Exploring the Electron Transfer Pathways in Photosystem I by High-Time-Resolution Electron Paramagnetic Resonance: Observation of the B-Side Radical Pair P₇₀₀⁺A_1B^– in Whole Cells of the Deuterated Green Alga Chlamydomonas reinhardtii at Cryogenic Temperatures

Abstract

Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-C2 symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P700(+)A1A(-) and P700(+)A1B(-) of the electron transfer chain. Results are presented first for the deuterated site-directed mutant PsaA-M684H in which electron transfer beyond the primary electron acceptor A0A on the PsaA branch of electron transfer is impaired. Analysis of the quantum oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the geometry of the B-side radical pair. The orientation of the g tensor of P700(+) in an external reference system is adapted from a time-resolved multifrequency EPR study of deuterated and 15N-substituted cyanobacteria (Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C.; Kothe, G. J. Am. Chem. Soc. 2001, 123, 4211-4222). Thus, we obtain the three-dimensional structure of the B-side radical pair following photoexcitation of PS I in its native membrane. The new structure describes the position and orientation of the reduced B-side quinone A1B(-) on a nanosecond time scale after light-induced charge separation. Furthermore, we present results for deuterated wild-type cells of C. reinhardtii demonstrating that both radical pairs P700(+)A1A(-) and P700(+)A1B(-) participate in the electron transfer process according to a mole ratio of 0.71/0.29 in favor of P700(+)A1A(-). A detailed comparison reveals different orientations of A1A(-) and A1B(-) in their respective binding sites such that formation of a strong hydrogen bond from A1(-) to the protein backbone is possible only in the case of A1A(-). We suggest that this is relevant to the rates of forward electron transfer from A1A(-) or A1B(-) to the iron-sulfur center F(X), which differ by a factor of 10. Thus, the present study sheds new light on the orientation of the phylloquinone acceptors in their binding pockets in PS I and the effect this has on function.

Species-dependent alteration of electron transfer in the early stages of charge stabilization in Photosystem I

“…Furthermore, the difference in the high-field emissive features in the two wild-type spectra implies that there may be a contribution from the B-branch radical pair in the spectrum of wild type C. reinhardtii. This is in line with two recent studies, which suggest that at low temperature a larger amount of B-branch electron transfer occurs in C. reinhardtii than in cyanobacterial PS I [11,12].…”

“…2, which shows a comparison of the protein-cofactor binding of the A 0A chlorophyll and the A 1A phylloquinone in the X-ray structure of wild type T. elongatus (top) and a structural model of the M688H PsaA variant (bottom). Differences in the behavior of these PS I variants at low temperature have also been reported between species [11,12]. An electron spin echo envelope modulation (ESEEM) study of whole cells of the wild type and M668H PsaB variant in Synechocystis sp.…”

“…PCC 6803 samples [19] but that both A-and B-branch radical pairs contribute to the low temperature spectra and OOPE traces from C. rheinhardtii wild type samples. [11,12,34] 4. Discussion…”

“…In a recent study, the structure of the B-branch radical P 700 + A 1B − was estimated from an analysis of Qband transient EPR data from the M688H PsaA variant in deuterated whole cells of C. reinhardtii [11]. Based on the geometry, it was suggested that the H-bond between A 1B and the protein backbone is weakened in the charge-separated state and that the faster rate of A 1 − to F X electron transfer in the B-branch compared to the A-branch may be due to a lowering of the reduction potential of A 1B as a result of the proposed weakening of the H-bond.…”

Directionality of Electron Transfer in Type I Reaction Center Proteins: High-Frequency EPR Study of PS I with Removed Iron–Sulfur Centers

“…This definitive observation of bidirectionality was later confirmed by a number of mutagenesis studies of Type I RCs. [21][22][23][24][25][26][27][28] In mutagenesis experiments, one of the ET pathways is shut down by altering the protein/cofactor structure, which leads to redirection of the ET from one branch to the other branch. This approach makes it impossible to determine the extent of asymmetry of ET along Aand B-branches.…”