Recruitment of a Foreign Quinone into the A1 Site of Photosystem I: II. STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF PHYLLOQUINONE BIOSYNTHETIC PATHWAY MUTANTS BY ELECTRON PARAMAGNETIC RESONANCE AND ELECTRON-NUCLEAR DOUBLE RESONANCE SPECTROSCOPY

Boris Zybailov,Art Van Der Est,Stephan G Zech,Christian Teutloff,T.Wade Johnson,Gaozhong Shen,Robert Bittl,Dietmar Stehlik,Parag R Chitnis,John H Golbeck

doi:10.1074/jbc.275.12.8531

Abstract

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance studies of the photosystem (PS) I quinone acceptor, A(1), in phylloquinone biosynthetic pathway mutants are described. Room temperature continuous wave EPR measurements at X-band of whole cells of menA and menB interruption mutants show a transient reduction and oxidation of an organic radical with a g-value and anisotropy characteristic of a quinone. In PS I complexes, the continuous wave EPR spectrum of the photoaccumulated Q(-) radical, measured at Q-band, and the electron spin-polarized transient EPR spectra of the radical pair P700(+) Q(-), measured at X-, Q-, and W-bands, show three prominent features: (i) Q(-) has a larger g-anisotropy than native phylloquinone, (ii) Q(-) does not display the prominent methyl hyperfine couplings attributed to the 2-methyl group of phylloquinone, and (iii) the orientation of Q(-) in the A(1) site as derived from the spin polarization is that of native phylloquinone in the wild type. Electron spin echo modulation experiments on P700(+) Q(-) show that the dipolar coupling in the radical pair is the same as in native PS I, i.e. the distance between P700(+) and Q(-) (25.3 +/- 0.3 A) is the same as between P700(+) and A(1)(-) in the wild type. Pulsed electron-nuclear double resonance studies show two sets of resolved spectral features with nearly axially symmetric hyperfine couplings. They are tentatively assigned to the two methyl groups of the recruited plastoquinone-9, and their difference indicates a strong inequivalence among the two groups when in the A(1) site. These results show that Q (i) functions in accepting an electron from A(0)(-) and in passing the electron forward to the iron-sulfur clusters, (ii) occupies the A(1) site with an orientation similar to that of phylloquinone in the wild type, and (iii) has spectroscopic properties consistent with its identity as plastoquinone-9.

Highlights

Electron paramagnetic resonance (EPR) and electronnuclear double resonance studies of the photosystem (PS) I quinone acceptor, A1, in phylloquinone biosynthetic pathway mutants are described
In PS I complexes, the continuous wave EPR spectrum of the photoaccumulated Q؊ radical, measured at Q-band, and the electron spin-polarized transient EPR spectra of the radical pair P700؉ Q؊, measured at X, Q, and W-bands, show three prominent features: (i) Q؊ has a larger g-anisotropy than native phylloquinone, (ii) Q؊ does not display the prominent methyl hyperfine couplings attributed to the 2-methyl group of phylloquinone, and (iii) the orientation of Q؊ in the A1 site as derived from the spin polarization is that of native phylloquinone in the wild type
The EPR and ENDOR spectroscopic results presented in this work provide strong support for the proposal that plastoquinone-9 is recruited by the menA and menB mutants as a substitute for the missing phylloquinone in PS I

Summary

Introduction

Electron paramagnetic resonance (EPR) and electronnuclear double resonance studies of the photosystem (PS) I quinone acceptor, A1, in phylloquinone biosynthetic pathway mutants are described. Pulsed electron-nuclear double resonance studies show two sets of resolved spectral features with nearly axially symmetric hyperfine couplings They are tentatively assigned to the two methyl groups of the recruited plastoquinone-9, and their difference indicates a strong inequivalence among the two groups when in the A1 site. Recent transient Qband EPR of P700ϩ A1Ϫ in PS I single crystals specify an angle of 65 Ϯ 20° between the quinone plane and the plane made up of the crystalline c axis (membrane normal) and the quinone carbonyl bond direction. We found that photoautotrophic growth under low light intensities and steady-state rates of flavodoxin reduction were relatively unaffected by the absence of phylloquinone To account for these results, we proposed that a foreign quinone, Q, had been recruited into the A1 site of PS I the menA and menB mutants. From EPR and ENDOR studies, we derive structural characteristics that are consistent with the identification of Q as plastoquinone-9 (Fig. 1)

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