structure of the PsaB phyllosemiquinone, and also prevents photoautotrophic growth.