EPR and ENDOR investigation of the primary electron acceptor radical anion QA.bul.- in iron-depleted photosystem II membrane fragments.

Abstract

Photosystem II (PS II) membrane fragments were treated with trypsin at pH = 7.4 followed by incubation with o-phenanthroline and lithium perchlorate. This procedure removes and/or decouples the non-heme Fe2+ associated with the quinones QA and QB in the PS II reaction center (RC). Treatment of such samples (referred to as iron-depleted) with sodium dithionite or illumination in the presence of dichlorophenol indophenol (DCIP) and sodium ascorbate yielded EPR spectra similar to those of the plastoquinone-9 (PQ-9) radical anion generated in organic solvents. Q-band EPR yielded the principal values of the g-tensor for PQ-9.- in 2-propanol and QA.- in PS II. Electron nuclear double resonance (ENDOR) experiments were performed both on PQ-9.- in vitro and on QA.- in the iron-depleted PS II samples. For the former a complete set of isotropic 1H hyperfine coupling constants and hyperfine tensors of the two methyl groups and the alpha-proton were obtained. On the basis of H/D exchange experiments two different hydrogen bonds could be detected in frozen solution that are formed between the carbonyl oxygens of the radical and protons from the surrounding solvent molecules. The hydrogen bond distances were estimated using the point-dipole model. 1H-ENDOR spectra of QA.- in iron-depleted PS II samples have been measured in buffers made in H2O and D2O. The spectrum in deuterated buffer allowed the determination of two different methyl group hyperfine tensors. Differences detected between the spectra in protonated and deuterated buffer reveal the hyperfine tensors of two exchangeable protons belonging to hydrogen bonds between the oxygens of QA and specific protein residues. The assignment of these hydrogen bonds in PS II is discussed and compared with the situation found in the bacterial reaction center.

Isolation and properties of PS II membrane fragments depleted of the non heme iron center

“…This finding indicates that the 'iron depleted' samples do not contain a non heme iron center which can be oxidized by Ka[Fe(CN)6] to Fe 3+ thereby acting as electron acceptor for very fast Q2" reoxidation. In perfect agreement with this conclusion, the same sample type was recently shown to lack any magnetic interaction of a non heme iron center with QA" [14]. Two basically distinct explanations can be offered for this effect: (i) the 'iron depletion' procedure really extracts the non heme iron from PS II or (ii) the Fe 2+ remains still bound but the micro environment is markedly changed so that the iron center is transferred from the high spin into the low spin (S = 0) diamagnetic state concomitant with a drastic increase of the redox potential that prevents its reoxidation by K3[Fe(CN)6].…”

“…Iron depleted samples were prepared as described in MacMillan et al [14]. After the final centrifugation step the samples were resuspended in 20 mM MES pH 6.5, 10 mM NaC1 and 30% (w/v) sucrose to chlorophyll concentrations of about 5 mg/ml.…”

“…The second procedure was first developed for isolated reaction centers from anoxygenic purple bacteria where the Fe 2÷ could be reversibly removed [13]. An application of this method in PS II appears to be more difficult ( [14] and references therein). Until now it was not even clear whether or not the Fe 2+ is really removed from PS II or simply transferred to the low spin state by the 'extraction procedure'.…”

“…The present study describes experiments to clarify this point and to optimize the preparation procedure. Based on M6ssbauer spectroscopic data it is unambiguously shown that a complete removal of the non heme iron center from PS II can be achieved by using twice the previously developed 'iron depletion' method [14].…”

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I

“…1). For the two CH 3 groups, different hyperfine couplings have been observed for plastoquinone-9 radical anions in liquid and in frozen alcoholic solution, as well as for the reduced primary acceptor Q A Ϫ in PS II (34). The inequivalence of the CH 3 groups was largest in frozen alcoholic solution with two hyperfine coupling tensors of A ʈ ϭ 8.58 MHz, A Ќ ϭ 5.29 MHz and A ʈ ϭ 6.68 MHz, A Ќ ϭ 3.70 MHz, respectively.…”