Irine P. Muhiuddin

Evidence from time resolved studies of the P700(.+)/A1(.-) radical pair for photosynthetic electron transfer on both the PsaA and PsaB branches of the photosystem I reaction centre.

Kinetic analysis using pulsed electron paramagnetic resonance (EPR) of photosynthetic electron transfer in the photosystem I reaction centres of Synechocystis 6803, in wild‐type Chlamydomonas reinhardtii, and in site directed mutants of the phylloquinone binding sites in C. reinhardtii, indicates that electron transfer from the reaction centre primary electron donor, P700, to the iron–sulphur centres, Fe–SX/A/B, can occur through either the PsaA or PsaB side phylloquinone. At low temperature reaction centres are frozen in states which allow electron transfer on one side of the reaction centre only. A fraction always donates electrons to the PsaA side quinone, the remainder to the PsaB side.

Photoaccumulation of the PsaB phyllosemiquinone in photosystem I of Chlamydomonas reinhardtii.

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700(z.rad;+). Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700(z.rad;+), and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.

Proton ENDOR spectroscopy of the anion radicals of the chlorophyll primary electron acceptors in type I photosynthetic reaction centres

AbstractChlorin anion radicals have been produced by photoaccumulation of photosystem I and the type I reaction centresof the anoxygenic bacteria Chlorobium limicola and Heliobacterium chlorum. Proton electron nuclear double resonance(ENDOR) spectrometry of the photoaccumulated radicals demonstrates that the photoaccumulation technique is re-ducing chlorophyll anions rather than bacteriochlorophyll anions, indicating that photoaccumulation specifically re-duces the primary electron acceptors (A 0 ) in these anoxygenic reaction centres and is not reducing the antennabacteriochlorophylls. Detailed analysis of the in vivo proton ENDOR spectra in comparison with in vitro (bacte-rio)chlorophyll and (bacterio)pheophytin anion radicals points up differences between the environment of the A 0 inphotosystem I and the anoxygenic type I reaction centres. 2003 Elsevier B.V. All rights reserved. 1. IntroductionReaction centres are integral membrane pig-ment–protein complexes that convert the electro-magnetic energy of sunlight into chemicalpotential energy, thereby powering most of thebiological activity on the planet [1]. Reactioncentres are classed into one of two types accordingto the identity of the terminal electron acceptors[2]. In one group these are four-iron/four-sulphurclusters (Fe

14N Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy of the Cation Radical P840+, the Primary Electron Donor of the Chlorobium limicola Reaction Center

The electronic structure of the oxidized primary chlorophyll electron donor, P840+., of the green sulfur bacterium Chlorobium limicola has been investigated using electron spin echo envelope modulation (ESEEM) spectroscopy. This ESEEM investigation of the electron spin density distribution in the radical cation P840+. in membranes isolated from C. limicola confirms that the electron spin is shared eqully between the two bacteriochlorophyll a molecules. Observation of the small hyperfine couplings to the ring nitrogens by ESEEM gives results that are in agreement with those obtained from ENDOR measurements (S. E. J. Rigby, R. Thapar, M. C. W. Evans and P. Heathcote, FEBS Lett. 350,24–28, 1994) of the large hyperfine couplings to the methyl group protons. These results in combination with the Raman spectroscopy of P840 (U. Feiler, D. Albouy, B. Robert and T. A. Mattioli, Biochemistry 34,11099–11105, 1995) all indicate that the reaction center of green sulfur photosynthetic bacteria is functionally a protein homodimer providing a symmetrical protein environment for the primary electron donor.

Studies on the Interaction between Tyrosine Yz and the Mn complex

We have shown that YZ interacts with the Mn cluster of the WOC in native and modified samples to give split electron paramagnetic resonance (EPR) signals at cryogenic temperatures, and that this places YZ close to the Mn cluster. This EPR signal can therefore be used in native samples to investigate YZ in normal turnover. Our latest results show that YZ in oxygen-evolving preparations can function down to at least 5K and suggest that this arises from tyrosinate-like behaviour of YZ. If YZ is effectively deprotonated, or if it were involved in a permanent hydrogen bond that is modified between YZ redox states, YZ would be able to be oxidised at temperatures where proton movements are prevented, as observed. YZ would be effectively negative in the reduced form and neutral when oxidised, accounting for EPR and electrochromic shift data. We therefore favour an electrostatically controlled electron transfer mechanism, where at physiological temperatures the changes in pKa induced by the initial oxidation of P680 cause a wave of deprotonation and progressive reduction in redox potential, firstly of YZ and then the WOC. YZ is therefore rapidly oxidised by P680+, and at the same time deprotonation from the WOC occurs to complete the oxidation of YZ and this deprotonation in turn lowers the redox potential in the Mn cluster, promoting its oxidation by YZ. Domino deprotonation through a hydrogen-bonding network leads to proton release into the lumen.

Studies of the protein binding pocket for naphthoquinones in type I (ferredoxin-reducing) reaction centres.

We have demonstrated [1] that photoaccumulation under reducing conditions at 205K produces an electron paramagnetic resonance (EPR) signal arising from a stable semiquinone form of the phylloquinone electron acceptor (A1•−) in Photosystem I (PSI) preparations. Electron Nuclear Double Resonance (ENDOR) studies of the phyllosemiquinone [2] have detected hyperfine couplings arising from protons, and thus given information on the electronic structure of the phyllosemiquinone in PSI.